I would like to know if this works on human.

what happens when the group neurons are not working. Can this problem be fixed to help with a person memory.

POINT 3

POINT TWO

PERHAPS POINT 1

if the new plan doesn't actually help the economy, it means nothing. given the state-GREAT DEPRESSION

Ignoring trolls hinges on the assumption that when trolls seek attention, but they'll lose interest when they are ignored. While this may be true for sometimes, ignoring continual harassment doesn't solve the underlying problem, especially when trolls escalate their behavior to force a reaction. It is insufficient in more serious cases of online harassment. This implies that if harassment continues, it’s due to the victim’s failure to “manage” their harasser.

They want to embarrass individuals in a lighthearted way. However, some pranks can cross a line, causing discomfort or distress, much like modern trolling, where the intent is to provoke a reaction, often at someone else's expense.

People make trolling for long and short times, but both to attract people's attention and emotional reactions. Trolling can significantly undermine constructive dialogue online by intentionally provoking negative emotions, often leading to conflict rather than meaningful exchange. This behavior not only derails conversations but can also create a toxic environment.

Button Clicked event Label: Menu Item View Conversations

Universaliteit gebaseerd op basisemoties: - Antecedenten: zelfde reactie op stimuli. - Fysiologie: overeenkomstige neurale paden bij bepaalde emoties. - Subjectieve ervaring: universele emotionele ervaring. - Herkenning: dezelfde emoties herkennen is vrijwel universeel. - Samenhang in emotionele reactiesystemen: compontenten van emotionele expressie (=gezicht, stem, etc.) zijn universeel. - Betekenis geven: universele reactie.

Sternberg's 3 vormige intelligentie: - Analytisch. - Creatief. - Praktisch.

Algemene intelligentiefactor (=G); - Verbaal redeneren. - Kwantitatief redeneren. - Abstract visueel redeneren. - Kortetermijngeheugen.

Flynn Effect=omgevingsfactoren die IQ verhogen over generaties heen.

Bell Curve=genetische factoren.

Redenen van culturele verschillen in cognitie:<br /> - Situationele factoren/ experimentele onderzoeksontwerpen. - Attributiebiases zijn universeler dan gedacht. - Analytische waarnemingen werden niet gekoppeld aan interne locus of contral/ holistische waarnemingen werden niet gekoppeld aan externe locus of control. - Sommige geheugendelen zijn universeler. - Trainingseffecten worden niet in overweging genomen.

3 dimensies symboliseren in 2: westerse culturen zien vaker in 2d; meer gewend aan afbeeldingen zoals bij deze illusies.

Ouch

I don't see Jordan Peterson's Self Authoring Program mentioned anywhere in here

This part shows that parasocial relationships can be both authentic and unauthetic, it really depends on the ethics and morale behind the person. Like the Mr Rogers situation, it also shows how the same thing can be considered both authentic or unauthentic based on the person viewing (Jessica vs the author)

This happens so often even with creators that don't have a big following, and I personally find this quite concerning. There are even individuals going as far as getting cosmetic surgery to look like a person or even finding their address.

The explanation of parasocial relationships impressed me the first time since it could explain why viewers feel like they "know" the public figure. Also, I feel surprised about that parasocial relationships appears from intimate-seeming interactions over time, often via media that simulate personal engagement. But in my perspective, this interactivity blurs the line between public and personal.

I suppose the different between authentic or inauthentic parasocial relationship in streamers-followers sense is whether the celebrity consider a specific follower as "fans". However, this judgement often based on whether followers spend enough money on streamers and streamers don't their fans' names (even username) in most circumstance. Therefore, I don't see any difference for follower between authentic and inauthentic parasocial relationship.

The relationship between streamers and their followers has became the one of the most common parasocial relationship, nowadays. In some sense, this kind of parasocial relationship was built by streamers on purpose. They will pick nicknames for their followers' group and responds to chat like they are talking like friends.

Joint Public Review:

Summary:

The authors present an intriguing investigation into the pathogenesis of Pol III variants associated with neurodegeneration. They established an inducible mouse model to overcome developmental lethality, administering 5 doses of tamoxifen to initiate the knock-in of the mutant allele. Subsequent behavioral assessments and histological analyses revealed potential neurological deficits. Robust analyses of the tRNA transcriptome, conducted via northern blotting and RNA sequencing, suggested a selective deleterious effect of the variant on the cerebrum, in contrast to the cerebellum and non-cerebral tissues. Through this work, the authors identified molecular changes caused by Pol III mutations, particularly in the tRNA transcriptome, and demonstrated its relative progression and selectivity in brain tissue. Overall, this study provides valuable insights into the neurological manifestations of certain genetic disorders and sheds light on transcripts/products that are constitutively expressed in various tissues.

Strengths:

The authors utilize an innovative mouse model to constitutively knock in the gene, enhancing the study's robustness. Behavioral data collection using a spectrometer reduces experimenter bias and effectively complements the neurological disorder manifestations. Transcriptome analyses are extensive and informative, covering various tissue types and identifying stress response elements and mitochondrial transcriptome patterns. Additionally, metabolic studies involving pancreatic activity and glucose consumption were conducted to eliminate potential glucose dysfunction, strengthening the histological analyses.

Comments on revised version from expert Editor #1:

The authors in the revised manuscript have effectively responded to all of the comments and suggestions raised by both reviewers. Overall, I find the revised version to be an important contribution to the field and the strength of evidence supporting the work's claims to be compelling.

Comments on revised version from expert Editor #2:

The authors have responded constructively to all the comments in the first round of reviews and clarified many issues in the manuscript. The current report represents a significant advance.

Comments on revised version from Reviewer #2:

The authors should include their clarifications of all concern raised by reviewer #2 (mentioned in the previous weaknesses) in the main text. They should consider including point #2 to point #10 in the main text (discussion section). The should highlight limitations of this study in discussion.

Also, they should clearly state that deciphering brain area specific behavioural deficits is beyond the scope of the manuscript with appropriate justification mentioned in the rebuttal letter.

I still do not agree with the author to state that "brain region-specific sensitivities to a defect in Pol III transcription". The changes are global and also not restricted to brain. Authors may consider restating this sentence. It is obvious that transcription defects related to tRNA production will lead to alteration in whole body physiology.

eLife Assessment

This study provides important insights into the mechanistic basis of neurological manifestations of RNA polymerase III-related disease by creating a mutant mouse to dissect transcriptional changes. The data provide compelling evidence for disease progression initiated by a global reduction in tRNA levels leading to integrated stress and innate immune responses and neuronal loss. The work will be of interest to those engaged in the study of chromosome biology, developmental biology and neurodegeneration.

Author response:

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

Reviewer #1 (Public Review):

Summary:

Moir, Merheb et al. present an intriguing investigation into the pathogenesis of Pol III variants associated with neurodegeneration. They established an inducible mouse model to overcome developmental lethality, administering 5 doses of tamoxifen to initiate the knock-in of the mutant allele. Subsequent behavioral assessments and histological analyses revealed potential neurological deficits. Robust analyses of the tRNA transcriptome, conducted via northern blotting and RNA sequencing, suggested a selective deleterious effect of the variant on the cerebrum, in contrast to the cerebellum and non-cerebral tissues. Through this work, the authors identified molecular changes caused by Pol III mutations, particularly in the tRNA transcriptome, and demonstrated its relative progression and selectivity in brain tissue. Overall, this study provides valuable insights into the neurological manifestations of certain genetic disorders and sheds light on transcripts/products that are constitutively expressed in various tissues.

Strengths:

The authors utilize an innovative mouse model to constitutively knock in the gene, enhancing the study's robustness. Behavioral data collection using a spectrometer reduces experimenter bias and effectively complements the neurological disorder manifestations. Transcriptome analyses are extensive and informative, covering various tissue types and identifying stress response elements and mitochondrial transcriptome patterns. Additionally, metabolic studies involving pancreatic activity and glucose consumption were conducted to eliminate potential glucose dysfunction, strengthening the histological analyses.

Weaknesses:

The study could have explored identifying the extent of changes in the tRNA transcriptome among different cell types in the cerebrum. Although the authors attempted to show the temporal progression of tRNA transcriptome changes between P42 and P75 mice, the causal link was not established. A subsequent rescue experiment in the future could address this gap.

Nonetheless, the claims and conclusions are supported by the presented data.

We thank Reviewer 1 for their thoughtful review and commentary.  We appreciate the reviewer’s finding that our “claims and conclusions are supported by the presented data.”   

We note that our findings on the temporal progression of transcriptional changes between P42 and P75 apply to both the Pol II and Pol III transcriptomes. Importantly, in the case of Pol III, only precursor and mature tRNAs are affected at P42 whereas at P75, numerous other Pol III transcripts are also changed.  We therefore attribute the changes in tRNA as being causal in disease initiation since this is the earliest  direct consequence of the Polr3a mutation.

To expand on the evidence demonstrating the progressive nature of Polr3-related disease in our mouse model, the revised manuscript includes new immunofluorescence data showing no change in microglial cell density in the cerebral cortex or the striatum at an early stage in the disease (Supplementary Fig. S6F, G).  This is in striking contrast to the findings at later times (P75) where the number of microglia increased significantly in the Polr3a mutant and exhibit an activated morphology (Fig. 4G,H).   

We agree with the reviewer that it will be interesting in the future to assess the impact of the Polr3a mutation in different neural cell types and to explore opportunities for suppressing disease phenotypes. 

Reviewer #2 (Public Review):

Summary:

The study "Molecular basis of neurodegeneration in a mouse model of Polr3 related disease" by Moir et.al. showed that how RNA Pol III mutation affects production, maturation and transport of tRNAs. Furthermore, their study suggested that RNA pol III mutation leads to behavioural deficits that are commonly observed in neurodegeneration. Although, this study used a mouse model to establish theses aspects, the study seems to lack a clear direction and mechanism as to how the altered level of tRNA affects locomotor behaviour. They should have used conditional mouse to delete the gene in specific brain area to test their hypothesis. Otherwise, this study shows a more generalized developmental effect rather than specific function of altered tRNA level. This is very evident from their bulk RNA sequencing study. This study provides some discrete information rather than a coherent story. My enthusiasm for publication of this article in eLife is dampened considering following reasons mentioned in the weakness.

Reviewer 2’s summary contains two misstatements: 

Moir et.al. showed that how RNA Pol III mutation affects production, maturation and transport of tRNAs.

Our experiments document the effect of a neurodegenerative disease-causing mutation in RNA polymerase III on the Pol III transcriptome with a particular focus on the tRNAome (i.e. the mature tRNA population). Experiments on the maturation and transport of tRNA were not performed as there was no indication that these processes might be negatively impacted at the earliest time point (P42). Additional comments about tRNA maturation and export are provided under points 8 and 9 (see below). 

The study seems to lack a clear direction and mechanism as to how the altered level of tRNA affects locomotor behaviour.

This comment misstates the purpose of our study while overlooking the important results. As stated in the abstract, our goal was to develop “a postnatal whole-body mouse model expressing pathogenic Polr3a mutations to examine the molecular mechanisms by which reduced Pol III transcription results primarily in central nervous system phenotypes.”

Accordingly, our work provides the first molecular analysis of RNA polymerase III transcription in an animal model of Polr3-related disease. The novelty and importance of the findings, as stated in the abstract, include the discovery that a global reduction in tRNA levels (and not other Pol III transcripts) at an early stage in the disease precedes the frank induction of integrated stress and innate immune responses, activation of microglia and neuronal loss at later times. These later events readily account for the observed neurobehavioral deficits that collectively include risk assessment, locomotor, exploratory and grooming behaviors. 

Strengths:

The study created a mouse model to investigate role of RNA PolIII transcription. Furthermore, the study provided RNA seq analysis of the mutant mice and highlighted expression specific transcripts affected by the RNA PolIII mutation.

Weaknesses:

(1) The abstract is not clearly written. It is hard to interpret what is the objective of the study and why they are important to investigate. For example: "The molecular basis of disease pathogenesis is unknown." Which disease? 4H leukodystrophy? All neurodegenerative disease?

We have modified the abstract to more clearly frame the objective of the study and its importance as reflected in the title “Molecular basis of neurodegeneration in a mouse model of Polr3-related disease”. We hope the reviewer will agree that the fourth sentence of the abstract, unchanged from the initial submission, clearly outlines the objective of the study.  

(2) How cerebral pathology and exocrine pancreatic atrophy are related? How altered tRNA level connects these two axes?

It is not known how cerebral pathology and exocrine pancreatic atrophy are related beyond their shared Pol III dysfunction in our mouse model of Polr3-related disease. We anticipate that altered tRNA levels connect these two axes. Indeed, the pancreas and the brain are both known to be highly sensitive to perturbations affecting translation (Costa-Mattioli and Walter, 2020 Science doi: 10.1126/science.aat5314). Changes to the tRNA population in the cerebrum and cerebellum of Polr3a mutant mice were extensively documented in the manuscript (e.g. Figs. 3, 5 and 6).  We also found reduced tRNA levels in the pancreas of the mutant mice but did not report these findings due to the absence of a stable reference transcript in total RNA from the atrophied pancreatic tissue, even at the earliest time point examined (P42). 

(3) Authors mentioned that previously observed reduction mature tRNA level also recapitulated in their study. Why this study is novel then?

Our study reports the novel finding that a pathogenic Polr3a mutation causes a global reduction in the steady state levels of mature tRNAs, i.e. the levels of all tRNA decoders were reduced with the vast majority these reaching statistical significance (Fig. 6D and 6F). In the introduction we refer to several studies that examined the effect of pathogenic Polr3 mutations on the levels of Pol III-derived transcripts. We noted that these studies examined only a small number of Pol III transcripts in CRISPR-Cas9 engineered cell lines, patient-derived fibroblasts and patient blood. Thus, no study until now has tested for or reported a global defect in the abundance of mature tRNAs in any model of Polr3-related disease. Moreover, no previous study of _Polr3_related disease has analyzed Pol III transcript levels in the brain or in any other tissue. 

(4) It is very intuitive that deficit in Pol III transcription would severely affect protein synthesis in all brain areas as well as other organs. Hence, growth defect observed in Polr3a mutant mice is not very specific rather a general phenomenon.

While we agree with the simple assumption that a “deficit in Pol III transcription likely would affect protein synthesis in all brain areas as well as other organs”, this turned out not to be the case. In fact, a novel finding of our study is that not all Polr3a mutant tissues show a translation stress response despite reduced Pol III transcription and reduced mature tRNA levels. This implies that in some tissues the reduction in tRNA levels caused by the Polr3a mutation is not sufficient to affect protein synthesis, at least to a point where the Integrated Stress Response is induced. The underlying basis for the growth deficit has not been defined in this work. However, we noted in the discussion that a growth defect was previously seen in mice where expression of the Polr3a mutation was restricted to the Olig2 lineage.  In the present postnatal whole-body inducible model, we anticipate that the diminished growth of the mice results from a combination of hormonal and nutritional deficits caused by cerebral and pancreatic dysfunction.

(5) Authors observed specific myelination defect in cortex and hippocampus but not in cerebellum. This is an interesting observation. It is important to find the link between tRNA removal and myelin depletion in hippocampus or cortex? Why is myelination not affected in cerebellum?

We agree that the specific myelin defect observed in the cortex and hippocampus, but not the cerebellum, is an interesting observation. Pol III dysfunction in this model and reduced tRNA levels are common to both cerebra and cerebella, yet the pathological consequences differ between these regions.  While we do not know why this is the case, the cells that oligodendrocytes support in these regions are functionally different. We suggest in the discussion that subtle defects in oligodendrocyte function in the cerebellum may be uncovered using more sensitive or specific assays than the ones we have employed to date.  In addition, consistent with our findings in other tissues where Pol III transcription and tRNA levels are reduced but phenotypes are lacking, we suggest that oligodendrocytes in the cerebellum may have a different minimum threshold for Pol III activity than in other regions of the brain. 

(6) How was the locomotor activity measured? The detailed description is missing. Also, locomotion is primarily cerebellum dependent. There is no change in term of growth rate and myelination in cerebellar neurons. I do not understand why locomotor activity was measured.

We used a behavioral spectrometer with video tracking and pattern-recognition software to quantify ~20 home cage-like behaviors, including locomotor activity, as part of our phenotypic characterization of the mice. This experimenter-unbiased approach reported several metrics of locomotion, specifically, total Track length (the total distance traveled in the instrument), Center Track length and the time spent running (Run Sum) and standing still (Still Sum) in a longitudinal study (Figs. 2A-C and Supplemental Fig. S3A-C). The Materials and Methods section on mouse behavior has been amended to provide a detailed description of these experiments. 

locomotion is primarily cerebellum dependen_t_

While we agree that the cerebellum plays a critical role in balance and locomotion, regions of the cerebrum that are affected in our mice, including the primary motor cortex and the basal ganglia (Fig. 4), also have important roles in locomotor activity and control. 

(7) The correlation with behavioural changes and RNA seq data is missing. There a number of transcripts are affected and mostly very general factors for cellular metabolism. Most of them are RNA Pol II transcribed. How a Pol III mutation influences RNA Pol II driven transcription? I did not find differential expression of any specific transcripts associated with behavioural changes. What is the motivation for transcriptomics analysis? None of these transcripts are very specific for myelination. It is rather a general cellular metabolism effect that indirectly influences myelination.

The differentially expressed mRNAs identified in our RNAseq analysis at P75 reflect both direct and secondary consequences of dysfunctional Pol III transcription on Pol II transcription. These effects can be achieved by multiple mechanisms. Induction of the Integrated Stress Response (ISR) due to insufficient tRNA can be considered a direct consequence of diminished Pol III transcription on Pol II transcription. An example of a secondary response is the activation of microglia and the innate immune response (which is known to accompany prolonged activation of the ISR), and the loss of neurons and oligodendrocytes. These changes are documented in Figs. 3 and 4. Importantly, loss of neurons, activated microglia and reduced oligodendrocyte numbers are each readily reconciled with changes in behavior.  

None of these transcripts are very specific for myelination 

The RNAseq data at P75 indicates only a modest reduction in oligodendrocyte-specific gene expression (as defined by single-cell RNAseq studies of purified cell populations, Mackenzie et al., 2018 Sci. Rep. doi: 10.1038/s41598-018-27293-5). Despite this, some oligodendrocytespecific transcripts with well-known roles in myelination were down-regulated in the Polr3a mutant (e.g. Plp1, Mog and Mobp). In addition, steroid synthesis pathway transcripts involved in the production of cholesterol, an abundant and essential component of myelin, were also downregulated (Supplementary Fig. S4E).

(8) What genes identified by transcriptomics analysis regulates maturation of tRNA? Authors should at least perform RNAi study to identify possible factor and analyze their importance in maturation of tRNA.

Of the many proteins involved in the maturation of tRNA (Phizicky and Hopper, 2023 RNA doi: 10.1261/rna.079620.123), RNAseq analysis at P75 identified only amino-acyl tRNA synthetases as being differentially-expressed (fold change >1.5, p adj. < 0.05, Table S1). These genes are canonical indicators of the ATF4-dependent Integrated Stress Response and their upregulation is widely interpreted as an attempt to restore efficient translation. In addition, our analysis of Pol III transcripts at P75 identified a reduction in the level of RppH1 (Fig. 3C), the RNA component of RNase P, which removes the 5’ leader of precursor tRNAs.  However, at P42, there was no effect on RppH1 abundance, or the expression of amino-acyl tRNA synthetase genes (Fig. 5C and Table S3).  Thus, an RNAi study to identify and analyze a possible factor involved in the maturation of tRNA is neither warranted nor relevant to the current body of work.

(9) What factors are influencing tRNA transport to cytoplasm? It may be possible that Polr3a mutation affect cytoplasmic transport of tRNA. Authors should study this aspect using an imaging experiment.

Our analysis of tRNA populations in this study employed total cellular RNA and thus reflect the abundance of mature tRNA from all cellular compartments. We have not assessed whether the reduction in tRNA abundance caused by the Polr3a mutation alters the dynamics of tRNA transport from the nucleus to the cytoplasm. However, we consider it highly unlikely that the Polr3a mutation would have a significant effect on cytoplasmic transport of tRNA. Imaging experiments along these lines are beyond the scope of the current study.

(10) Does alteration of cytoplasmic level of tRNA affects translation? Author should perform translation assay using bio-orthoganal amino acid (AHA) labelling.

It is not known whether the reduced tRNA levels affect translation globally in the Polr3a mutant, but we predict that this may not be the case. Since tissues (heart and kidney) and brain regions (cerebrum and cerebellum) that share a decrease in tRNA abundance do not share activation of the Integrated Stress Response (a reporter of aberrant translation), we anticipate that effects on translation may be limited to specific regions or cell populations and to specific mRNAs within these cells. The current study provides the foundation for further work to address these questions.

Reviewer #1 (Recommendations For The Authors):

Below are a few comments, mostly regarding typographical errors, presentation, and clarity, that we believe would enhance this manuscript:

On the heatmaps generated, it would be ideal to place "WT" before "KI," with "WT" on the left. This will maintain consistency with the rest of the manuscript, where "WT" conditions precede "KI" conditions, as observed in the bar graphs and dot plots.

All heatmaps have been remade with WT on the left and KI on the right to maintain consistency throughout the manuscript. 

Authors mentioned in several instances (Discussion Pg 19 Line 2, for instance) the analysis of changes in the "Pol II transcriptome." Is this a typographical error?

The reference to the Pol II transcriptome is not a typographical error (Discussion Pg 19 Line2). Here and elsewhere in the manuscript, we are distinguishing between changes to the Pol III transcriptome and the timing of subsequent changes to the Pol II transcriptome. The text has been edited to clarify this relationship in several places.   

(1) Introduction, Page 4, last paragraph.

Analysis of the Pol III transcriptome reveals a common decrease in pre-tRNA and mature tRNA populations and few if any changes among other Pol III transcripts across multiple tissues. Analysis of the Pol II transcriptome reveals activation of the integrated stress response in cerebra but not in other surveyed tissues.

(2) Results, page 8, 2nd paragraph

To investigate the molecular changes to Pol III transcript levels caused by the Polr3a mutation and any secondary effects on the Pol II transcriptome, we initially focused on the cerebra of adult mice at P75.

(3) Discussion, Page 19, second paragraph

Pol III dysfunction and the reduction in the cerebral tRNA population at P42 coincides with behavioral deficits and precedes substantial downstream alterations in the Pol II transcriptome, which include induction of an innate immune response (IR) and an ISR, and indicators of neurodegeneration (i.e., activation of cell death pathways and loss of mitochondrial DNA). These findings suggest a causal role for the lower tRNA abundance and/or altered tRNA profile in disease progression.

In supplementary figure 1, authors validated the expression of their systems using flow cytometry and observed a high level of recombination frequency in different tissue types. Can the flow cytometry data distinguish between cell types within the cerebrum (neurons/microglia/astrocytes)?

The flow cytometry experiments reported in Supplementary Fig. S1 used a dual tdTomato-EGFP reporter to assess recombination. The cerebral and cerebellar samples were gated on fluorescence from endogenous expression of tdTomato (red), EGFP (green) and DAPI (blue) staining. In principle, flow cytometry could be used to distinguish between cell types within the cerebrum (neurons/microglia/astrocytes). However,  this would require (i) an antibody to a cell surface marker on the cell type of interest and (ii) a fluorescent probe conjugated to the primary antibody or a fluorescent secondary antibody that is spectrally well resolved from the emission spectra of tdTomato, eGFP and DAPI.

Results section 1: Is there any particular reason why P28 was chosen as the commencement of tamoxifen injection?

P28 was chosen so that any effect of the Polr3a mutation on development and differentiation would be limited in the tissues we examined. 

Fig 1C: The number of asterisks does not match between the graph and the figure legend.

Fig. 1C has been corrected to match the number of asterisks in the graph and figure legend.

Results section 3:

This section seemed a little brief, especially when compared to the depth of the succeeding sections. Authors can state in greater detail which behaviors were quantified. In S3A-C, my understanding is that the animals were placed in an open-field test. This procedure can be briefly mentioned in the methods, as well as in the main manuscript text.

In the legends of S3, a bracket is missing for "(D-F)" on line 5. Additionally, the alignment of legends for each bar graph could be consistent for all graphs except under the condition of spatial constraint.

Detailed methods pertaining to the measurement and calculation of home cage-like behaviors reported by the behavioral spectrometer have been added to the Methods section on Mouse Behavior. 

In the Results, Figs. S3A-C show anxiety-like behaviors which measure the number and duration of visits and the distance traveled  in a 15 cm2  central area of the arena. Figs. 2A-C show locomotor behaviors including Tracklength, Run sum and Still sum. The open field-like behavior is reported as total Tracklength in the behavioral spectrometer, i.e. the total distance travelled in the arena. This is now more clearly described in  the main manuscript and the Methods section. “overall locomotor activity was decreased in Polr3a-tamKI mice as indicated by the reduced track length at P42, P49, P56 and P63 (Fig. 2A).” 

The legend of S3, now has the missing bracket "(D-F)" on line 5. 

The legends within each bar graph are now consistent and aligned as much as spatial constraints allow.

Results section 4:

Similar to our earlier questions for S1, is it possible to distinguish samples derived from different cell types (neurons/glia)? In figure 4, this is mainly done post-hoc, based on the known gene expression. Maybe the authors could discuss this small limitation? In Fig S4C, the color contrast for the heatmap legend needs to be corrected.

It is not possible to accurately distinguish different neural cell sub-types, such as different types of neurons, or different types of oligodendrocytes in bulk RNAseq. Hence, we have reported only high confidence correlations based on known gene expression signatures (Fig. 4). We discuss only the data for which we can draw confident conclusions. The heatmap and legend in Fig. S4C has been amended. 

Results section 5:

In figure S5A, the alignment of asterisk significance markers could be adjusted.

Asterisks have been realigned in Fig. S5A

Reviewer #2 (Recommendations For The Authors):

Methods Section should include detailed procedure.

A detailed description of the methods pertaining to the measurement and calculation of behaviors using the behavioral spectrometer has been added to the Methods section.

Statistical tests should have detailed information

Statistical tests are detailed in the Methods section “Statistical Analysis”. Additional details pertaining to calculations of behavioral data have been added to the “Mouse behavior” section of the Methods.

This line kind of insists the idea that authenticity can vary based on the person, and that some people consider something to be unauthentic while others believe the same ting is.

lonelygirl15 was one of the first major examples of the "Unreality" genre on the internet, following the footsteps of Blair Witch Project and inspiring future works like Slenderman. The appeal of unreality is similar to satire, tricking the audience into believing what they were watching was real, but for suspense or horror instead of comedy. But understandably, for those who aren't "in on it" can feel betrayed if what they were being fed was a lie, especially in the infancy of a new medium (online video).

This reminds me of an internet celebrity I knew before. She uploaded a video about picking up elementary school students' homework in Paris, and this video caused a big sensation on the video website. But later it was discovered that her video was self-directed and self-acted. She gained huge traffic with a video, but in the end her social platform account was blocked for spreading false facts.

Long-standing social relations have led to the result that direct communication between people and cooperation is based on honesty, so that society can develop in the long term, therefore, when one of the two parties has a problem with honesty, it will immediately lead to a breakdown of the relationship and the transaction stops, which is why the society has to make rules to restrain people.

When people have realized that the girl's behavior is acting, virtual and not real, why do they continue to watch while allowing the girl's channel to continue to explode in popularity, my personal opinion is that on the internet, the line of judging authenticity has become relatively blurred as most of the views are that most of the content on the internet is virtual, and that when the girl's behavior doesn't live up to the fraudulent When the girl's behavior does not amount to fraud, people will choose to forgive her.

I find it scary that the federal minimum wage in 2012 was $7.25 per hour, which only amounts to $13,930 a year. It seems unfair that someone working full-time would still struggle to provide for their family. This also makes me think about the impact on children in those families, who might miss out on opportunities because their parents simply can't afford things like education or extracurricular activities. It shows how important it is to have a fair wage that allows people to meet their basic needs.

La necesidad de la inversión en planificación de la comunicación a largo plazo, obliga a la empresa a exprimir la comunicación para generar nuevas capacidades y a acercar cada vez mas en lo posible a la empresa con perspectiva en comunicación aplicada a requerimientos del mundo actual, estratégicamente.

I think that its funny when social media sites put in so much effort to enforcing policies against using emojis and non offensive symbols but struggle to remove hate crimes or cyberbullying even when it is reported.

I also think that it is important for users to have the option of anonymity because it allows users to express themselves freely. However, there is catch because anonymity could also encourage criminal behavior because there is no way of tracing things back to a real person. Anonymity should be carefully controlled by allowing partial anonymity.

It is for this reason I feel that people are resistant to ID requirements to sign up for a social media. Many countries are considering requiring a driver's license or ID to access 18+ or even 13+ material. While even an email could possibly provide a name via a thorough investigation, it's still a manual barrier that prevents a hacker to easily dox large groups of people. However providing legal name and address to a third-party platform holder, even if secure, gives each user 1 less layer of protection in case of a data breach. (Not to mention that people already view platform holders as THE antagonistic force.)

It's important to think about the circumstances in which this genuine behavior occurs. People can express their "true" opinions and sentiments in an anonymous setting, but there may be no real repercussions. In a more accountable, face-to-face setting, impulsive, raw emotion may not always be indicative of an individual's basic principles, and the lack of responsibility may make it difficult to distinguish between the two types of self-expression.

Anonymity can both discourage authenticity and encourage inauthentic behavior, depending on the context and the individuals involved. On one hand, anonymity can create a sense of freedom, allowing people to express thoughts and opinions they might suppress in real life due to fear of judgment or consequences. On the other hand, the lack of accountability that comes with anonymity can also lead to inauthentic or even harmful behavior.

Its pretty wild to me that @Sciencing_BI was able to lie about what they were doing for so long without being caught. Today, if you claimed to be a professor on X, everyone would go fact check it and i feel like instantly it would be caught and wouldnt gain much attention.

I find it interesting that person faked being a ASU professor and went so far into the lie that people believed it. This conveys the importance of authentication real human beings when creating social media accounts. If this had been something even more serious like a bomb threat being posted on social media, a simple tweet could lead to wars.

I feel like authenticity is ver important in social media. For example when people argue on social media or when they see they are wrong in an argument the jump to saying stuff that is not authentic pulling sources that are not real and making stuff up, which is very deceiving for people trying to make that connection on social media

I feel like this issue is magnified in today's information environment. People portray themselves online, which often leads to a disconnect that can damage relationships and self-perception. We need more authentic interactions and more honesty between people.

I have had a Schrodinger's asshole account harass me as an Asian individual during Covid one time and he pulled this move after I took matters into my own hands. I don't see too many of these types of accounts anymore, but there were a lot of them back in 2020.

This reminds me of Kpop fans who buy robots to like their idols' posts in order to make their idols look more popular. Because people have a herd mentality, when people find that an idol's post has a lot of likes, they may choose to like it as well.

I remember the feature on twitter where it would show you which device the author was posting from. I never saw value in it but in this situation it seems like it was useful to see which of the tweets are coming from his team or him specfically. I find it to be a little funny how trumps post were more angry while his team was more calm and basically cleaning up his mess.

I believe that the contrast between the 2 different types of tweets, the ones made on Android and Apple truly highlight how the tone and the message can really differ depending on who is managing the account. This difference also shows how data analysis can play a role in finding patterns in tweets and forms of communication. It also can highlight the difference between personal and campaign driven tweets and posts.

I thought this was interesting just because of all the misinformation that led Trumps twitter to get banned in the first place, bots could've been the reason for reinstating him. it's just an interesting thought of how so much cannot be trusted from the internet. Even previously stated, with the Android and iPhone, it's hard to tell what's true and what's not.

studets ned to understand their rights and be able to recognize if they are in the right or wrong within the case

Basically saying don't go and ask your friends from another school that has taken that course for answers because that is cheating

Students are not allowed to back out of the university in fera, and should take full responsibility for their actions and be thoughrough and honest.

after learning about all of the horrible effects of hazing I am glad it is talked about in the code of conduct

If the student disagrees with the allegation, they can submit an appeal that can be reviewed

cultures with high power distance tend to have hierarchical structures and clear authority roles, while low power distance cultures value equality and open dialogue

I believe that code-switching and our public personas allow us to navigate different social contexts and situations that we may be put in while also still being able to express authentic versions of ourselves. Adjusting our behavior to match different social situations reflects our understanding of social expectations and our ability to communicate in different ways.

the paragraph introduces an important and complex issue concisely. Adding a few more details about its effects on behavior, specific examples, and emotional impact could make it even more insightful.

ok

here

!

!

Mr. Denneny's archive is now at Brown University

As written it will return NA for entire sample_segment column. Instead put left_join(tbi$hh, by = c("survey_year", "hh_id")) %>%

If you join it like this, survey_year will become survey_year.x. Instead do left_join(tbi$hh, by = c("hh_id", "survey_year")) %>%

If you join it like this, survey_year will become survey_year.x. Instead do left_join(tbi$hh, by = c("hh_id", "survey_year")) %>%

If you join it like this, survey_year will become survey_year.x. Instead do left_join(tbi$hh, by = c("hh_id", "survey_year")) %>%

mode_type = values_list[variable_unified == "mode_type", value_upcoded],

i like the phrase

Author response:

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

Reviewer #1 (Recommendations For The Authors): 

Figures 1 and 2. How do the authors know that the lysine mutations are specific to constitutive activity and not because it is causing the channel to be now voltage sensitive? 

As shown in the revised Figs. 1b, S2a, and 3b, TMEM16F I521K/M522K, TMEM16F I521E, and TMEM16A I546K/I547K spontaneously expose PS, respectively. Neither membrane depolarization nor calcium stimulation was introduced under these conditions and the cells were grown in calcium-free media after transfection to limit calcium-dependent activation. Our new experiments further demonstrate that TMEM16F T526K (Fig. 1b) and TMEM16A E551K (Fig. 3b), which are further away from the activation gate, exhibit either strongly attenuated or lack spontaneous lipid scrambling activity. According to these results, the gain-of-function mutants (TMEM16F

I521K/M522K/I521E and TMEM16A I546K/I547K) are indeed constitutively active. This constitutive scramblase activity is not due to a gain of voltage sensitivity as ion channel activity is also minimal around the resting membrane potential of a HEK cell (Fig. 1d, e and Fig. 3d, e).

The authors see very large currents of 5 -10 nA in their electrophysiology experiments in Figures 2D and 3D. I understand that Figure 2D are whole-cell recordings but are the authors confident that the currents that they are recordings from the mutants are indeed specific to TMEM16A. More importantly, in Figure 3D they see 3-5nA currents in insideout patches, which is huge. They have no added divalent in their bath solution, which could lead to larger single-channel amplitudes, but 3-5nA seems excessive. Some control to demonstrate that these are indeed OSCA1.2 currents is important. 

TMEM16A and TMEM16F are well-known for their high cell surface expression. Therefore, the current amplitude is usually huge even in excised inside-out or outside-out patches—please see our previous publications for details: 1) 10.1016/j.cell.2012.07.036, 2) 10.7554/eLife.02772, 3) 10.1038/s41467-019-11784-8, 4) 10.1038/s41467-019-09778-7, 5) 10.1016/j.celrep.2020.108570, 6) 10.1085/jgp.202012704, and 7) 10.1085/jgp.202313460. 

HEK293 cells do not have endogenous TMEM16A (https://doi.org/10.1038/nature07313, 10.1016/j.cell.2008.09.003 , DOI: 10.1126/science.1163518). It therefore serves as a widely used cell line for studying TMEM16A biophysics. As overexpressing the WT control barely elicited any obvious current in 0 Ca2+ (Fig. 3d), there is no doubt that the large outward-rectifying current (hallmark of CaCC) in the revised Fig. 3d (previous Fig. 2D) was elicited from the mutant TMEM16A channels. The strong outward rectification also rules out the possibility of this being leak current.

Regarding Fig. 4d (previous Fig. 3D), OSCA1.2 has excellent surface expression as shown in Fig. 4b. OSCA1.2 also has much higher single channel conductance (121.8 ± 3.4 pS, 10.7554/eLife.41844) than TMEM16A (~3-8 pS) and TMEM16F (<1 pS). Therefore, recording nA OSCA1.2 current from excised patches is normal given larger OSCA1.2 current at depolarized voltages than the current recorded at hyperpolarized voltages (please see our explanation in the next response). As the reviewer pointed out, lack of divalent ions in our experimental conditions may also partially contribute to the large conductance. To further verify, we conducted mock transfection recordings (please see Author response image 1 below). WT- but not mock (GFP)transfected cells gave rise to large current, further supporting that the recorded current was indeed through OSCA1.2. 

Author response image 1.

Representative inside-out currents for mock (GFP)- and OSCA1.2 WT-transfected cells. OSCA1.2 is responsible for nA currents elicited by the pressure and voltage protocols shown.

Figure 3D and 5D. Most of the traces and current quantification is done at positive potentials and is outward current. Do the authors observe inward currents? It is difficult to judge by the figures since currents are so large. OSCA/TMEM63s are cationic channels and all published data on these channels have demonstrated robust inward currents at negative, physiologically relevant potentials. The lack of inward currents but only large outward currents suggests that these mutations could be doing something else to the channel. 

Yes. We indeed observe inward current at negative holding potentials under pressure clamp (Author response image 2). However, mechanosensitive OSCA and TMEM63A channels are also voltage dependent. Their outward current is an order of magnitude larger at depolarized voltages (e.g., Author response image 2, also 10.7554/eLife.41844, see Fig. 1H). 

Author response image 2.

Voltage-dependent rectification of OSCA1.2 current. a. Representative OSCA1.2 trace (bottom) elicited by a voltage-ramp under -50 mmHg (top). b. The difference in inward and outward current amplitudes. 

We found that quantifying the OSCA1.2 outward current has advantages over the inward current. Usually, using the gold standard pressure clamp protocol at negative holding voltages, peak inward current amplitude is quantified. However, OSCA inward current quickly inactivates (10.7554/eLife.41844, see Fig. 1C). This makes robust quantification and comparison with mutant channels difficult. Holding the membrane at a constant pressure and measuring OSCA1.2 G-V overcomes these issues associated with the classical inward current measurements. The large depolarization-driven outward current does not inactivate, and robust tail current (Response Fig. 1, 2) allows us to construct G-V relationships. We found quantifying mutants’ voltage dependence at constant pressure is more consistent than quantifying pressure dependence at constant voltage. These advantages make our new protocol preferable to the commonly used gold standard pressure clamp protocol for characterizing and comparing the gating mutations identified in this manuscript. 

Figure 3 and 5. Why are mechanically activated currents being recorded at random pressure stimuli (-50 mmHg for OSCA) and (-80 mmHg for Tmem63a)? The gold standard in the field is to run an entire pressure response curve. Given that only outward currents are observed at membrane potentials +120mV and above at 0mmHg, this questions whether they are indeed constitutively active. 

As we explained in the previous response, both voltage and membrane stretch activate OSCA/TMEM63A channels. We found measuring voltage dependence under constant pressure provided more consistent quantification than the gold standard pressure response protocol. This may be due to the variability of applied membrane tension under repeated stretches versus the more consistent applied voltage. Additionally, we chose -50 mmHg and -80 mmHg to reflect the reported differences in half-maximal pressures between OSCA1.2 and TMEM63A (e.g., P50 ~55 mmHg for 1.2 and ~61 mmHg for 63A in 10.7554/eLife.41844 versus ~86 mmHg for 1.2 and -123 mmHg for 63A in 10.1016/j.neuron.2023.07.006).

We also used higher pressure in cell attached mode to increase TMEM63A current amplitudes, which are usually tiny.  We have updated our method section (Lines 329334) to further clarify why we used these protocols. 

Please note that in TMEM16 proteins, ions and lipids might not always co-transport.

This means that under certain conditions, only one type of substrate may go through. For instance, in WT TMEM16F, Ca2+ stimulation can easily trigger PS exposure at resting membrane potential. No ionic currents are elicited until strong depolarization is applied. Similarly, the TMEM16F GOF mutations spontaneously transport lipids, leading to loss of lipid asymmetry (Fig. 1b, c). However, in 0 Ca2+, these TMEM16F mutant channels still need strong depolarization for ion conduction (Fig. 1d, e). Although the detailed mechanism still needs to be further investigated, the OSCA1.2 and TMEM63A GOF mutations share similar features with TMEM16 proteins, exhibiting ion conduction under high pressures and depolarizing voltages, yet constitutively active scrambling.  

Some clarity is needed for their choice of residues. I understand that a lot of this is also informed by the structures of these ion channels. According to the alignment shown in Supplementary Figure 1, they chose LA for OSCA1.2, which is in line with the IM (TMEM16F) and II(TMEM16A) residues but for Tmem63a they chose the hydrophobic gate residue W and S. Was the A476 tested? Also, OSCA1.2 already has a K in the hydrophobic gating residue region. How do the authors reconcile this with their model? 

We appreciate this critical comment. We have included the characterization of TMEM63A A476K (Fig. 6, corresponding to M522 in 16F, I547 in 16A, and A439 in OSCA1.2). Interestingly, A476K transfected cells did not show obvious spontaneous PS exposure yet exhibited a modest shift in V50 comparable to W472K and S475K. These differences may reflect the high-tension activated nature of the TMEM63 proteins (10.1016/j.neuron.2023.07.006) as compared to OSCA1.2, where the corresponding mutation (A439K, Fig. 4b, c) showed very little spontaneous activity and required hypotonic stimulation to promote more robust PS exposure (Fig. 5). 

Furthermore, as we showed in Figs. 1b-c and 3b-c, there is a lower limit (towards the Cterminus) of the TM 4 lysine mutation effect, which becomes insufficient to cause a constitutively open pore for spontaneous lipid scrambling. It is possible that TMEM63A A476K represents the lower limit of TM 4 mutations that can convert TMEM63A into a spontaneous lipid scramblase.  

Regarding OSCA1.2 K435 and TMEM63A W472, these sites correspond to the hydrophobic gate residues on TM 4 in TMEM16F (F518, Fig. 1a) and TMEM16A (L543, Fig. 3a) so it is unsurprising to us that a lysine mutation at this site causes constitutive scramblase activity in TMEM63A (Fig. 6b, c). For OSCA1.2, it is more intriguing since this residue is already a lysine (K435). In Supplementary Fig. 5 our new experiments show that neutralizing K435 with leucine (K435L) in the background of L438K significantly attenuates spontaneous PS exposure from ~63% PS positive for L438K alone (two lysine residues) to ~31% for K435L/L438K (one lysine). One the other hand, the K435L mutation by itself is also insufficient to induce PS exposure. Therefore, the endogenous lysine at residue 435 has an additive effect on the spontaneous scramblase activity of L438K. We believe the explanation for this result lies in experiments conducted in model transmembrane helices, which have shown that stacking hydrophilic side chains within the membrane interior promotes trans-bilayer lipid flipping (see 10.1248/cpb.c22-00133). 

These same studies also support our observation (10.1038/s41467-019-09778-7) that highly hydrophilic side chains (such as lysine or glutamic acid) accelerate trans-bilayer lipid flipping more effectively than hydrophobic side chains such as isoleucine or alanine (Author response image 3, see also 10.1021/acs.jpcb.8b00298).

Author response image 3.

Trans-bilayer lipid flipping rates (kflip) accelerate with increasing side chain hydropathy for a residue placed in the center of a model transmembrane helical peptide

How do the authors know that osmotic shock is indeed activating OSCA1.2 and TMEM63A? If they can record from the channels then electrophysiology data that confirms activation of the channel in the presence of hypoosmotic shock will strengthen the osmolarity active scramblase activity demonstrated in Figure 4. So far, there is conclusive data showing that they are mechanically activated but conclusive electrophysiological data for OSCA/TMEM63 osmolarity activation is not described yet, including the reference (38) they indicate in line 132. Although osmotic shock can perturb mechanical properties of the membrane it can also activate volume-regulated anion channels, which are also present in HEK cells. 

Thank you for raising this important question. While reference 38, (now reference 39) shows direct electrophysiological evidence of hypertonicity-induced current (e.g., Fig. 4 f, g, i, and j in 10.1038/nature13593), direct electrophysiological evidence that OSCA/TMEM63 can be activated by hypotonic stimulation is still missing. To address this question, we conducted whole-cell patch clamp experiments on mocktransfected and OSCA1.2 WT-transfected cells stimulated with 120 mOsm/kg hypotonic solution, comparable to the same conditions as hypotonic-induced scrambling shown in Fig. 5. As shown in Supplementary Fig. 6, our whole-cell recording detected a slowly evolving yet robust outward rectifying current in OSCA1.2-transfected cells, which was not observed in mock transfected cells. 

To avoid the contamination from endogenous SWELL osmo-/volume-regulated chloride channels, our new experiment used 140 mM Na gluconate to replace NaCl in both the pipette and the bath solution. Because SWELL/VRAC channels are minimally permeable to gluconate anions (e.g., 10.1007/BF00374290), we conclude that hypotonic stimulation can indeed activate OSCA1.2 albeit with perhaps lower efficiency compared to mechanical stimulation.  

Minor comments 

What is the timeline for the scramblase assay for all the experiments (except Figure 4)? How long is the AnnexinV incubated before imaging? 

Thank you for pointing out this point where we have not provided sufficient detail. Cells were imaged in the scramblase assay (including in Fig. 4, now revised Fig. 5) in AnnexinV-containing buffer immediately and without a formal incubation period because AnnexinV binding to exposed PS proceeds rapidly. We have included additional detail in the methods section to eliminate any confusion (Lines 310-312).

In some places of the document, it says OSCA/TMEM63, and in other places, it is denoted as TMEM63/OSCA. The literature so far has always called the family OSCA/TMEM63- please stay consistent with the field. 

Thank you for pointing this out, we have corrected these instances to be consistent with the field.   

Reviewer #2 (Recommendations For The Authors): 

(1) The authors' statement that the channel/scramblase family members have a relatively low "energetic barrier for scramblase" activity needs further support. While mutating the hydrophobic channel gate certainly could destabilize ion conduction to cause a GOF effect on channel activity, it is still not clear why scramblase activity, which is tantamount to altered permeation, happens in the mutant channels. Are permeation and channel gating (opening) coupled in these channels? If so, what is the basis for the coupling? Is scramblase activity only observed when the gating is destabilized or are they separable? 

We appreciate these great questions. For the question about the ‘energetic barrier’ statement, please see our response to point (3) where we have carried out MD simulations of the OSCA1.2 WT and L438K mutant to provide insight into how the permeation pathway is altered by these mutations. 

Regarding why TMEM16A can be converted into a scramblase, we use the extensively studied TMEM16 proteins as examples to improve our current understanding of OSCA/TMEM63 proteins. For further details please see our original paper (10.1038/s41467-019-09778-7) and our review (10.3389/fphys.2021.787773), which are summarized as follows: 

(1) The “neck region”, consisting of the exofacial halves of TMs 3-6, form the poregate region for both ion and lipid permeation (Author response image 4B). In the closed state, the neck region is constricted and TMs 4 and 6 interact with each other, preventing substrate permeation. The hydrophobic inner activation gate that we identified (10.1038/s41467-019-09778-7) resides right underneath the inner mouth of the neck region, controlling both ion and lipid permeation scrambling. 

(2) Based on our functional observations and the available scramblase structures of TMEM16 proteins in multiple conformations, we proposed a clamshell-like gating model to describe TMEM16 lipid scrambling (Author response image 4D). According to this model, Ca2+-induced conformational changes weaken the TM 4/6 interface. This promotes the separation of the two transmembrane segments, analogous to the opening of a clam shell, allowing a membrane-spanning groove to facilitate permeation of the lipid headgroup.

(3) For the CaCC, TMEM16A, Ca2+ binding dilates the pore. However, the binding energy likely cannot open the TM 4/6 interface at the neck region so, in the absence of groove formation, only Cl- ions but not lipids can permeate. (Pore dilation model, Author response image  4C). 

(4) Introducing charged residues near the inner activation gate disrupts the neck region, potentially by weakening the hydrophobic interactions between TMs 4 and 6. This mutational effect results in constitutively active TMEM16F scramblases and enables spontaneous lipid permeation in the TMEM16A CaCC. 

(5) In our revision, we tested additional mutations with different side chain properties (Supplementary Fig. 2), validating previous findings by us (10.1038/s41467-01909778-7) and others (10.1038/s41467-022-34497-x) that gate disruption increases with the side chain hydropathy of the mutation. 

(6) We further extended lysine mutations to two helical turns below the inner activation gate on TM 4 and identified a lower limit for mutation-induced spontaneous scramblase activity in TMEM16F and TMEM16A (Figs. 1b, c and 3b, c, respectively). Together, all these points lend additional support to our proposed gating models for TMEM16 proteins, which we postulate may also relate to the OSCA/TMEM63 family based on the evidence provided in our manuscript.

Author response image 4

Model of gating (and regulatory) mechanisms in the TMEM16 family. (B) overall architecture and proposed modules, (C) pore-dilation gating model for CaCCs, (D) Clamshell gating model for CaPLSases.

Regarding the relationship between ion and lipid permeation through TMEM16 scramblases, the following is the summary of our current understanding: 

(1) Functionally, ion and lipid permeation are not necessarily obligatory to each other. This is evidenced by our previous biophysical characterizations of TMEM16F ion channel and lipid scramblase activities. Ca2+ can trigger TMEM16F lipid scrambling at resting membrane potentials, however, Ca2+ alone is insufficient to record TMEM16F current. Strong membrane depolarization synergistically with elevated intracellular Ca2+ is required to activate ion permeation. Based on these observations, we postulate that ions and lipids may have different extracellular gates, despite sharing an inner activation gate (10.1038/s41467-019-09778-7). Ca2+ alone may sufficiently open the inner gate (and extracellular gate) for lipids, whereas depolarization is likely required to open the extracellular gate and allow ion flux. Further structure-function studies are needed to test this hypothesis. 

(2) Structurally, the open conformation of TMEM16 scramblases such as the fungal orthologs and human TMEM16K (Supplementary Fig. 1 b-d) are widely open, which allows lipid and ion co-transport. Ion and lipid co-transport has also been demonstrated in various MD simulations (e.g., 10.7554/eLife.28671, 10.3389/fmolb.2022.903972, and 10.1038/s41467-021-22724-w)

(3) Functionally, we (10.1085/jgp.202012704) and others (10.7554/eLife.06901.001) have measured dual recording of channel and scramblase activities, also demonstrating that ions and lipids are co-transported simultaneously when the proteins are fully activated.

(4) In this manuscript, we also provide multiple examples (TMEM16F in Fig. 1, TMEM16A in Fig. 3, OSCA1.2 in Fig. 4, and TMEM63A in Fig. 6) of mutations showing spontaneous phospholipid scramblase activities, yet their channel activities require strong depolarization or, in the case of TMEM63A, high pressures to be elicited.

Together, this new evidence further supports our hypothesis that there might be multiple gates for ion and lipid permeation, in addition to the shared inner gate we previously identified. We hope these detailed explanations help convey the intricacy of these intriguing questions. Of course, future studies are needed to test our hypothesis and elucidate the complex relationship between ion and lipid permeation of these proteins. 

(2) One weakness in the experimental approach is the very limited number of substitutions used to infer the conclusion regarding the energetic barrier and other conclusions relating to scramblase activity. Additional substitutions of charged and polar amino acids at the hydrophobic gate would be helpful in illuminating the molecular determinants of the GOF phenotype and also reveal varying patterns of lipid permeation which could be enormously informative. These additional mutations for analysis of TMEM16F and OSCA should be added to the study. 

We appreciate these great suggestions which were shared by multiple reviewers. We have included our duplicated response below.

“Response to reviewers 2 & 3: In our 2019 paper (10.1038/s41467-019-09778-7), we have systematically tested the side chain properties at the inner activation gate of TMEM16F on lipid scrambling activity (Response Fig. 6) and, since then, these results have been supplemented by others as well (10.1038/s41467-022-34497-x). In summary, mutating the inner activation gate residues to polar or charged residues generally results in constitutively activated scramblases without requiring Ca2+ (Fig 5a in 10.1038/s41467-019-09778-7). Because these residues form a hydrophobic gate, introducing smaller side chains via alanine substitution are also gain-of-function with the Y563A mutant as well as the F518A/Y563A/I612A variant being constitutively active (Fig. 3a in 10.1038/s41467-019-09778-7). Meanwhile, mutating these gate residues to hydrophobic amino acids causes no change for I612W, a slight gain-of-function for F518W, slight loss-of-function of F518L, and complete loss-of-function for Y563W (Fig. 4b in 10.1038/s41467-01909778-7). These findings clearly demonstrate that the side-chain properties are critical for regulating the gate opening. Charged mutations including lysine and glutamic acid are the most effective to promote gate opening (Fig 5a in 10.1038/s41467-019-09778-7).

Similarly, others have observed that side chain hydropathy at the F518 site in TMEM16F correlates with shifts in the Ca2+ EC50 (Fig. 2 of 10.1038/s41467-022-34497-x). Note that this publication resolved the structure of the TMEM16F F518H mutant, revealing a previously unseen conformation that we have highlighted in Supplementary Fig. 1e and discussed in lines 235-238. Please also see our response to Reviewer #1 above, where we discuss discoveries in model transmembrane helical peptide systems showing that transbilayer lipid flipping rates correlate with side chain hydropathy (Author response image 3), distance between stacked hydropathic residues (schematic in 10.1248/cpb.c22-00133), and even helical angle between stacked side chains (not show). 

Following the reviewers’ suggestions, we have tested additional mutations in alternative locations and with different side chains.  

(1) We have added data for TMEM16F I521A and I521E to demonstrate a similar effect of alternative side chains to what has previously been reported by us and others. We found that I521A failed to show spontaneous scrambling activity (Supplementary Fig. 2), yet I521E (Supplementary Fig. 2) is a constitutively active lipid scramblase, similar to I521K (Fig. 1). This further demonstrates that gate disruption correlates with the side chain hydropathy and that this site lines a critical gating interface.

(2) We also added lysine mutations two helical turns below the conserved inner activation gate for TMEM16F T526 (Fig. 1), TMEM16A E551 (Fig. 3). We found that there is indeed a lower limit for the observed effect in TMEM16, where lysine mutations no longer induce spontaneous lipid scrambling activity. This indicates that when TM 4/6 interaction is weaker toward intracellular side (Figs. 1a, 3a), the TM 4 lysine mutation loses the ability to promoting lipid scrambling by disrupting the TM 4/6 interface to enable clamshell-like opening of the permeation pathway. 

(3) We added a TMEM16F lysine mutation on TM 6 at residue I611 (Fig. 2). Similar to I612K (Response Fig. 6), I611K also leads to spontaneous lipid scrambling and enhanced channel activity in the absence of calcium (Fig. 2). This shows that charged mutations along TM 6 can also promote lipid scrambling, strengthening our model that hydrophobic interactions along the TM 4/6 interface are critical for gating and lipid permeation.”

(3) Related to the above point, it would be enormously useful to perform even limited computational modelling to support the "energetic barrier" statement. Specifically, can the authors model waters in the putative pore to examine water occupancy in the WT and mutant channels to better understand how the barrier for ions and lipids is altered in the TMEM16? 

We appreciate this suggestion and have now conducted atomistic MD simulations of OSCA1.2 WT and L438K mutant for ~1 μs (Supplementary Fig. 4). The simulations revealed, elevated water occupancy in the pore region of the L438K mutant, likely due to a widening at the TM 4/6 interface. Conversely, the WT interface remained constricted, largely disallowing water occupancy. These computational results support our previously proposed clamshell-like gating model for TMEM16 scramblases and provide strong support that the L438K mutation is disrupting the interaction of the TM 4/6 interface, in turn reducing the energetic barrier for both ion and lipid permeation. 

(4) I am puzzled about the ability of OSCA and the TMEM63 proteins which are cation channels to conduct negatively charged lipids. How can the pore be selective for cations and yet permeate negatively charged molecules when lipids are presented? 

This is a great question. TMEM16 scramblase (as well as other known scramblases, such as the Xkr and Opsin families) are surprisingly non-selective to phospholipids (all major phospholipid species, not just anionic lipids like PS). It is still debated whether lipid headgroups indeed insert into an open pore or hydrophilic groove (Response Fig. 5), or if they may traverse the bilayer by the so-called ‘out-of-groove’ model. Regardless of the model, the consensus is that Ca2+-induced conformational changes catalyze lipid permeation and the mutations we have introduced are designed to mimic these conformational changes by separating the TM 4/6 interface.

Additionally, TMEM16F channel activity was first characterized as cation non-selective (10.1016/j.cell.2012.07.036), similar to OSCA/TMEM63s, which may even exhibit some chloride permeability (10.7554/eLife.41844.001). Thus, it appears as though scramblase activity is agnostic to headgroup charge and compatible with both a mutant anion channel (TMEM16A) and mutant cation channels (TMEM16F, OSCA1.2, and TMEM63A), however, more detailed structural, functional, and computational studies are needed to further clarify ion and lipid co-transport mechanisms.  

(5) Do pore blockers like Gd3+ which block permeation also inhibit the scramblase activity of the mutant channels? This should be tested for the mutant channels. 

While extracellular Gd3+ has been previously reported as an inhibitor of OSCA1.2 (10.7554/eLife.41844.001), we did not observe this effect (Author response image 5), but instead saw inhibition by intracellular Gd3+ (Author response image 6). Given this discrepancy, we did not test Gd3+ inhibition of the OSCA1.2 scramblases, but instead tested Ani9, a paralog-specific inhibitor of TMEM16A, on the TMEM16A I546K gain-offunction and found it attenuated both ion channel and phospholipid scramblase activities (Supplementary Fig. 3).

Author response image 5.

200 µM Gd3+ext fails to inhibit OSCA1.2 currents in cell-attached patches. Pressure-elicited peak currents (n=6 each). Statistical test is an unpaired Student’s t-test.

Author response image 6.

200 µM Gd3+int completely inhibits OSCA1.2 currents in inside-out patches. (a) representative traces in before (black), during (red), and after (blue) Gd3+ application. (b) Representative application timecourse. (c) Quantification of peak currents (n=8 each). Statistical test is one-way ANOVA.

Minor: 

- Some of the current amplitudes shown in Figures 2 and 3 are enormous. Is liquid junction potential corrected in these experiments? If not, it would be preferable to correct this to avoid voltage errors. 

Thanks for the question. The large current amplitude is due to 1) great surface expression of the proteins; 2) large single channel conductance of OSCA channels, 3) much larger current at positive voltages for OSCA channels. Our control experiment showed that WT TMEM16A at 0 Ca2+ did not give rise to any current (Fig. 3d), further demonstrating that the large current was not due to liquid junction potential. For the OSCA recordings, we also did not observe current in mock-transfected cells, further excluding the possible interference of liquid junction potential (Response Fig. 1)

- Related, authors could consider adding some evidence using selective pharmacology to support the conclusions that the observed currents arise from TMEM or OSCA channels. 

Thanks for the suggestion. As mentioned above, we have added experiments with Ani9, a specific inhibitor of TMEM16A, in Supplementary Fig. 3. We found that Ani9 robustly attenuated both ion channel and phospholipid scramblase activities for the TMEM16A I546K gain-of-function mutant. This is also consistent with our previous publication (10.1038/s41467-019-09778-7), where Ani9 efficiently inhibited the TMEM16A L534K mutant scramblases. Additionally, we have provided mock controls (Response Fig. 1, Fig. 6d, e) to show that the observed currents are indeed attributable to OSCA1.2 and TMEM63A.

Reviewer #3 (Recommendations For The Authors): 

Given that the authors postulate that the introduction of a positive charge via the lysine side chain is essential to the constitutive activity of these proteins, additional mutation controls for side chain size (e.g. glutamine/methionine) or negative charge (e.g. glutamic acid), or a different positive charge (i.e. arginine) would have strengthened their argument. To more comprehensively understand the TM4/TM6 interface, mutations at locations one turn above and one turn below could be studied until there is no phenotype. In addition, the equivalent mutations on the TM6 side should be explored to rule out the effects of conformational changes that arise from mutating TM4 and to increase the strength of evidence for the importance of side-chain interactions at the TM6 interface. 

We appreciate these great suggestions which were shared by multiple reviewers. We have included our previous responses below.

“Response to reviewers 2 & 3: In our 2019 paper (10.1038/s41467-019-09778-7), we have systematically tested the side chain properties at the inner activation gate of TMEM16F on lipid scrambling activity (Response Fig. 6) and, since then, these results have been supplemented by others as well (10.1038/s41467-022-34497-x). In summary, mutating the inner activation gate residues to polar or charged residues generally results in constitutively activated scramblases without requiring Ca2+ (Fig 5a in 10.1038/s41467-019-09778-7). Because these residues form a hydrophobic gate, introducing smaller side chains via alanine substitution are also gain-of-function with the Y563A mutant as well as the F518A/Y563A/I612A variant being constitutively active (Fig. 3a in 10.1038/s41467-019-09778-7). Meanwhile, mutating these gate residues to hydrophobic amino acids causes no change for I612W, a slight gain-of-function for F518W, slight loss-of-function of F518L, and complete loss-of-function for Y563W (Fig. 4b in 10.1038/s41467-01909778-7). These findings clearly demonstrate that the side-chain properties are critical for regulating the gate opening. Charged mutations including lysine and glutamic acid are the most effective to promote gate opening (Fig 5a in 10.1038/s41467-019-09778-7).

Similarly, others have observed that side chain hydropathy at the F518 site in TMEM16F correlates with shifts in the Ca2+ EC50 (Fig. 2 of 10.1038/s41467-022-34497-x). Note that this publication resolved the structure of the TMEM16F F518H mutant, revealing a previously unseen conformation that we have highlighted in Supplementary Fig. 1e and discussed in lines 235-238. Please also see our response to Reviewer #1 above, where we discuss discoveries in model transmembrane helical peptide systems showing that transbilayer lipid flipping rates correlate with side chain hydropathy (Author response image 3), distance between stacked hydropathic residues (schematic in 10.1248/cpb.c22-00133), and even helical angle between stacked side chains (not show). 

Following the reviewers’ suggestions, we have tested additional mutations in alternative locations and with different side chains.  

(1) We have added data for TMEM16F I521A and I521E to demonstrate a similar effect of alternative side chains to what has previously been reported by us and others. We found that I521A failed to show spontaneous scrambling activity (Supplementary Fig. 2), yet I521E (Supplementary Fig. 2) is a constitutively active lipid scramblase, similar to I521K (Fig. 1). This further demonstrates that gate disruption correlates with the side chain hydropathy and that this site lines a critical gating interface.

(2) We also added lysine mutations two helical turns below the conserved inner activation gate for TMEM16F T526 (Fig. 1), TMEM16A E551 (Fig. 3). We found that there is indeed a lower limit for the observed effect in TMEM16, where lysine mutations no longer induce spontaneous lipid scrambling activity. This indicates that when TM 4/6 interaction is weaker toward intracellular side (Figs. 1a, 3a), the TM 4 lysine mutation loses the ability to promoting lipid scrambling by disrupting the TM 4/6 interface to enable clamshell-like opening of the permeation pathway. 

(3) We added a TMEM16F lysine mutation on TM 6 at residue I611 (Fig. 2). Similar to I612K (Response Fig. 6), I611K also leads to spontaneous lipid scrambling and enhanced channel activity in the absence of calcium (Fig. 2). This shows that charged mutations along TM 6 can also promote lipid scrambling, strengthening our model that hydrophobic interactions along the TM 4/6 interface are critical for gating and lipid permeation.”

The experiments for OSCA1.2 osmolarity effects on gating and scramblase in Figure 4 could be improved by adding different levels of osmolarity in addition to time in the hypotonic solution.

We thank the reviewer for this excellent suggestion. We extensively tested this idea and found evidence (Response Fig. 10) that intermediate osmolarity (220 and 180 mOso/kg) also can enhance the scramblase activity of the A439K mutant, albeit to a milder extent compared to 120 mOso/kg stimulation. This suggests that swellinginduced membrane stretch may proportionally induce A439K activation and lipid scrambling. Due to the relatively mild sensitivity of OSCA to osmolarity and the variations induced by the experimental conditions, we believe it is better to not include this data to avoid overclaiming. We hope the reviewer would agree. 

Author response image 7.

AnV intensities of WT- and A439K-transfected cells after 10 minutes of hypotonic stimulation at the listed osmolarities.

Some confocal images appear to be rotated relative to each other (e.g. Figures 2b and 3b).

Thank you for identifying these errors, they are corrected in the revision.

eLife Assessment

This important study advances our understanding of the mechanisms controlling lipid flux and ion permeation in the TMEM16 and OSCA/TMEM63 family channels. The study provides compelling new evidence indicating that side chains along the TM4/6 interface play a key role in gating lipid and ion fluxes in these channels. The authors suggest that the transmembrane channel/scramblase family proteins may have originally functioned as scramblases but lost this capacity over evolution.

Reviewer #1 (Public review):

Summary:

TMEM16, OSCA/TMEM63, and TMC belong to a large superfamily of ion channels where TMEM16 members are calcium activated lipid scramblases and chloride channels, whereas OSCA/TMEM63 and TMCs are mechanically activated ion channels. In the TMEM16 family, TMEM16F is a well characterized calcium activated lipid scramblase that play an important role in processes like blood coagulation, cell death signaling, and phagocytosis. In a previous study the group has demonstrated that lysine mutation in TM4 of TMEM16A can enable the calcium activated chloride channel to permeate phospholipids too. Based on this they hypothesize that the energy barrier for lipid scramblase in these ion channels is low, and that modification in the hydrophobic gate region by introducing a charged side chain between TM4/6 interface in TMEM16 and OSCA/TMEM63 family can allow lipid scramblase. In this manuscript, using scramblase activity via Annexin V binding to phosphatidylserine, and electrophysiology, the authors demonstrate that lysine mutation in TM4 of TMEM16F and TMEM16A can cause constitutive lipid scramblase activity. The authors then go on to show that analogous mutations in OSCA1.2 and TMEM63A can lead to scramblase activity. The revised version does a thorough characterization of residues that form the hydrophobic gate region in TM4/6 of this superfamily of channels. Their results indicated that disrupting the TM4/6 interaction can reduce energy barrier for this channels to scramblase lipids.

Strengths:

Overall, the authors introduce an interesting concept that this large superfamily can permeate ions and lipids.

Weaknesses:

none noted in the revised version.

Reviewer #2 (Public review):

This focused study by Lowry and colleagues that identifies a key molecular motif that controls ion permeation vs combined ion permeation and lipid transport in three families of channel/scramblase proteins, in TMEM16 channels, in the plant-expressed and stress-gated cation channel OSCA, and in the mammalian homolog and mechanosensitive cation channel, TMEM63. Between them, these three channels share low sequence similarity and have seemingly differing functions, as anion (TMEM16 channels), or stress-activated cation channels (OSCA/TMEM63). The study finds that in all three families, mutating a single hydrophobic residue in the ion permeation pathway of the channels confers lipid transport through the pores of the channels, indicating that TMEM16 and related OSCA and TMEM63 channels have a conserved potential for both ion and lipid permeation. The authors interpret the findings as revealing that these channel/scramblase proteins have a relatively low "energetic barrier for scramblase" activity. The experiments are done with a high level of rigor and the revised paper is very well written and addresses the previous concerns.

Reviewer #3 (Public review):

This study was focused on the conserved mechanisms across the Transmembrane Channel/Scramblase superfamily, which includes members of the TMEM16, TMEM63/OSCA, and TMC families. In previous work, the authors have studied the role of the inner activation gate of these proteins. Here, the authors show that the introduction of mutations at the TM4-TM6 interface, which are close to the inactivation gate, can disrupt gating and confer scramblase activity to non-scramblases proteins.

Overall, the confocal imaging experiments, patch clamping experiments, and data analysis are performed well and in line with standard methods. The molecular dynamics simulation work is focused but adds supportive evidence to their findings. Although there could have been more extensive molecular analysis to bolster the authors' arguments on the role of the TM4-TM6 interface (e.g. evaluate effects of size/hydrophobicity, double mutants, cross-linking, more in-depth simulation data), there is adequate evidence to conclude that certain residues at this interface is critical to ion conduction and phospholipid scramblase activity. The data presented only adds incremental depth of knowledge for each individual channel, but together, they show this to be true for conserved TM4 residues across TMEM16F, TMEM16A, OSCA1.2, and TMEM63A proteins. This breadth of data is a major strength of this paper, and provides strong evidence for a coupled pathway for ion conduction and phospholipid transport, though the underlying biophysical mechanism is still speculative and remains to be elucidated.

Change to "... a non-empty set of vectors" for clarity?

HEENT = everything normal, non-remkarable

Entendimento do STF: é devido o pagamento de honorários sucumbenciais à Defensoria Pública, quando representa parte vencedora em demanda ajuizada contra qualquer ente público, inclusive aquele que integra → ⚠ cancelamento da Súmula 421 do STF

This statement is deeply impactful as it highlights the pain and alienation that Helena, as an Asian American, has experienced due to being grouped together with others based on superficial perceptions and stereotypes. It emphasizes how often people don't take the time to understand individual differences in Asian American communities, leading to feelings of being misunderstood and isolated. Helena's experience demonstrates the emotional toll and the profound sense of exclusion that can result from such stereotyping and ignorance.

I’m all for recognising that racist attitudes in children aren’t harmless. It makes me afraid of these behaviors being unchecked without early intervention. Children do experience this period of development of learning from and imitating. If we simply reject them as childish in the sense of not learning anything, then we are cutting off critical opportunities to teach them how to embrace difference.

Concentration is measured in percentage, not g/mL (which represents density).

Successful Secretary Presented by Royal Office Typewriters. A Thomas Craven Film Corporation Production, 1966. https://www.youtube.com/watch?v=If5b2FiDaLk.

Script: Lee Thuna<br /> Educational Consultant: Catharine Stevens<br /> Assistant Director: Willis F. Briley<br /> Design: Francisco Reynders<br /> Director & Producer: Carl A. Carbone<br /> A Thomas Craven Film Corporation Production

"Mother the mail"

gendered subservience

"coding boobytraps"

"I think you'll like the half sheet better. It is faster." —Mr. Typewriter, timestamp

A little bit of the tone of "HAL" from 2001: A Space Odyssey (1968). This is particularly suggestive as H.A.L. was a one letter increment from I.B.M. and the 1966 Royal 660 was designed to compete with IBM's Selectric

This calm voice makes suggestions to a secretary while H.A.L. does it for a male astronaut (a heroic figure of the time period). Suddenly the populace feels the computer might be a bad actor.

"We're living in an electric world, more speed and less effort."—Mr. Typewriter<br /> (techno-utopianism)

I find it odd that sometimes white Americans frame Asian Americans in such a way that makes them seem superior to other people of colour, particularly African Americans, as an extension of maintaining control over them. And yes, this strategy can divide minority groups into competing or competing for status or recognition in a white world order. Asian Americans being "nearer to whites" is often driven by a combination of academic and economic accomplishments that contrast the resentment against structural inequality among African Americans and other minorities who have enjoyed less.

I see that a number of Asian American researchers have indeed played a significant role in explaining Asian Americans’ adaptive disadvantages. It has been an innovator in illuminated racism and the oppressions faced by this community. Moreover, the fact that we’re less likely to explicitly cite whites as critical to generating and maintaining racist spaces parallels a trend in scholarship generally.

Something i found interesting was something is the way people still discriminate or push aside others peoples history which is still as important as any other history. I'm happy to see that in the school i work in because we are a diverse school, we tend to incorporate everyones culture, and we have the kids do culture projects in which they present where they're from and parents bring in food from their culture in which everyone gets to try.

The fact that the reading calls out the standards Asian groups have to meet because it's a standard is interesting. It is something i've always hear of what people say from these groups, but the Asians i've met have always been the most hard working, but over work themselves through pressure because of their parents of their enviroment.

This is something sad but important to know because suicide groups do not just focus on a certain groups but it can be any one generally. Here specifically talking about the Asian American groups, as of what i know or learned from, these groups are held to a high standard and discipline where it is sad seeing the results of it.

author provided sources, is planned

Testing ideas

Deserved if you messed something this important up

That he is ready for halloween

This quote explains how dedicated the character was to their Halloween costume.

he was so excited to show off his costume.

the halloween party ain't till next week. so he embbrassed himself??

有点特别

Noch nicht peer-reviewte Studien sprechen dafür, dass die globale Erhitzung die Niederschläge durch den Hurrikan Milton um 20 und seine Windgeschwindigkeit um 10% gesteigert hat. Dles führte zu etwa doppelt so großen Zerstörungen als bei einem nicht von der Erhitzung beteuerten Sturm gleicher Seltenheit. https://www.nytimes.com/2024/10/11/climate/milton-climate-change.html

https://www.hostingadvice.com/how-to/best-mediawiki-hosting/

Miles touches on the interplay of knowledge written down on index cards and the knowledge which is kept only in one's mind. Some practitioners in the space from 2013-2024 seem to imply that they're writing almost everything out in far deeper detail than Miles would indicate. In his incarnation, much of the knowledge might be more quickly indicated by a short sentence or heading which the brain can associate to longer explanations.

This sort of indexing is akin to some of the method potentially seen in Marshall Mathers' zettelkasten.

I'm creating a tag here for "card index for productivity" to track the idea of productivity in writing which I'm specifically using separately from the tag "card index as productivity system" which is used to describe their use for project tracking systems in systems like GTD, Memindex, etc.

How would degranulation limit inflammation? The MCs are releasing pro-inflammatory cytokines, as you pointed out.

Consider including a quantification of the increase reported by Esaulova et al? It's always difficult to evaluate the importance of a finding like this without some appreciation of the magnitude of the change.

in reply to Cesar A. Hidalgo at https://x.com/realAnthonyDean/status/1844409919161684366

via Anthony Dean @realAnthonyDean

Advanced Typing: Duplicating and Manuscript. Vol. MN-1512d, 1943. https://www.youtube.com/watch?v=7ve5JnTUzvo.

Stencils

Before writing stencils, be sure to clean your type. (Don't use liquid solvent.)

Be sure to place the cushion sheet properly behind the stencil.

Place the paper bail rollers at the extreme left and right of the stencil to prevent them from marking the master.

For errors, rub individual characters separately with a burnisher using a circular motion.

Hectograph masters, Hectograph ribbon (ditto ribbon)

Wax pencils

Typefaces

20% more type on a page with elite than 10 inch pica.

Pica allows approximately 26-40 lines on standard letterhead giving 300-450 words to a page.

Special characters: - o for degrees ' and " for feet and inches or minutes and seconds along with superscript - division: - backspace colon - pound sterling: L backspace f - exclamation point: period backspace ' - equal sign: hyphen backspace variable hyphen - paragraph mark: P backspace I

proofreaders' marks<br /> # followed by a number is used to mean insert that number of spaces

Centering timestamp 19:37

https://www.calligraphr.com/en/

Can be used to digitize typewriter typefaces.

ethical vs. exemplary prophet

Prophet can demand obedience on higher beings behalf or spread doctrine through exemplary lifestyles

Prophets require a mystical doctrine alongside magical acts- which mystagogues (or magicians) lack

prophets gather people around them? Mystagouges don't?

from internet- teacher or producer of mystical doctrine

No matter the semblance, religious truth and salvations defines prophetic schools from the most abstract schools of thought

philosophy or efforts towards social reform unattached to a doctrine can not be considered prophetic teachings

Not protecting human rights for the sake of human rights

teacher of ethics outgrowth of prophets??

social reform all has charismatic orientation

Jesus not concerned with social reform but other propehts of different religions were

Most prophets trying to explain God's wrath through social injustice, not initiate social reform

all Israelite social reforms was for the sake of foreign policy

but, this behavior is not common among prophets

Moses transitioned from a prophet to lawgiver, finding solutions to economic conflict and oriented Israelites as a nation towards a god.

prophets are characteristically, not capitalizing on their skills and abilities

Although not magicians, magic almost always involved in proving prophets legitimacy

Prophets orientation is to a doctrine nit magic, self-interested supernatural actions

Priest gains credibility through servitude Prophet gains credibility through personal revelations and interpretations as well as charisma

what we care about- what makes a prophet and what makes a priest?

position of individual within the movement of a religion not relevant

fascinating- everything would go bad in a very physical and material sense if belief in whatever no longer existed, promotes people to give

pop is the og designation of successor

power to absolve people from sins passed down from ___ to the office individuals, bishop or priest

same groups different fonts? can't attain there power through rational or simply traditional means, charismatic involved somehow

charisma can only remain anti-economic for so long

charismatic admin can become bureacratic

charismatic admin can tax or require fees of members

begin to build other economic structures, less based on charismatic power

charismatic power transfers to traditionalized hereditary power over time

all charisma must be awakened (differ from like a monk)

charisma can be transferable- making the charisma an object independent of the individual

sometimes charisma is hereditary

can be designated by larger community

Glocal (=globale en lokale elementen): - Etic: woordenboeken, eigenschappen reduceren via vertrouwdheid, persoonlijkheidseigenschappen verzamelen via andere instrumenten. - Emic: aanvullende metingen ter reducatie/ andere woorden mogelijk verwijderen, kwalitatieve gegevens via gesproken eigenschappen, en gegevens vergelijken met andere culturen.

9 clusters van SAPI: 1. Gewetensvolheid. 2. Emotionele stabiliteit. 3.. Extraversie. 4. Ontspanning. 5. Integriteit. 6. Intellect. 7. Openheid. 8. Relatieharmonie. 9. Zachtmoedigheid.

Doelen van SAPI-project: - Inheemse theoretische model van persoonlijkheid ontwikkelen. - Persoonlijkheidstest ontwikkelen die onbevooroordeeld is.

4 factoren van zelfbeschrijving: - Sociale potentie. - Afhankelijkheid. - Clementie (=zachtaardigheid). - Interpersoonlijke verbondenheid.

Perspectief van zowel binnenuit als buitenaf; hoe iemand iets zelf ervaart maar ook hoe iemand anders naar bepaalde realiteit kijkt.

Emic=perspectief van binnenuit; hoe iemand iets zelf ervaart, specifiek voor bepaalde groep.

Selectieve migratie van vergelijkbare individuen= (woon)plaatsen opzoeken op basis van vergelijkbare mensen (=qua persoonlijkheid).

Cultuur maakt persoonlijkheid= gedrag aanleren door wat om je heen hierover gedeeld wordt.

Twijfelpunten emic approach: - Methoden zijn vaak niet rigoureus (=streng) genoeg. - Nieuwe instrument heroverwegen; incrementele validiteit.

Emic=perspectief van binnenuit... m=meubels.

Etic=perspectief van buitenaf... t=tuin.

Twijfelpunten etic approach: - Oorspronkelijke Engelse instrumenten leiden tot etnocentrisme (=minder kijken naar andere culturen). - Vergelijken en overeenkomsten zoeken leidt tot belangrijke details overslaan (=blinde vlek).

Morris & Peng; Aquarium onderzoek: - Chinese prikkel: situationele verklaring=vis achtervolgd. - Amerikaanse prikkel: dispositionele verklaring= vis leider. - Controle groep: tussenin...

Dispositions=persoonlijke staat..

Prejudice=vooroordelen.

took out the paragraph with they keyboard shortcuts. they don't work on Mac.

direct UN def violation

Crazy Horse and Spotted Tail came to represent two traditions of Indigenous resistance; one would follow a path of armed struggle, the other the path of diplomacy.

very eye catching statement

horrible person

I assume that the "not required" means it was waived.

OK WHOA .... so far, far from random selection. So the whole idea that "they did better" mgiht have been because htey were in this cohort.

Welp, ql1 was the more "mathy' version.

yup!!!

I bet it was procedural.

Interesting. Very interensting. Would they have done differently?

(what's recitation?)

**RIGHT **

Amen.

More interesting language but boy do I appreciate the "alluring." And hey, it's "successful"! THe CA law is like the IL law.

... so it improves student attitude....

Think of the lots and lots of meetings discussing this :P Note it doesn't say "just use a calculator to figure out percents."

(yup, challenging.... )

lol not loaded language or anything

"room for improvement..." yes.

Beautiful vision.

(Having a human voice read the words would be even more compelling.)

Author response:

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

Public Reviews:

Reviewer #2 (Public review):

Weaknesses:

The authors have clarified that the first features available for each patient have been used. However, they have not shown that these features did not occur before the time of post-stroke epilepsy. Explicit clarification of this should be performed.

The data utilized in our analysis were collected during the first examination or test conducted after the patients' admission. We specifically excluded any patients with a history of epilepsy, ensuring that all cases of epilepsy identified in our study occurred after admission. Therefore, the features we analyzed were collected after the patients' admission but prior to the onset of post-stroke epilepsy.

Reviewer #3 (Public review):

Weaknesses:

The writing of the article may be significantly improved.

Although the external validation is appreciated, cross-validation to check robustness of the models would also be welcome.

Thank you for your helpful advice.  Performing n-fold cross-validation is a crucial step to ensure the reliability and robustness of the reported results, especially when dealing with the datasets which don't have sufficient quantity.   We revised our code and did a 5 fold cross-validation version ,it didn’t have much promote（because our model has reach the auc of 0.99）.Considering that we have sufficient quantity of more than 20000 records, we think split the dataset by 7:3 and train the model is enough for us. We have uploaded the code of 5 fold cross-validation version and ploted the 5 fold test roc  on GitHub at https://github.com/conanan/lasso-ml/lasso_ml_cross_validation.ipynb as an external resource. We  trained the 5 fold average model and ploted the 5 fold test roc curves, the results show some improvement, but it is not substantial because the best model are still tree models in the end.

External validation results may be biased/overoptimistic, since the authors informed that "The external validation cohort focused more on collecting positive cases 80 to examine the model's ability to identify positive samples", which may result in overoptimistic PPV and Sensitivity estimations. The specificity for the external validation set has not been disclosed.

Thank you for your valuable feedback regarding the external validation results. We appreciate your concerns about potential bias and overoptimism in our estimations of positive predictive value (PPV) and sensitivity.

To clarify, we have uploaded the code for external validation on GitHub at https://github.com/conanan/lasso-ml. The results indicate that the PPV is 0.95 and the specificity is 0.98.

While we focused on collecting more positive cases due to their lower occurrence rate, this approach allows us to better evaluate the model's ability to predict positive samples, which is crucial in clinical settings. We believe that emphasizing positive cases enhances the model's utility for practical applications(So a little overoptimism is acceptable ).

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

Public Reviews:

Reviewer #1 (Public Review):

Weaknesses 1:

The methodology needs further consideration. The Discussion needs extensive rewriting.

Thanks for your advice, we have revised the Discussion

Reviewer #2 (Public Review):

Weaknesses 2:

There are many typos and unclear statements throughout the paper.

There are some issues with SHAP interpretation. SHAP in its default form, does not provide robust statistical guarantees of effect size. There is a claim that "SHAP analysis showed that white blood cell count had the greatest impact among the routine blood test parameters". This is a difficult claim to make.

Thank you for your suggestion that the SHAP analysis is really just a means of interpreting the model.  In our research, we compared the SHAP analysis with traditional statistical methods, such as regression analysis.  We found the SHAP results to be consistent with the statistical results from the regression for variables like white blood cell count (see Table 1). This alignment leads us to believe the SHAP analysis is providing reliable insights in this context

The Data Collection section is very poorly written, and the methodology is not clear.

Thanks for your advice, we have revised the Data Collection section.

There is no information about hyperparameter selection for models or whether a hyperparameter search was performed. Given this, it is difficult to conclude whether one machine learning model performs better than others on this task.

Thank you for the advices of performing hyperparameter. We used the package of sklearn, xgboost, lightgbm of python 3.10 to construct the model and  didn’t change the default settings before. It is not proper and may lead to  less certain conclusions. Now we carry out grid search to select and optimize hyperparameters and they make the model better. The best model is still RF.

The inclusion and exclusion criteria are unclear - how many patients were excluded and for what reasons?

The procedure of selection is in figure1. Total there are 42079 records from the stroke database, 24733 patients were diagnosed as ischemic stroke or lacular stoke with new onset. Then we excluded hemorrage stroke(4565),history of stroke(2154), TIA(3570), unclear cause stroke(561) and records who missed important data(6496). Then we excluded patients whose seizure might be attributed to other potential causes (brain tumor, intracranial vascular malformation, traumatic brain injury,etc)(865). Then we exclude patient who had a seizure history(152) or died in hospital (1444). Then we excluded patients who were lost in follow-up (had no outpatient records and can’t contact by phone )or died within 3 months of the stroke incident(813). Finally 21459 cases are involved in this research.

There is no sensitivity analysis of the SMOTE methodology: How many synthetic data points were created, and how does the number of synthetic data points affect classification accuracy?

Thanks for your remind, we have accept these advice and change the SMOTE to SMOTEENN (Synthetic Minority Over-sampling Technique combined with Edited Nearest Neighbors) technique to resample an imbalanced dataset for machine learning. The code is

smoteenn = SMOTEENN(samplingstrategy='auto', randomstate=42)

the SMOTEENN class comes from the imblearn library. The samplingstrategy='auto' parameter tells the algorithm to automatically determine the appropriate sampling strategy based on the class distribution. The randomstate=42 parameter sets a seed for the random number generator, ensuring reproducibility of the results.

Did the authors achieve their aims? Do the results support their conclusions?

Yes, we have achieve some of the aims of predicting PSE while still leave some problem.

The paper does not clarify the features' temporal origins. If some features were not recorded on admission to the hospital but were recorded after PSE occurred, there would be temporal leakage.

The data used in our analysis is from the first examination or test conducted after the patients' admission, retrieved from a PostgreSQL database. First, we extracted the initial admission date for patients admitted due to stroke. Then, we identified the nearest subsequent examination data for each of those patients.

The sql code like follows:

SELECT TO_DATE(condition_start_date, 'DD-MM-YYYY') AS DATE

FROM diagnosis

WHERE person_id ={} and (condition_name like '%梗死%' or condition_name like '%梗塞%') and(condition_name like '%脑%'or condition_name like '%腔隙%'))

order by DATE limit 1

The authors claim that their models can predict PSE. To believe this claim, seeing more information on out-of-distribution generalisation performance would be helpful. There is limited reporting on the external validation cohort relative to the reporting on train and test data.

Thank you for the advice. The external validation is certainly very important, but there have been some difficulties in reaching a perfect solution.  We have tried using open-source databases like the MIMIC database, but the data there does not fit our needs as closely as the records from our own hospital.  The MIMIC database lacks some of the key features we require, and also lacks the detailed patient follow-up information that is crucial for our analysis.   Given these limitations, we have decided to collect newer records from the same hospitals here in Chongqing.  We believe this will allow us to build a more comprehensive dataset to support robust external validation.  While it may not be a perfect solution, gathering this additional data from our local healthcare system is a pragmatic step forward.   Looking ahead, we plan to continue expanding this Chongqing-based dataset and report on the results of the greater external validation in the future.  We are committed to overcoming the challenges around data availability to strengthen the validity and generalizability of our research findings.

For greater certainty on all reported results, it would be most appropriate to perform n-fold cross-validation, and report mean scores and confidence intervals across the cross-validation splits

Thank you for your helpful advice. Performing n-fold cross-validation is a crucial step to ensure the reliability and robustness of the reported results, especially when dealing with the datasets which don't have sufficient quantity. While we have sufficient quantity of more than 20000 records, so we think split the dataset by 7:3 and train the model is enough for us. We revised our code and did a 5 fold cross-validation version ,it had little promote（because our model has reach the auc of 0.99）, we may use this great technique in our next study if there is not enough cases.

Additional context that might help readers

The authors show force plots and decision plots from SHAP values. These plots are non-trivial to interpret, and the authors should include an explanation of how to interpret them.

Thank you for your helpful advice. It is a great improve for our draft, we have added the explanation that we use the force plot of the first person to show the influence of different features of the first person, we can see that long APTT time contribute best to PSE, then the AST level and others, the NIHSS score may be low and contribute opposite to the final result. Then the decision plot is a collection of model decisions that show how complex models arrive at their predictions

Reviewer #3 (Public Review):

Weaknesses3:

There are issues with the readability of the paper. Many abbreviations are not introduced properly and sometimes are written inconsistently. A lot of relevant references are omitted. The methodological descriptions are extremely brief and, sometimes, incomplete.

Thanks for your advice, we have revised these flaws.

The dataset is not disclosed, and neither is the code (although the code is made available upon request). For the sake of reproducibility, unless any bioethical concerns impede it, it would be good to have these data disclosed.

Thank you for your recommendations. We have made the code available on GitHub at https://github.com/conanan/lasso-ml. While the data is private and belongs to the hospital. Access can be requested by contacting the corresponding author to apply from the hospitals and specifying the purpose of inquiry.

Although the external validation is appreciated, cross-validation to check the robustness of the models would also be welcome.

Thank you for your valuable advice. Performing n-fold cross-validation is crucial for ensuring the reliability and robustness of results, especially with limited datasets. However, since we have over 20,000 records, we believe that a 70:30 split for training and testing is sufficient.

We revised our code and implemented 5-fold cross-validation, which provided minimal improvement, as our model has already achieved an AUC of 0.99. We plan to use this technique in future studies if we encounter fewer cases.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

My comments include two parts:

(1) Methodology<br /> a-This study was based on multiple clinical indicators to construct a model for predicting the occurrence of PSE. It involved various multi-class indicators such as the affected cortical regions, locations of vascular occlusion, NIHSS scores, etc. Only using the SHAP index to explain the impact of multi-class variables on the dependent variable seems slightly insufficient. It might be worth considering the use of dummy variables to improve the model's accuracy.

Thank you for the detailed feedback on the study methodology. The SHAP analysis is really just a means of interpreting the model, which we compared with the combination of SHAP and traditional statistics, so we think SHAP analysis is reliable in this research. We have used the dummy variables, expecially when dealing with the affected cortical regions, locations of vascular occlusion, for example if frontal region is involved the variable is 1. But they have less impact in the machine learning model

b-The study used Lasso regression to select 20 features to build the model. How was the optimal number of 20 features determined?

Lasso regression is a commonly used feature screening method. Since we extract information from the database and try to include as many features as possible, the cross-verification curve of lasso regression includes 78 features best, but it will lead to too complex model. We select 10,15,20,25,30 features for modeling according to the experiment. When 20 features are found, the model parameters are good and relatively concise. Improve the number of features contribute little to the model effect, decrease the number of features influence the concise of model ,for example the auc of the model with 15 features will drop under 0.95. So we finally select 20 features.

c-The study indicated that the incidence rate of PSE in the enrolled patients is 4.3%, showing a highly imbalanced dataset. If singly using the SMOTE method for oversampling, could this lead to overfitting?

Thanks for your remind, singly using the SMOTE method for oversampling is inproper. Now we have find this improvement and change the SMOTE to SMOTEENN (Synthetic Minority Over-sampling Technique combined with Edited Nearest Neighbors) technique to resample an imbalanced dataset for machine learning. First, oversampling with SMOTE and then undersampling with ENN to remove possible noise and duplicate samples. The code is

smoteenn = SMOTEENN(sampling_strategy='auto', random_state=42)

the SMOTEENN class comes from the imblearn library. The sampling_strategy='auto' parameter tells the algorithm to automatically determine the appropriate sampling strategy based on the class distribution. The random_state=42 parameter sets a seed for the random number generator, ensuring reproducibility of the results.

(2) Clinical aspects:

Line 8, history of ischemic stroke, this is misexpression, could be: diagnosis of ischemic stroke.

Line 8, several hospitals, should be more exact; how many?

Line 74 indicates that the data are from a single centre, this should be clarified.

Line 4 data collection: The criteria read unclear; please clarify further.

Thanks for your remind, we have revised the draft and correct these errors.

Line 110, lab parameters: Why is there no blood glucose?

Because many patients' blood sugar fluctuates greatly and is easily affected by drugs or diet, we finally consider HBA1c as a reference index by asking experts which is more stable.

Line 295, The author indicated that data lost; this should be clarified in the results part, and further, the treatment of missing data should be clarified in the method part.

Thanks for your remind, we have revised the draft and correct these errors.

I hope to see a table of the cohort's baseline characters. The discussion needs extensive rewriting; the author seems to be swinging from the stoke outcome and the seizure, sometimes losing the target.

Figure1 is the procedure of the selection of patients. Table1 contains the cohort's baseline characters

For the swinging from the stoke outcome and the seizure, that is because there are few articles on predicting epilepsy directly by relevant indicators, while there are more articles on prognosis. So we can only take epilepsy as an important factor in prognosis and comprehensively discuss it, or we can't find enough articles and discuss them

Reviewer #2 (Recommendations For The Authors):

There are typos and examples of text that are not clear, including:

"About the nihss score, the higher the nihss score, the more likely to be PSE, nihss score has a third effect just below white blood cell count and D-dimer."

"and only 8 people made incorrect predictions, demonstratijmng a good predictive ability of the model."

"female were prone to PSE"

" Waafi's research"

"One-heat' (should be one-hot)

Thanks for your remind, we have revised the draft and correct these errors.

The Data Collection section is poorly written, and the methodology is not clear. It would be much more appropriate to include a table of all features used and an explanation of what these features involve. It would also be useful to see the mean values of these features to assess whether the feature values are reasonable for the dataset.

Thanks for your remind. All data are from the first examination or test after admission, presented through the postgresql database . First we extract the first date of the patients who was admitted by stroke ,then we extract informations from the nearest examination from the admission. We extract by the SQL code by computer instead of others who may extract data by manual so we get as much data as possible other than only get the features which was reported before .The table of all features used and their mean±std is in table1.

The paper does not clarify the features' temporal origins. If some features were not recorded on admission to the hospital but were recorded after PSE occurred, there would be temporal leakage. I would need this clarified before believing the authors achieved their claims of building a predictive model.

All relevant index results were from the first examination after admission, and the mean standard deviation was listed in the statistical analysis section in table1.

The authors claim that their models can predict PSE. To believe this claim, seeing more information on out-of-distribution generalisation performance would be helpful. There is limited reporting on the external validation cohort relative to the reporting on train and test data.

Thank you for the advice, the external validation is very important but there are some difficulties to reach a perfect one. We have tried some of the open source database like the mimic database ,but these data don't fit our request because they don't have as much features as our hospital and lack of follow-up of the relevant patients. In the end we collected the newer records in the same hospitals in Chongqing and we will collect more and report a greater external validation in the future.

For greater certainty on all reported results, It would be most appropriate to perform n-fold cross-validation, and report mean scores and confidence intervals across the cross-validation splits.

Thank you for your helpful advice. Performing n-fold cross-validation is a crucial step to ensure the reliability and robustness of the reported results, especially when dealing with the datasets which don't have sufficient quantity. While we have sufficient quantity of more than 20000 records, so we think split the dataset by 7:3 and train the model is enough for us. We revised our code and did a 5 fold cross-validation version ,it had little promote, we will use this great technique in our next study.

The authors show force plots and decision plots from SHAP values. These plots are non-trivial to interpret, and the authors should include an explanation of how to interpret them.

It is a great improve for our draft, we have added the explanation we use the force plot of the first person to show the influence of different features of the first person, we can see that long APTT time contribute best to PSE, then the AST level and others, the NIHSS score may be low and contribute lower to the final result. Then the decision plot is a collection of model decisions that show how complex models arrive at their predictions

Reviewer #3 (Recommendations For The Authors):

Abbreviations should not be defined in the abstract )or only in the abstract).

Please explicit what are the purposes of the study you are referring to in "Currently, most studies utilize clinical data to establish statistical models, survival analysis and cox regression."

Authors affirm: "there is still a relative scarcity of research 49 on PSE prediction, with most studies focusing on the analysis of specific or certain risk factors ." This statement is especially curious since the current study uses risk factors as predictors.

It is not clear to me what the authors mean by "No study has proposed or established a more comprehensive and scientifically accurate prediction model." The authors do not summarize the statistical parameters of previously reported model, or other relevant data to assess coverage or validity (maybe including a Table summarizing such information would be appropriate. In any case, I would try to omit statements that imply, to some extent, discrediting previous studies without sufficient foundation.

"antiepileptic drugs" is an outdated name. Please use "antiseizure medications"

Thanks for your remind, we have revised the draft and correct these errors.

The authors say regarding missing data that they "filled the data of the remaining indicators with missing values of more than 1000 cases by random forest algorithm". Please clarify what you mean by "of more than 1000 cases." Also, provide details on the RF model used to fill in missing data.

Thanks for your remind. "of more than 1000 cases" was a wrong sentence and we have corrected it. Here is the procedure, first we counted the values of all laboratory indicators for the first time after stroke admission( everyone who was admitted because of stroke would perform blood routine , liver and kidney function and so on), excluded indicators with missing values of more than 10%, and filled the data of the remaining indicators with missing values by random forest algorithm using the default parameter. First, we go through all the features, starting with the one with the least missing (since the least accurate information is needed to fill in the feature with the least missing). When filling in a feature, replace the missing value of the other feature with 0. Each time a regression prediction is completed, the predicted value is placed in the original feature matrix and the next feature is filled in. After going through all the features, the data filling is complete.

Please specify what do you mean by negative group and positive group, Avoid tacit assumptions.

Thanks for your remind, we have revised the draft and correct these errors.

Please provide more details (and references) on the smote oversampling method. Indicate any relevant parameters/hyperparameters.

Thanks for your remind, we have accept these advice and change the SMOTE to SMOTEENN (Synthetic Minority Over-sampling Technique combined with Edited Nearest Neighbors) technique to resample an imbalanced dataset for machine learning. The code is

smoteenn = SMOTEENN(sampling_strategy='auto', random_state=42)

the SMOTEENN class comes from the imblearn library. The sampling_strategy='auto' parameter tells the algorithm to automatically determine the appropriate sampling strategy based on the class distribution. The random_state=42 parameter sets a seed for the random number generator, ensuring reproducibility of the results.

The methodology is presented in an extremely succinct and non-organic manner (e.g., (Model building) Select the 20 features with the largest absolute value of LASSO." Please try to improve the narrative.

Lasso regression is a commonly used feature screening method. Since we extract information from the database and try to include as many features as possible, the cross-verification curve of lasso regression includes 78 features best, but it will lead to too complex model. We select 10,15,20,25,30 features for modeling according to the experiment. When 20 features are found, the model parameters are good and relatively concise. Improve the number of features contribute little to the model effect, decrease the number of features influence the concise of model ,for example the auc of the model with 15 features will drop under 0.95. So we finally select 20 features.

Many passages of the text need references. For example, those that refer to Levene test, Welch's t-test, Brier score, Youden index, and many others (e.g., NIHSS score). Please revise carefully.

Thanks for your remind, we have revised the draft and correct these errors.

"Statistical details of the clinical characteristics of the patients are provided in the table." Which table? Number?

Thanks for your remind, we have revised the draft and correct these errors， it is in table1.

Many abbreviations are not properly presented and defined in the text, e.g., wbc count, hba1c, crp, tg, ast, alt, bilirubin, bua, aptt, tt, d_dimer, ck. Whereas I can guess the meaning, do not assume everyone will. Avoid assumptions.

ROC is sometimes written "ROC" and others, "roc." The same happens for PPV/ppv, and many other words (SMOTE; NIHSS score, etc.).

Please rephrase "ppv value of random forest is the highest, reaching 0.977, which is more accurate for the identification of positive patients(the most important function of our models).". PPV always refer to positive predictions that are corroborated, so the sentences seem redundant.

Thanks for your remind, we have revised the draft and correct these errors.

What do you mean by "Complex algorithms". Please try to be as explicit as possible. The text looks rather cryptic or vague in many passages.

Thanks for your remind, "Complex algorithms" is corrected by machine learning.

The text needs a thorough English language-focused revision, since the sense of some sentences is really misleading. For instance "only 8 people made incorrect predictions,". I guess the authors try to say that the best algorithm only mispredicted 8 cases since no people are making predictions here. Also, regarding that quote... Are the authors still speaking of the results of the random forest model, which was said to be one of the best performances?

Thanks for your remind, we have revised the draft and correct these errors.

The authors say that they used, as predictors "comprehensive clinical data, imaging data, laboratory test data, and other data from stroke patients". However, the total pool of predictors is not clear to me at this point. Please make it explicit and avoid abbreviations.

Thanks for your remind, we have revised the draft and correct these errors.

Although the authors say that their code is available upon request, I think it would be better to have it published in an appropriate repository.

Thanks for your remind, we showed our code at  https://github.com/conanan/lasso-ml.

YouTube channel link is broken

freedom

eLife Assessment

This study aims to analyse the effect of polymorphism on meiotic recombination in subspecies of Saccharomyces. The detection of reciprocal and non-reciprocal events is based on sequencing the haploid products of meiosis, and frequencies are compared between strains having introgressed genomic segments and strains lacking such segments. Unfortunately, the method used are inadequate for quantifying the non-reciprocal events.

Reviewer #1 (Public review):

Summary:

The authors explored how the presence of interspecific introgressions in the genome affects the recombination landscape. This research aims to shed light on the genetic phenomena influencing the evolution of introgressed regions. However, it is important to note that the study is based on examining only one generation, which limits the scope for making broad evolutionary conclusions. In this study, yeast hybrids with large introgressions (ranging from several to several dozen percent of the chromosome length) from another yeast species were crossed. The products of meiosis were then isolated and sequenced to examine the genome-wide distribution of both crossovers (COs) and noncrossovers (NCOs). The authors found a significant reduction in the frequency of COs within the introgressed regions, which is a phenomenon well-documented in various systems. They also report that introgressed regions exhibit an increased frequency of NCOs. Unfortunately, this conclusion seems flawed, as there is no accurate method for correcting the detection level of NCOs when the compared regions (introgressed and non-introgressed) differ drastically in SNP density. The authors further confirmed that introgressions significantly limit the local shuffling of genetic information, and while NCOs contribute slightly to this shuffling, they do not compensate for the loss of CO recombination. This is widely known fact.

In summary, the study makes a limited contribution to the understanding of how polymorphism impacts meiotic recombination. The conclusion regarding the increase in NCO frequency in polymorphic regions is likely incorrect.

Reviewer #3 (Public review):

When members of two related but diverged species mate, the resulting hybrids can produce offspring where parts of one species' genome replace those of the other. These "introgressions" often create regions with a much greater density of sequence differences than are normally found between members of the same species. Previous studies have shown that increased sequence differences, when heterozygous, can reduce recombination during meiosis specifically in the region of increased difference. However, most of these studies have focused on crossover recombination, and have not measured noncrossovers. The current study uses a pair of Saccharomyces uvarum crosses: one between two natural isolates that, while exhibiting some divergence, do not contain introgressions; the other is between two fermentation strains that,<br /> when combined, are heterozygous for 9 large regions of introgression that have much greater divergence than the rest of the genome. The authors wished to determine if introgressions differently affected crossovers and noncrossovers, and, if so, what impact that would have on the gene shuffling that occurs during<br /> meiosis.

While both crossovers and noncrossovers were measured, assessing the true impact of increased heterology (inherent in heterozygous introgressions) is complicated by the fact that the increased marker density in heterozygous introgressions also increases the ability to detect noncrossovers. The authors now use a revised correction aimed at compensating for this difference, and based on that correction, conclude that, while as expected crossovers are decreased by increased sequence heterology, noncrossovers neither increase nor decrease substantially. They then show that genetic shuffling overall is substantially reduced in regions of heterozygous introgression, which is not surprising given that one type of event is reduced and the other remains at similar levels. However, the correction currently used remains poorly justified, tests of its validity are not presented. Thus, the only possibly novel conclusion, that noncrossovers are less affected by heterology than crossovers, remains to be adequately tested.

In conclusion, of the three main conclusions as stated in the abstract, one (that crossovers go down) has been shown in many systems, one (that noncrossovers increase) is wrong, and the third (that allele shuffling is reduced) is obvious. Given this, the impact of this work on the field will be minimal at best, and negative to the extent that readers are led astray.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The authors investigated how the presence of interspecific introgressions in the genome affects the recombination landscape. This research was intended to inform about genetic phenomena influencing the evolution of introgressed regions, although it should be noted that the research itself is based on examining only one generation, which limits the possibility of drawing far-reaching evolutionary conclusions. In this work, yeast hybrids with large (from several to several dozen percent of the chromosome length) introgressions from another yeast species were crossed. Then, the products of meiosis were isolated and sequenced, and on this basis, the genome-wide distribution of both crossovers (COs) and noncrossovers (NCOs) was examined. Carrying out the analysis at different levels of resolution, it was found that in the regions of introduction, there is a very significant reduction in the frequency of COs and a simultaneous increase in the frequency of NCOs. Moreover, it was confirmed that introgressions significantly limit the local shuffling of genetic information, and NCOs are only able to slightly contribute to the shuffling, thus they do not compensate for the loss of CO recombination.

Strengths:

- Previously, experiments examining the impact of SNP polymorphism on meiotic recombination were conducted either on the scale of single hotspots or the entire hybrid genome, but the impact of large introgressed regions from another species was not examined. Therefore, the strength of this work is its interesting research setup, which allows for providing data from a different perspective.

- Good quality genome-wide data on the distribution of CO and NCO were obtained, which could be related to local changes in the level of polymorphism.

Weaknesses:

(1)  The research is based on examining only one generation, which limits the possibility of drawing far-reaching evolutionary conclusions. Moreover, meiosis is stimulated in hybrids in which introgressions occur in a heterozygous state, which is a very unlikely situation in nature. Therefore, I see the main value of the work in providing information on the CO/NCO decision in regions with high sequence diversification, but not in the context of evolution.

While we are indeed only examining recombination in a single generation, we respectfully disagree that our results aren't relevant to evolutionary processes. The broad goals of our study are to compare recombination landscapes between closely related strains, and we highlight dramatic differences between recombination landscapes. These results add to a body of literature that seeks to understand the existence of variation in traits like recombination rate, and how recombination rate can evolve between populations and species. We show here that the presence of introgression can contribute to changes in recombination rate measured in different individuals or populations, which has not been previously appreciated. We furthermore show that introgression can reduce shuffling between alleles on a chromosome, which is recognized as one of the most important determinants for the existence and persistence of sexual reproduction across all organisms. As we describe in our introduction and conclusion, we see our experimental exploration of the impacts of introgression on the recombination landscape as complementary to studies inferring recombination and introgression from population sequencing data and simulations. There are benefits and challenges to each approach, but both can help us better understand these processes. In regards to the utility of exploring heterozygous introgression, we point out that introgression is often found in a heterozygous state (including in modern humans with Neanderthal and/or Denisovan ancestry). Introgression will always be heterozygous immediately after hybridization, and depending on the frequency of gene flow into the population, the level of inbreeding, selection against introgression, etc., introgression will typically be found as heterozygous.

- The work requires greater care in preparing informative figures and, more importantly, re-analysis of some of the data (see comments below).

More specific comments:

(1) The authors themselves admit that the detection of NCO, due to the short size of conversion tracts, depends on the density of SNPs in a given region. Consequently, more NCOs will be detected in introgressed regions with a high density of polymorphisms compared to the rest of the genome. To investigate what impact this has on the analysis, the authors should demonstrate that the efficiency of detecting NCOs in introgressed regions is not significantly higher than the efficiency of detecting NCOs in the rest of the genome. If it turns out that this impact is significant, analyses should be presented proving that it does not entirely explain the increase in the frequency of NCOs in introgressed regions.

We conducted a deeper exploration of the effect of marker resolution on NCO detection by randomly removing different proportions of markers from introgressed regions of the fermentation cross in order to simulate different marker resolutions from non-introgressed regions. We chose proportions of markers that would simulate different quantiles of the resolution of non-introgressed regions and repeated our standard pipeline in order to compare our NCO detection at the chosen marker densities. More details of this analysis have been added to the manuscript (lines 188-199, 525-538). We confirmed the effect of marker resolution on NCO detection (as reported in the updated manuscript and new supplementary figures S2-S10, new Table S10) and decided to repeat our analyses on the original data with a more stringent correction. For this we chose our observed average tract size for NCOs in introgressed regions (550bp), which leads to a far more conservative estimate of NCO counts (As seen in the updated Figure 2 and Table 2). This better accounts for the increased resolution in introgressed regions, and while it's possible to be more stringent with our corrections, we believe that further stringency would be unreasonable. We also see promising signs that the correction is sufficient when counting our CO and NCO events in both crosses, as described in our response to comment 39 (response to reviewer #3).

(2) CO and NCO analyses performed separately for individual regions rarely show statistical significance (Figures 3 and 4). I think that the authors, after dividing the introgressed regions into non-overlapping windows of 100 bp (I suggest also trying 200 bp, 500 bp, and 1kb windows), should combine the data for all regions and perform correlations to SNP density in each window for the whole set of data. Such an analysis has a greater chance of demonstrating statistically significant relationships. This could replace the analysis presented in Figure 3 (which can be moved to Supplement). Moreover, the analysis should also take into account indels.

We're uncertain of what is being requested here. If the comment refers to the effect of marker density on NCO detection, we hope the response to comment 2 will help resolve this comment as well. Otherwise, we ask for some clarification so that we may correct or revise as appropriate.

(3) In Arabidopsis, it has been shown that crossover is stimulated in heterozygous regions that are adjacent to homozygous regions on the same chromosome (http://dx.doi.org/10.7554/eLife.03708.001, https://doi.org/10.1038/s41467-022-35722-3).

This effect applies only to class I crossovers, and is reversed for class II crossovers (https://doi.org/10.15252/embj.2020104858, https://doi.org/10.1038/s41467-023-42511-z). This research system is very similar to the system used by the authors, although it likely differs in the level of DNA sequence divergence. The authors could discuss their work in this context.

We thank the reviewer for sharing these references. We have added a discussion of our work in the context of these findings in the Discussion, lines 367-376.

Reviewer #2 (Public Review):

Summary:

Schwartzkopf et al characterized the meiotic recombination impact of highly heterozygous introgressed regions within the budding yeast Saccharomyces uvarum, a close relative of the canonical model Saccharomyces cerevisiae. To do so, they took advantage of the naturally occurring Saccharomyces bayanus introgressions specifically within fermentation isolates of S. uvarum and compared their behavior to the syntenic regions of a cross between natural isolates that do not contain such introgressions. Analysis of crossover (CO) and noncrossover (NCO) recombination events shows both a depletion in CO frequency within highly heterozygous introgressed regions and an increase in NCO frequency. These results strongly support the hypothesis that DNA sequence polymorphism inhibits CO formation, and has no or much weaker effects on NCO formation. Eventually, the authors show that the presence of introgressions negatively impacts "r", the parameter that reflects the probability that a randomly chosen pair of loci shuffles their alleles in a gamete.

The authors chose a sound experimental setup that allowed them to directly compare recombination properties of orthologous syntenic regions in an otherwise intra-specific genetic background. The way the analyses have been performed looks right, although this reviewer is unable to judge the relevance of the statistical tests used. Eventually, most of their results which are elegant and of interest to the community are present in Figure 2.

Strengths:

Analysis of crossover (CO) and noncrossover (NCO) recombination events is compelling in showing both a depletion in CO frequency within highly heterozygous introgressed regions and an increase in NCO frequency.

Weaknesses:

The main weaknesses refer to a few text issues and a lack of discussion about the mechanistic implications of the present findings.

- Introduction

(1) The introduction is rather long. | I suggest specifically referring to "meiotic" recombination (line 71) and to "meiotic" DSBs (line 73) since recombination can occur outside of meiosis (ie somatic cells).

We agree and have condensed the introduction to be more focused. We also made the suggested edits to include “meiotic” when referring to recombination and DSBs.

(2) From lines 79 to 87: the description of recombination is unnecessarily complex and confusing. I suggest the authors simply remind that DSB repair through homologous recombination is inherently associated with a gene conversion tract (primarily as a result of the repair of heteroduplex DNA by the mismatch repair (MMR) machinery) that can be associated or not to a crossover. The former recombination product is a crossover (CO), the latter product is a noncrossover (NCO) or gene conversion. Limited markers may prevent the detection of gene conversions, which erase NCO but do not affect CO detection.

We changed the language in this section to reflect the reviewer’s suggestions.

(3) In addition, "resolution" in the recombination field refers to the processing of a double Holliday junction containing intermediates by structure-specific nucleases. To avoid any confusion, I suggest avoiding using "resolution" and simply sticking with "DSB repair" all along the text.

We made the suggested correction throughout the paper.

(4) Note that there are several studies about S. cerevisiae meiotic recombination landscapes using different hybrids that show different CO counts. In the introduction, the authors refer to Mancera et al 2008, a reference paper in the field. In this paper, the hybrid used showed ca. 90 CO per meiosis, while their reference to Liu et al 2018 in Figure 2 shows less than 80 COs per meiosis for S. cerevisiae. This shows that it is not easy to come up with a definitive CO count per meiosis in a given species. This needs to be taken into account for the result section line 315-321.

This is an excellent point. We added this context in the results (lines 180-187).

(5) In line 104, the authors refer to S. paradoxus and mention that its recombination rate is significantly different from that of S. cerevisiae. This is inaccurate since this paper claims that the CO landscape is even more conserved than the DSB landscape between these two species, and they even identify a strong role played by the subtelomeric regions. So, the discussion about this paper cannot stand as it is.

We agree with the reviewer's point. We also found that the entire paragraph was unnecessary, so it and the sentence in question have been removed.

(6) Line 150, when the authors refer to the anti-recombinogenic activity of the MMR, I suggest referring to the published work from Martini et al 2011 rather than the not-yet-published work from Copper et al 2021, or both, if needed.

Added the suggested citation.

Results

(7) The clear depletion in CO and the concomitant increase in NCO within the introgressed regions strongly suggest that DNA sequence polymorphism triggers CO inhibition but does not affect NCO or to a much lower extent. Because most CO likely arises from the ZMM pathway (CO interference pathway mainly relying on Zip1, 2, 3, 4, Spo16, Msh4, 5, and Mer3) in S. uvarum as in S. cerevisiae, and because the effect of sequence polymorphism is likely mediated by the MMR machinery, this would imply that MMR specifically inhibits the ZMM pathway at some point in S. uvarum. The weak effect or potential absence of the effect of sequence polymorphism on NCO formation suggests that heteroduplex DNA tracts, at least the way they form during NCO formation, escape the anti-recombinogenic effect of MMR in S. uvarum. A few comments about this could be added.

We have added discussion and citations regarding the biased repair of DSB to NCO in introgression, lines 380-386.

(8) The same applies to the fact that the CO number is lower in the natural cross compared to the fermentation cross, while the NCO number is the same. This suggests that under similar initiating Spo11-DSB numbers in both crosses, the decrease in CO is likely compensated by a similar increase in inter-sister recombination.

Thank you to the reviewer for this observation. We agree that this could explain some differences between the crosses.

(9) Introgressions represent only 10% of the genome, while the decrease in CO is at least 20%. This is a bit surprising especially in light of CO regulation mechanisms such as CO homeostasis that tends to keep CO constant. Could the authors comment on that?

We interpret these results to reflect two underlying mechanisms. First, the presence of heterozygous introgression does reduce the number of COs. Second, we believe the difference in COs reflects variation in recombination rate between strains. We note that CO homeostasis need not apply across different genetic backgrounds. Indeed, recombination rate is appreciated to significantly differ between strains of S. cerevisiae (Raffoux et al. 2018), and recombination rate variation has been observed between strains/lines/populations in many different species including Drosophila, mice, humans, Arabidopsis, maize, etc. We reference S. cerevisiae strain variability in the Introduction lines 128-130, and have added context in the Results lines 180-187, and Discussion lines 343-350.

(10) Finally, the frequency of NCOs in introgressed regions is about twice the frequency of CO in non-introgressed regions. Both CO and NCO result from Spo11-initiating DSBs.

This suggests that more Spo11-DSBs are formed within introgressed regions and that such DSBs specifically give rise to NCO. Could this be related to the lack of homolog engagement which in turn shuts down Spo11-DSB formation as observed in ZMM mutants by the Keeney lab? Could this simply result from better detection of NCO in introgressed regions related to the increased marker density, although the authors claim that NCO counts are corrected for marker resolution?

The effect noted by the reviewer remains despite the more conservative correction for marker density applied to NCO counts (as described in the response to Reviewer 1, comment #2). Given that CO+NCO counts in introgressed regions are not statistically different between crosses, it is likely that these regions are simply predisposed to a higher rate of DSBs than the rest of the genome. This is an interesting observation, however, and one that we would like to further explore in future work.

(11) What could be the explanation for chromosome 12 to have more shuffling in the natural cross compared to the fermentation cross which is deprived of the introgressed region?

We added this text to the Results, lines 323-327, "While it is unclear what potential mechanism is mediating the difference in shuffling on chromosome 12, we note that the rDNA locus on chromosome 12 is known to differ dramatically in repeat content across strains of S. cerevisiae (22–227 copies) (Sharma et a. 2022), and we speculate that differences in rDNA copy number between strains in our crosses could impact shuffling."

Technical points:

(12) In line 248, the authors removed NCO with fewer than three associated markers.

What is the rationale for this? Is the genotyping strategy not reliable enough to consider events with only one or two markers? NCO events can be rather small and even escape detection due to low local marker density.

We trust the genotyping strategy we used, but chose to be conservative in our detection of NCOs to account for potential sequencing biases.

(13) Line 270: The way homology is calculated looks odd to this reviewer, especially the meaning of 0.5 homology. A site is either identical (1 homology) or not (0 homology).

We've changed the language to better reflect what we are calculating (diploid sequence similarity; see comment #28). Essentially, the metric is a probability that two randomly selected chromatids--one from each parent--will share the same nucleotide at a given locus (akin to calculating the probability of homozygous offspring at a single locus). We average it along a segment of the genome to establish an expected sequence similarity if/when recombination occurs in that segment.

(14) Line 365: beware that the estimates are for mitotic mismatch repair (MMR). Meiotic MMR may work differently.

We removed the citation that refers exclusively to mitotic recombination. The statement regarding meiotic recombination is otherwise still reflective of results from Chen & Jinks-Robertson

(15) Figure 1: there is no mention of potential 4:0 segregations. Did the authors find no such pattern? If not, how did they consider them?

The program we used to call COs and NCOs (ReCombine's CrossOver program) can detect such patterns, but none were detected in our data.

Reviewer #3 (Public Review):

When members of two related but diverged species mate, the resulting hybrids can produce offspring where parts of one species' genome replace those of the other. These "introgressions" often create regions with a much greater density of sequence differences than are normally found between members of the same species. Previous studies have shown that increased sequence differences, when heterozygous, can reduce recombination during meiosis specifically in the region of increased difference. However, most of these studies have focused on crossover recombination, and have not measured noncrossovers. The current study uses a pair of Saccharomyces uvarum crosses: one between two natural isolates that, while exhibiting some divergence, do not contain introgressions; the other is between two fermentation strains that, when combined, are heterozygous for 9 large regions of introgression that have much greater divergence than the rest of the genome. The authors wished to determine if introgressions differently affected crossovers and noncrossovers, and, if so, what impact that would have on the gene shuffling that occurs during meiosis.

(1) While both crossovers and noncrossovers were measured, assessing the true impact of increased heterology (inherent in heterozygous introgressions) is complicated by the fact that the increased marker density in heterozygous introgressions also increases the ability to detect noncrossovers. The authors used a relatively simple correction aimed at compensating for this difference, and based on that correction, conclude that, while as expected crossovers are decreased by increased sequence heterology, counter to expectations noncrossovers are substantially increased. They then show that, despite this, genetic shuffling overall is substantially reduced in regions of heterozygous introgression. However, it is likely that the correction used to compensate for the effect of increased sequence density is defective, and has not fully compensated for the ascertainment bias due to greater marker density. The simplest indication of this potential artifact is that, when crossover frequencies and "corrected" noncrossover frequencies are taken together, regions of introgression often appear to have greater levels of total recombination than flanking regions with much lower levels of heterology. This concern seriously undercuts virtually all of the novel conclusions of the study. Until this methodological concern is addressed, the work will not be a useful contribution to the field.

We appreciate this concern. Please see response to comments #2 and #38. We further note that our results depicted in Figure 3 and 4 are not reliant on any correction or comparison with non-introgressed regions, and thus our results regarding sequence similarity and its effect on the repair of DSBs and the amount of genetic shuffling with/without introgression to be novel and important observations for the field.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) Line 149 - this sentence refers to a mixture of papers reporting somatic or meiotic recombination and as these processes are based on different crossover pathways, this should not be mixed. For example, it is known that in Arabidopsis MSH2 has a pro-crossover function during meiotic recombination.

Corrected

(2) What is unclear to me is how the crosses are planned. Line 308 shows that there were only two crosses (one "natural" and one "fermentation"), but I understand that this is a shorthand and in fact several (four?) different strains were used for the "fermentation cross". At least that's what I concluded from Fig. 1B and its figure caption. This needs to be further explained. Were different strains used for each fermentation cross, or was one strain repeated in several crosses? In Figure 1, it would be worth showing, next to the panel showing "fermentation cross", a diagram of how "natural cross" was performed, because as I understand it, panel A illustrates the procedure common to both types of crosses, and not for "natural cross".

We thank the reviewer for drawing our attention to confusion about how our crosses were created. We performed two crosses, as depicted in Figure 1A. The fermentation cross is a single cross from two strains isolated from fermentation environments. The natural cross is a single cross from two strains isolated from a tree and insect. Table S1 and the methods section "Strain and library construction" describe the strains used in more detail. We modified Figure 1 and the figure legend to help clarify this. See also response to comment #37.

(3) The authors should provide a more detailed characterization of the genetic differences between chromosomes in their hybrids. What is the level of polymorphism along the S. uvarum chromosomes used in the experiments? Is this polymorphism evenly distributed? What are the differences in the level of polymorphism for individual introgressions? Theoretically, this data should be visible in Figure 2D, but this figure is practically illegible in the present form (see next comment).

As suggested, we remade Figure 2D to only include chromosomes with an introgression present, and moved the remaining chromosomes to the supplements (Figure S11). The patterns of markers (which are fixed differences between the strains in the focal cross) should be more clear now. As we detail in the Methods line 507-508, we utilized a total of 24,574 markers for the natural cross and 74,619 markers for the fermentation cross (the higher number in the fermentation cross being due to more fixed differences in regions of introgression).

(4) Figure 2D should be prepared more clearly, I would suggest stretching the chromosomes, otherwise, it is difficult to see what is happening in the introgression regions for CO and NCO (data for SNPs are more readable). Maybe leave only the chromosomes with introgressions and transfer the rest to the supplement?

See previous comment.

(5) How are the Y scales defined for Figure 2D?

Figure 2D now includes units for the y-axis.

(6) Are increases in CO levels in fermentation cross-observed at the border with introgressions? This would indicate local compensation for recombination loss in the introgressed regions, similar to that often observed for chromosomal inversions.

We see no evidence of an increase in CO levels at the borders of introgressions, neither through visual inspection or by comparing the average CO rate in all fermentation windows to that of windows at the edges of introgressions. This is included in the Discussion lines 360-366, "While we are limited in our interpretations by only comparing two crosses (one cross with heterozygous introgression and one without introgression), these results are in line with findings in inversions, where heterozygotes show sharp decreases in COs, but the presence of NCOs in the inverted region (Crown et al., 2018; Korunes & Noor, 2019). However, unlike heterozygous inversions where an increase in COs is observed on freely recombining chromosomes (the inter-chromosomal effect), we do not see an increase in COs on the borders flanking introgression or on chromosomes without introgression."

(7) Line 336 - "We find positive correlations between CO counts..." - you should indicate here that between fermentation and natural crosses, it was quite hard for me to understand what you calculated.

We corrected the language as suggested.

(8) The term "homology" usually means "having a common evolutionary origin" and does not specify the level of similarity between sequences, thus it cannot be measured. It is used incorrectly throughout the manuscript (also in the intro). I would use the term "similarity" to indicate the degree of similarity between two sequences.

We corrected the language as suggested throughout the document.

(9) Paragraph 360 and Figure 3 - was the "sliding window" overlapping or non-overlapping?

We added clarifying language to the text in both places. We use a 101bp sliding window with 50bp overlaps.

(10) Line 369 - what is "...the proportion of bases that are expected to match between the two parent strains..."?

We clarified the language in this location, and hopefully changes associated with the comment about sequence similarity will make the comment even clearer in context.

(11) Line 378 - should it refer to Figure S1 and not Figure 4?

Corrected.

(12) Line 399 - should refer to Figure 4, not Figure 5.

Corrected

(13) Line 444-449 - the analysis of loss of shuffling in the context of the location of introgression on the chromosome should be presented in the result section.

We shifted the core of the analysis to the results, while leaving a brief summary in the discussion.

(14) The authors should also take into account the presence of indels in their analyses, and they should be marked in the figures, if possible.

We filtered out indels in our variant calling. However, we did analyze our crosses for the presence of large insertions and deletions (Table S2), which can obscure true recombination rates, and found that they were not an issue in our dataset.

Reviewer #2 (Recommendations For The Authors):

This reviewer suggests that the authors address the different points raised in the public review.

(1) This reviewer would like to challenge the relevance of the r-parameter in light of chromosome 12 which has no introgression and still a strong depletion in r in the fermentation cross.

We added this text to the Results, lines 377-381, "While it is unclear what potential mechanism is mediating the difference in shuffling on chromosome 12, we note that the rDNA locus on chromosome 12 is known to differ dramatically in repeat content across strains of S. cerevisiae (22–227 copies) (Sharma et a. 2022), and we speculate that differences in rDNA copy number between strains in our crosses could impact shuffling."

(2) This reviewer insists on making sure that NCO detection is unaffected by the marker density, notably in the highly polymorphic regions, to unambiguously support Figure 1C.

We've changed our correction for resolution to be more aggressive (see response to comment #2), and believe we have now adequately adjusted for marker density (see response to comment #38).

Reviewer #3 (Recommendations For The Authors):

I regret using such harsh language in the public review, but in my opinion, there has been a serious error in how marker densities are corrected for, and, since the manuscript is now public, it seems important to make it clear in public that I think that the conclusions of the paper are likely to be incorrect. I regret the distress that the public airing of this may cause. Below are my major concerns:

(1) The paper is written in a way that makes it difficult to figure out just what the sequence differences are within the crosses. Part of this is, to be frank, the unusual way that the crosses were done, between more than one segregant each from two diploids in both natural and fermentation cases. I gather, from the homology calculations description, that each of these four diploids, while largely homozygous, contained a substantial number of heterozygosities, so individual diploids had different patterns of heterology. Is this correct? And if so, why was this strategy chosen? Why not start with a single diploid where all of the heterologies are known? Why choose to insert this additional complication into the mix? It seems to me that this strategy might have the perverse effect of having the heterology due to the polymorphisms present in one diploid affect (by correction) the impact of a noncrossover that occurs in a diploid that lacks the additional heterology. If polymorphic markers are a small fraction of total markers, then this isn't such a great concern, but I could not find the information anywhere in the manuscript. As a courtesy to the reader, please consider providing at the beginning some basic details about the starting strains-what is the average level of heterology between natural A and natural B, and what fraction of markers are polymorphic; what is the average level of heterology between fermentation A and fermentation B in non-introgressed regions, in introgressed regions, and what fraction of markers are polymorphic? How do these levels of heterology compare to what has been examined before in whole-genome hybrid strains? It also might be worth looking at some of the old literature describing S. cerevisiae/S. carlsbergensis hybrids.

We thank the reviewer for drawing our attention to confusion about the cross construction. These crosses were conducted as is typical for yeast genetic crosses: we crossed 2 genetically distinct haploid parents to create a heterozygous diploid, then collected the haploid products of meiosis from the same F1 diploid. Because the crosses were made with haploid parents, it is not possible for other genetic differences to be segregating in the crosses. We have revised Figure 1 and its caption to clarify this. Further details regarding the crosses are in the Methods section "Strain and library construction" and in Supplemental Table S1. We only utilized genetic markers that are fixed differences between our parental strains to call CO and NCO. As we detail in the Methods line 507-508, we utilized a total of 24,574 markers for the natural cross and 74,619 markers for the fermentation cross (the higher number in the fermentation cross being due to more fixed differences in regions of introgression). We additionally revised Figure 2D (and Figure S11) to help readers better visualize differences between the crosses.

(2) There are serious concerns about the methods used to identify noncrossovers and to normalize their levels, which are probably resulting in an artifactually high level of calculated crossovers in Figure 2. As a primary indication of this, it appears in Figure 2 that the total frequency of events (crossovers + noncrossovers) in heterozygous introgressed regions are substantially greater than those in the same region in non-introgressed strains, while just shifting of crossovers to noncrossovers would result in no net increase. The simplest explanation for this is that noncrossovers are being undercounted in non-introgressed relative to introgressed heterozygous regions. There are two possible reasons for this: i. The exclusion of all noncrossover events spanning less than three markers means that many more noncrossovers in introgressed heterozygous regions than in non-introgressed. Assuming that average non-homology is 5% in the former and 1% in the latter, the average 3-marker event will be 60 nt in introgressed regions and 300 nt in non-introgressed regions - so many more noncrossovers will be counted in introgressed regions. A way to check on this - look at the number of crossover-associated markers that undergo gene conversion; use the fraction that involves < 3 markers to adjust noncrossover levels (this is the strategy used by Mancera et al.). ii. The distance used for noncrossover level adjustment (2kb) is considerably greater than the measured average noncrossover lengths in other studies. The effect of using a too-long distance is to differentially under-correct for noncrossovers in non-introgressed regions, while virtually all noncrossovers in heterozygous introgressed regions will be detected. This can be illustrated by simulations that reduce the density of scored markers in heterozygous introgressed regions to the density seen in non-introgressed regions. Because these concerns go to the heart of the conclusions of the paper, they must be addressed quantitatively - if not, the main conclusions of the paper are invalid.

We adjusted the correction factor (See also response to comment #2) and compared the average number of CO and NCO events in introgressed and non-introgressed regions between crosses (two comparisons: introgression CO+NCO in natural cross vs introgression CO+NCO in fermentation cross; non-introgression CO+NCO in natural cross vs non-introgression CO+NCO in fermentation cross). We found no significant differences between the crosses in either of the comparisons. This indicates that the distribution of total events is replicated in both crosses once we correct for resolution.

(3) It is important to distinguish the landscape of double-strand breaks from the landscape of recombination frequencies. Double-strand breaks, as measured by uncalibrated levels of Spo11-linked oligos, is a relative number - not an absolute frequency. So it is possible that two species could have a similar break landscape in terms of topography but have absolute levels higher in one species than in the other.

We agree with this statement, however, we have removed the relevant text to streamline our introduction.

(4) Lines 123-125. Just meiosis will produce mosaic genomes in the progeny of the F1; further backcrossing will reduce mosaicism to the level of isolated regions of introgression.

Adjusted the language to be more specific.

(5) Please provide actual units for the Y axes in Figure 2D.

We have corrected the units on the axes.

(6) Tables (general). Are the significance measures corrected for multiple comparisons?

In Table 3, the cutoff was chosen to be more conservative than a Bonferroni corrected alpha=0.01 with 9 comparisons (0.0011). In text, any result referred to as significant has an associated hypothesis test with a p-value less than its corresponding Bonferroni-corrected alpha of 0.05. This has been clarified in the caption for Table 3 and in the text where relevant.

Para criar atividades de aprendizagem em ambientes digitais temos de combinar objetivos pedagógicos com as oportunidades oferecidas pela tecnologia, promovendo interação e colaboração entre os estudantes. Acho essencial definir metas claras e propor atividades relevantes que incentivem uma participação ativa,Os momentos síncronos e assíncronos, permite um maior envolvimento dos estudantes, Uma aprendizagem ativa, baseada na resolução problemas, também deve ser incentivada, com desafios práticos que permitam aplicar o conhecimento de forma crítica. Devemos contar com um feedback contínuo, tanto por parte do professor como dos colegas, que ajuda os estudantes a atingir uma aprendizagem mais rica e reflexiva. Para garantir a inclusão e acessibilidade, devemos criar atividades flexíveis, que se adaptem às necessidades de todos os estudantes. Ao usarmos ferramentas tecnológicas adequadas facilitamos a criação de um ambiente interativo e estimulante.

Task and Talk is Clearly based in the Lewis signaling game. However this setup seems to lead to entangled solutions at least for the minimal number of required signals.<br /> A needs to send one of three signals - the one he does not need. Then B needs to send one of four symbols for the state of one then the other (order is selected by symmetry breaking and learned thoroug reinfocement.) In this minimal version the signals are reused. If agent A has 8 signals he can respond fully without needed a second round.

so 64 states and an state space that both symmetrical and has three normal subgroups - so agent could learn to choose composable representations if one provides that right incentives to pick these over other equilibria.

so AFAIK this is what we expect in RL - a quick and efficent solution not the Oxford English dictionary. Or that solving a maze should be done with style.

so they just mean compsitional language

This paragraph effectively captures the tension between authenticity and corporate control in social media management. It highlights the duality where an employee may express genuine thoughts but remains bound to the corporation's directives, making their authenticity conditional and fragile.

The xyz window opens up. add screen shot of that window (without clicking any of the options first)

I am not sure how much detail we want to give here. Not sure how frequently this feature is being used. If we want to give more info, we could list the steps for each edit action. They differ a lot and have some slightly tricky steps. Especially the last step differs (sometimes it's enough to click Apply, sometimes it's necessary to click Save Edits

We are not sure in what cases already inserted images are affected.

La Importancia de una Calefacción Eficiente

Eficiencia Energética y Sostenibilidad

Las soluciones modernas de calefacción, como bombas de calor y sistemas de aislamiento avanzados, permiten un consumo energético reducido, mejorando el confort y minimizando el impacto ambiental. Adoptar prácticas conscientes, como el uso adecuado de termostatos y la optimización de la ventilación, complementa estos esfuerzos.

Mantenimiento y Reparación

El mantenimiento regular de sistemas de calefacción es esencial para garantizar su eficiencia. La limpieza de radiadores, revisión de tuberías y mantenimiento de calderas son cruciales para prevenir fallos y optimizar el rendimiento.

Tipos de Sistemas de Calefacción

Los sistemas disponibles incluyen calderas de gas, bombas de calor y calefacción eléctrica. La elección del sistema adecuado depende del consumo energético, emisiones, costos de mantenimiento y necesidades específicas del hogar.

Aislamiento y Ventilación

Un buen aislamiento y una ventilación eficiente son vitales para reducir el consumo energético y mantener la salud del hogar. Controlar la humedad también ayuda a evitar problemas estructurales y de salud.

Consejos Prácticos

Los termostatos inteligentes optimizan el uso de la calefacción, programando su funcionamiento de manera eficiente y evitando el desperdicio de energía. Invertir en estos dispositivos contribuye a reducir significativamente las facturas energéticas.

En conjunto, el artículo enfatiza la importancia de una combinación de tecnologías avanzadas y prácticas adecuadas para lograr una calefacción eficiente y sostenible.

Carlo Buontempo, der Leiter des europäischen Copernicus-Programms, setzt sich in einem Interview der Repubblica mit dem bei Klimaleugner:innen beliebten Argument auseinander, früher sei es auch oft heiß gewesen. Er verweist auf öffentlich zugängliche Daten zur globalen Erhitzung wie die von ERA5.

https://www.repubblica.it/green-and-blue/dossier/negazionisti-climatici/2024/10/07/news/carlo_buontempo_nella_storia_mai_temperature_cosi_alte-423532658/

ERA5 Climate Data Store: https://cds.climate.copernicus.eu/datasets/reanalysis-era5-single-levels?tab=overview

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Oh this was such a nice project... so sad it dissappeared...

Not sure I am following this step...

Experiments both make existingn 'natural' systems more visible while providing new imaginaries. Quite similar

Sounds similar to how we perceive experimental publishing

The managers of water companies.

They could formulate corresponding strategic plans for the enterprise in a more macro-level.

Customers.

They will pay more attention to the conservation and use of water resources and provide necessary and timely feedback and suggestions to relevant departments to ensure the maximum water quality safety.

5

I also wonder about this on the departmental level. Some faculty will be more comfortable or experienced with these approaches than others, so, I think it's important for chairs and directors to be having these conversations with faculty as well.