Technology is biased against those who look different than the people creating it.

But why didn't they listen to Gebru when she warned about it?

Fired for speaking out against the bias is terrible for Google's image.

A.I. is biased against women, going off the work of men on the internet.

A.I. will not fairly represent every group, because they haven't allowed every group to be apart of the discussion.

She doesn't have to deal with the anymore and is happy as she was being used as an tool

Causal loop models and other models with recording of conversations with transcripts and summaries. AI can help with the summaries.

Multiple scales is the approach that most concerns and interests me. Robin Asby has done interesting conceptual work here--extending Stafford Beer's thinking and models.

Even though plays are around 2 hours, everything within a play is chosen intentionally, leaving only the parts that move the story forward. Every information is purposefully written there to give the maximum information about a character or the plot. Similarly with poems, where each word is chosen with lots of thought, so that each sentence can have the maximum amount of information.

good principle

this is also so much less emotionally overwhelming

Meaning that the way in which we can say they are identical is of the same sense, and therefore is not as problematic as first proposed.

eLife Assessment

This manuscript describes an AI-automated microscopy-based approach to characterize both bacterial and host cell responses associated with Shigella infection of epithelial cells. The methodology is compelling and should be helpful for investigators studying a variety of intracellular pathogens. The authors have acquired important findings regarding host and bacterial responses in the context of infection, which should be followed up with further mechanistic-based studies.

Reviewer #2 (Public review):

Summary:

Septin caging has emerged as one of the innate immune response of eukaryotic cells to infections by intracellular bacteria. This fascinating assembly of eukaryotic proteins into complex structures restricts bacteria motility within the cytoplasm of host cells, thereby facilitating recognition by cytosolic sensors and components of the autophagy machinery. Given the different types of septin caging that have been described thus far, a single cell, unbiased approach to quantify and characterise septin recruitment at bacteria is important to fully grasp the role and function of caging. Thus, the authors have developed an automated image analysis pipeline allowing bacterial segmentation and classification of septin cages that will be very useful in the future, applied to study the role of host and bacterial factors, compare different bacterial strains or even compare infections by clinical isolates.

Strengths:

The authors developed a solid pipeline that has been thoroughly validated. When tested on infected cells, automated analysis corroborated previous observations and allowed the unbiased quantification of the different types of septin cages as well as the correlation between caging and bacterial metabolic activity. This approach will prove an essential asset in the further characterisation of septin cages for future studies.

Weaknesses:

As the main aim of the manuscript is to described the newly developed analysis pipeline, the results illustrated in the manuscript are essentially descriptive. The developed pipeline seems exceptionally efficient in recognising septin cages in infected cells but its application for a broader purpose or field of study remains limited.

Reviewer #4 (Public review):

Summary

In this study, López-Jiménez and colleagues demonstrate the utility of using high-content microscopy in dissecting host and bacterial determinants that play a role in the establishment of infection using Shigella flexneri as a model. The manuscript nicely identifies that infection with Shigella results in a block to DNA replication and protein synthesis. At the same time, the host responds, in part, via the entrapment of Shigella in septin cages.

Strengths:

The main strength of this manuscript is its technical aspects. They nicely demonstrate how an automated microscopy pipeline coupled with artificial intelligence can be used to gain new insights regarding elements of bacterial pathogenesis, using Shigella flexneri as a model system. Using this pipeline enabled the investigators to enhance the field's general understanding regarding the role of septin cages in responding to invading Shigella. This platform should be of interest to those who study a variety of intracellular microbial pathogens.

Another strength of the manuscript is the demonstration - using cell biology-based approaches- that infection with Shigella blocks DNA replication and protein synthesis. These observations nicely dovetail with the prior findings of other groups. Nevertheless, their clever click-chemistry-based approaches provide visual evidence of these phenomena and should interest many.

Weaknesses:

There are two main weaknesses of this work. First, the studies are limited to findings obtained using a single immortalized cell line. It is appreciated that HeLa cells serve as an excellent model for studying aspects of Shigella pathogenesis and host responses. However, it would be nice to see that similar observations are observed with an epithelial cell line of intestinal, preferably colonic origin, and eventually, with a non-immortalized cell line, although it is appreciated that the latter studies are beyond the scope of this work.

The other weakness is that the studies are minimally mechanistic. For example, the investigators have data to suggest that infection with Shigella leads to an arrest in DNA replication and protein synthesis; however, no follow-up studies have been conducted to determine how these host cell processes are disabled. Interestingly, Zhang and colleagues recently identified that the Shigella OspC effectors target eukaryotic translation initiation factor 3 to block host cell translation (PMID: 38368608).

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public Review):

Summary:

In this study, López-Jiménez and colleagues demonstrated the utility of using high-content microscopy in dissecting host and bacterial determinants that play a role in the establishment of infection using Shigella flexneri as a model. The manuscript nicely identifies that infection with Shigella results in a block to DNA replication and protein synthesis. At the same time, the host responds, in part, via the entrapment of Shigella in septin cages.

Strengths:

The main strength of this manuscript is its technical aspects. They nicely demonstrate how an automated microscopy pipeline coupled with artificial intelligence can be used to gain new insights regarding elements of bacterial pathogenesis, using Shigella flexneri as a model system. Using this pipeline enabled the investigators to enhance the field's general understanding regarding the role of septin cages in responding to invading Shigella. This platform should be of interest to those who study a variety of intracellular microbial pathogens.

Another strength of the manuscript is the demonstration - using cell biology-based approaches- that infection with Shigella blocks DNA replication and protein synthesis. These observations nicely dovetail with the prior findings of other groups. Nevertheless, their clever click-chemistry-based approaches provide visual evidence of these phenomena and should interest many.

We thank the Reviewer for their enthusiasm on technical aspects of this paper, regarding both the automated microscopy pipeline coupled with artificial intelligence and the click-chemistry based approaches to dissect DNA replication and protein synthesis by microscopy.

Weaknesses:

There are two main weaknesses of this work. First, the studies are limited to findings obtained using a single immortalized cell line. It is appreciated that HeLa cells serve as an excellent model for studying aspects of Shigella pathogenesis and host responses. However, it would be nice to see that similar observations are observed with an epithelial cell line of intestinal, preferably colonic origin, and eventually, with a non-immortalized cell line, although it is appreciated that the latter studies are beyond the scope of this work.

The immortalized cell line HeLa is widely regarded as a paradigm to study infection by Shigella and other intracellular pathogens. However, we agree that future studies beyond the scope of this work should include other cell lines (eg. epithelial cells of colonic origin, macrophages, primary cells). 

The other weakness is that the studies are minimally mechanistic. For example, the investigators have data to suggest that infection with Shigella leads to an arrest in DNA replication and protein synthesis; however, no follow-up studies have been conducted to determine how these host cell processes are disabled. Interestingly, Zhang and colleagues recently identified that the Shigella OspC effectors target eukaryotic translation initiation factor 3 to block host cell translation (PMID: 38368608). This paper should be discussed and cited in the discussion.

We appreciate the Reviewer’s concern about the lack of follow up work on observations of host DNA and protein synthesis arrest upon Shigella infection, which will be the focus of future studies. We acknowledge the recent work of Zhang et al. (Cell Reports, 2024) considering their similar results on protein translation arrest, and this reference has been more fully discussed in the revised version of the manuscript.

Reviewer #2 (Public Review):

Summary:

Septin caging has emerged as one of the innate immune responses of eukaryotic cells to infections by intracellular bacteria. This fascinating assembly of eukaryotic proteins into complex structures restricts bacteria motility within the cytoplasm of host cells, thereby facilitating recognition by cytosolic sensors and components of the autophagy machinery. Given the different types of septin caging that have been described thus far, a single-cell, unbiased approach to quantify and characterise septin recruitment at bacteria is important to fully grasp the role and function of caging. Thus, the authors have developed an automated image analysis pipeline allowing bacterial segmentation and classification of septin cages that will be very useful in the future, applied to study the role of host and bacterial factors, compare different bacterial strains, or even compare infections by clinical isolates.

Strengths:

The authors developed a solid pipeline that has been thoroughly validated. When tested on infected cells, automated analysis corroborated previous observations and allowed the unbiased quantification of the different types of septin cages as well as the correlation between caging and bacterial metabolic activity. This approach will prove an essential asset in the further characterisation of septin cages for future studies.

We thank the Reviewer for their positive comments, and for highlighting the strength of our imaging and analysis pipeline to analyse Shigella-septin interactions.

Weaknesses:

As the main aim of the manuscript is to describe the newly developed analysis pipeline, the results illustrated in the manuscript are essentially descriptive. The developed pipeline seems exceptionally efficient in recognising septin cages in infected cells but its application for a broader purpose or field of study remains limited.

The main objective of this manuscript is the development of imaging and analysis tools to study Shigella infection, and in particular, Shigella interactions with the septin cytoskeleton. In future work we will provide more mechanistic insight with novel experiments and broader applicability, using different cell lines (in agreement with Reviewer 1), mutants or clinical isolates of Shigella and different bacteria species (eg. Listeria, Salmonella, mycobacteria).

Reviewer #3 (Public Review):

Summary:

The manuscript uses high-content imaging and advanced image-analysis tools to monitor the infection of epithelial cells by Shigella. They perform some analysis on the state of the cells (through measurements of DNA and protein synthesis), and then they focus on differential recruitment of Sept7 to the bacteria. They link this recruitment with the activity of the bacterial T3SS, which is a very interesting discovery. Overall, I found numerous exciting elements in this manuscript, and I have a couple of reservations. Please see below for more details on my reservations. Nevertheless, I think that these issues can be addressed by the authors, and doing so will help to make it a convincing and interesting piece for the community working on intracellular pathogens. The authors should also carefully re-edit their manuscript to avoid overselling their data (see below for issues I see there). I would consider taking out the first figure and starting with Figure 3 (Figure 2 could be re-organized in the later parts)- that could help to make the flow of the manuscript better.

Strengths:

The high-content analysis including the innovative analytical workflows are very promising and could be used by a large number of scientists working on intracellular bacteria. The finding that Septins (through SEPT7) are differentially regulated through actively secreting bacteria is very exciting and can steer novel research directions.

We thank the Reviewer for their constructive feedback and excitement for our results, including our findings on T3SS activity and Shigella-septin interactions. In accordance with the Reviewer’s comments, we avoid overselling our data in the revised version of the manuscript.

Weaknesses:

The manuscript makes a connection between two research lines (1: Shigella infection and DNA/protein synthesis, 2: regulation of septins around invading Shigella) that are not fully developed - this makes it sometimes difficult to understand the take-home messages of the authors.

We agree that the manuscript is mostly technical and therefore some of our experimental observations would benefit from follow up mechanistic studies in the future. We highlight our vision for broader applicability in response to weaknesses raised by Reviewer 2.

It is not clear whether the analysis that was done on projected images actually reflects the phenotypes of the original 3D data. This issue needs to be carefully addressed.

We agree with the Reviewer that characterizing 3D data using 2D projected images has limitations.

We observe an increase in cell and nuclear surface that does not strictly imply a change in volume. This is why we measure Hoechst intensity in the nucleus using SUM-projection (as it can be used as a proxy of DNA content of the cell). However, we agree that future use of other markers (such as fluorescently labelled histones) would make our conclusions more robust.

Regarding the different orientation of intracellular bacteria, we agree that investigation of septin recruitment is more challenging when bacteria are placed perpendicular to the acquisition plane. In a first step, we trained a Convolutional Neural Network (CNN) using 2D data, as it is easier/faster to train and requires fewer annotated images. In doing so, we already managed to correctly identify 80% of Shigella interacting with septins, which enabled us to observe higher T3SS activity in this population. In future studies, we will maximize the 3D potential of our data and retrain a CNN that will allow more precise identification of Shigella-septin interactions and in depth characterization of volumetric parameters.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) To conclude that cell volume is indeed increased, the investigators should consider staining the cells with markers that demarcate cell boundaries and/or are confined to the cytosol, i.e., a cell tracker dye.

Staining using our SEPT7 antibody enables us to define cell boundaries for cellular area measurements (Novel Figure 1 - figure supplement 1A). However, we agree with the Reviewer that staining cells with additional markers (such as a cell tracker dye) would be required to conclude that cell volume is increased. We therefore adjust our claims in the main text (lines 107-115 and 235-246).

(2) Line 27: I understand what is meant by "recruited to actively pathogenic bacteria with increased T3SS activation." However, one could argue that there are many different roles of the intracytosolic bacteria in pathogenesis in terms of pathogenesis, not just actively secreting effectors.

T3SS secretion by cytosolic bacteria is tightly regulated and both T3SS states (active, inactive) likely contribute to the pathogenic lifestyle of S. flexneri. In agreement with this, we removed this statement from the manuscript (lines 27, 225 and 274).

(3) Line 88: Please clarify in the text that HeLa cells are being studied.

We explicitly mention that the epithelial cell line we study is HeLa in the main text (line 93), in addition to the Materials and methods (line 328).

(4) Line 97: is it possible to quantify the average distance of the nuclei from the cell perimeter? This would help provide some context as to what it means to be a certain distance from the nucleus, i.e., is there another way to point out that distance from nuclei correlates with movement inward post-invasion at the periphery?

To provide more context to the inward movement of bacteria to the cell centre, we provide calculations based on measurements in Figure 1G, I. If we approximate geometric shape of both cells and nucleus to a circle, the median radius of a HeLa cell is 31.1 µm² (uninfected cell) and 36.3 µm² (infected cell). Similarly, the median radius of the nucleus is 22.2 µm² (uninfected cell) and 24.57 µm² (infected cell).

However, we note that Figure 1F shows distance of bacteria to the centroid of the cell, which is the geometric centre of the cell, and which does not necessarily coincide with the geometric centre of the nucleus. We also note that nuclear area increases with infection (in a bacterial dose dependent manner). Finally, we note that these measurements are performed on max projections of 3D Z-stacks. In this case we cannot fully appreciate distance to the nucleus for bacteria located above it.

(5) Lines 212-213 - there is no Figure 9A, B - I think this should be Figure 7A, B.

Text has been updated (lines 216-217).

Reviewer #2 (Recommendations For The Authors):

Testing the analysis pipeline as a proof-of-concept question such as the comparison of caging around the laboratory strain as compared to one or a few clinical isolates or mutants of interest would help stress the relevance of this new, remarkable tool.

We thank the Reviewer for their enthusiasm.

Future research in the Mostowy lab will capitalise on the high-content tools generated here to explore the frequency and heterogeneity of septin cage entrapment for a wide variety of S. flexneri mutants and Shigella clinical isolates.

The sentence in line 215 ends with "in agreement with" followed by a reference.

Text has been updated (line 219).

The sentence in line 217 on the correlation between caging and T3SS is not very clear.

Text has been clarified (lines 221-223).

There is a typo in line 219 : "protrusSions"

Text has been updated (line 223).

Reviewer #3 (Recommendations For The Authors):

Major points

The quantitative analysis approach in Figure 1 has multiple issues. Some examples:<br /> (1) How was the cell area estimated? Normally, a marker for the whole cell (CellMask or similar) or cells expressing GFP would be good indicators. Here it is not clear to me what was done.

The cell area was estimated using SEPT7 antibody staining which is enriched under the cell cortex. CellProfiler was used to segment cells based on SEPT7 staining, using a propagation method from the identified nucleus based on Otsu thresholding. To provide more clarity on how this was performed, we now include a new figure (Figure 1- figure supplement 1A) showing a representative image of HeLa cells stained with SEPT7 and the corresponding cell segmentation performed with CellProfiler software, together with an updated figure legend explaining the procedure (lines 784–787).

(2) The authors use Hoechst and integrated z-projections (Figure 1 S1) as a proxy to estimate nuclear volume. Hoechst staining depends on the organization of the DNA within the nucleus and I find that the authors need to do better controls to estimate nuclear size - this would be possible with cells expressing fluorescently labeled histones, or even better with a fluorescently tagged nuclear pore/envelope marker. The current quantification approach is misleading.

We understand Reviewer #3’s concerns about using Hoechst staining as a proxy of nuclear volume, due to potential differences in DNA organisation within the nucleus.

Following the recommendation of Reviewer #3 in the following point 3, text has been updated (lines 107–115 and 235-246).

(3) Was cell density assessed for the measurements? If cells are confluent, bacteria could spread between cells within 3 hrs, if cells are less dense, this does not occur. When epithelial cells are infected for some hours, they have the tendency to round up a bit (and to appear thicker in z), but a bit smaller in xy. My suggestion to the authors (as they use these findings to follow up with experiments on the underlying processes) would be to tone down their statements - eg, Hoechst staining could be simply indicated as altered, but not put in a context of size (this would require substantial control experiments).

Local cell density was not directly measured, but the experiment was set up to infect at roughly 80% confluency (cells were seeded at 10⁴ cells/well 2 days prior to infection in a 96-well microplate, as described in the Materials and methods section) and to ensure bacterial spread between cells.

In agreement with Reviewer #3 we tone down statements in the main text (see response to point 2 above).

In addition, I found Figure 1 (and parts of Figure 2) disconnected from the rest of the manuscript, and it may even be an idea to take it out of the manuscript (that could also help to deal with my feedback relating to Figure 1). I would suggest starting the manuscript with the current Figure 3 and building the biological story with a stronger focus on SEPT7 (and its links with T3 secretion and actively pathogenic bacteria) from there on. As it stands, the two parts of the manuscript are not well connected.

We carefully considered this comment but following revisions we have not reorganised the manuscript. We believe that high-content characterisation of S. flexneri infection in Figure 1 and 2 provides insightful information about changes in host cells in response to infection. Following this, we move onto characterising intracellular bacteria (and in particular those entrapped in septin cages) in the second part of the manuscript (Figure 3-7). Similar methods were used to analyse both host and bacterial cells and results obtained offer complementary views on host-pathogen interactions.

My major reservation with the experimental work of the current version of the manuscript relates to Figure 5: The analysis of the septin phenotypes in Figure 5 seems to be problematic - to me, it appears that analysis and training were done on projected image stacks. As bacteria are rod-shaped their orientation in space has an enormous impact on how the septin signal appears in a projection - this can lead to wrong interpretation of the phenotypes. The authors need to do some quantitative controls analyzing their data in 3D. To be more clear: the example "tight" (second row) shows a bacterium that appears short. It may be that it's actually longer if one looks in 3D, and the septin signal could possibly fall in the category "rings" or even "two poles".

The deep learning training and subsequent analysis of septin-cage entrapment is done on projected Z-stacks, which presents limitations. Future work in the Mostowy lab will exploit this first study and dive deeper into 3D aspects of the data.

To address Reviewer #3’s concern, we include a sentence explaining that this analysis was performed using 2D max projections (lines 708 and 724), as well as acknowledging its limitations in the main text (lines 259-262).

Minor points

The scale bar in Fig 1 is very thin.

We corrected the scale bar in Fig. 1 to make it more visible.

Could it be that Figure 1F is swapped with Figure1E in the description?

Descriptions for Figure 1E and F are correct.

Line 27: what does "actively pathogenic bacteria" mean? I propose to change the term.

We agree with Reviewer #3 that “actively pathogenic bacteria” should be removed from the text. This update is also in agreement with Reviewer #1 (see Reviewer #1 point 2).

Line 28: "dynamics" can be confusing as it relates to dynamic events imaged by time-lapse.

Although we are making a snapshot of the infection process at 3 hpi, we capture asynchronous processes in both host and bacterial cells (eg. host cells infected with different bacterial loads, bacterial cells undergoing actin polymerisation or septin cage entrapment). We agree that we are not following dynamics of full events over time. However, our high content approach enables us to capture different stages of dynamic processes. To avoid confusion, we replace “dynamics” by “diverse interactions” (line 28), and we discuss the importance of follow-up studies studying microscopy timelapses (line 274).

Paragraph 59 following: the concept of heterogeneity was investigated in some detail for viral infection by the Pelkmans group (PMID: 19710653) using advanced image analysis tools. Advanced machine-learning-based analysis was then performed on Salmonella invasion by Voznica and colleagues (PMID: 29084895). It would be great to include these somewhat "old" works here as they really paved the way for high-content imaging, and the way analyses were performed then should be also discussed in light of how analyses can be performed now with the approaches developed by the authors.

We agree. These landmark studies have now been included in the main text (lines 71-74).

Line 181: I do not know what "morphological conformations" means, perhaps the authors can change the wording or clarify.

We substituted the phrase “morphological conformations” by “morphological patterns” to improve clarity in the main text (lines 185).

The authors claim (eg in the abstract) that they are measuring the dynamic infection process. To me, it appears that they look at one time-point, so no dynamic information can be extracted. I suggest that the authors tone down their claims.

Please note our response above (Minor points, Line 28) which also refers to this question.

Try to check in with Kris for further feedback and also on my progress in the course

avoid the use of AI in large writing quantities, instead use applications to correct grammar to help with developing writing skills

Be sure to be prepared for peer review days to receive points for the day

Try to finish assignments the earliest time possible and get started on upcoming assignments to stay on track to avoid stress!

My exact thoughts as a film student, critical thinking in literature felt like just a GE requirement I felt I just needed to pass but this course I feel like it's something I can take with me in the future and not just in my career.

reminds me of a quote I resonate with, by Aldo Leopold, is "One of the penalties of an ecological education is that one lives alone in a world of wounds". I think fearlessly embracing the burning world is a powerful statement

reminds me of a recent conversation I had with friends that conceptualizes mans dominion over nature that is mentioned in the bible. I think our western religious traditions are starkly different than the place based relations the author is referring to

Everyone is unique and because of this uniqueness each person's brain reacts differently when they write.

it's good to reread it two times so that you don't miss anything important like a date or name and or a key word that is in the question that could cause you to have to change your answer.

eLife Assessment

This study presents a computational-experimental workflow for optimizing RNA aptamers targeting SARS-CoV-2 RBD. While the integrated approach combining docking, molecular dynamics, and experimental validation shows some promise, the useful findings are limited by the extremely weak binding affinities (>100 µM KD) and restriction to a single target system. The evidence is incomplete, with experimental design issues in the antibody competition assays and a lack of specificity testing undermining confidence in the conclusions.

Reviewer #1 (Public review):

Summary:

In this study, the authors attempt to devise general rules for aptamer design based on structure and sequence features. The main system they are testing is an aptamer targeting a viral sequence.

Strengths:

The method combines a series of well-established protocols, including docking, MD, and a lot of system-specific knowledge, to design several new versions of the Ta aptamer with improved binding affinity.

Weaknesses:

The approach requires a lot of existing knowledge and, importantly, an already known aptamer, which presumably was found with Selex. In addition, although the aptamer may have a stronger binding affinity, it is not clear if any of it has any additional useful properties such as stability, etc.

Reviewer #2 (Public review):

Summary:

This manuscript proposes a workflow for discovering and optimizing RNA aptamers, with application in the optimization of a SARS-CoV-2 RBD. The authors took a previously identified RNA aptamer, computationally docked it into one specific RBD structure, and searched for variants with higher predicted affinity. The variants were subsequently tested for RBD binding using gel retardation assays and competition with antibodies, and one was found to be a stronger binder by about three-fold than the founding aptamer.

Overall, this would be an interesting study if it were performed with truly high-affinity aptamers, and specificity was shown for RBD or several RBD variants.

Strengths:

The computational workflow appears to mostly correctly find stronger binders, though not de novo binders.

Weaknesses:

(1) Antibody competition assays are reported with RBD at 40 µM, aptamer at 5 µM, and a titration of antibody between 0 and 1.2 µg. This approach does not make sense. The antibody concentration should be reported in µM. An estimation of the concentration is 0-8 pmol (from 0-1.2 µg), but that's not a concentration, so it is unknown whether enough antibody molecules were present to saturate all RBD molecules, let alone whether they could have displaced all aptamers.

(2) These are not by any means high-affinity aptamers. The starting sequence has an estimated (not measured, since the titration is incomplete) KD of 110 µM. That's really the same as non-specific binding for an interaction between an RNA and a protein. This makes the title of the manuscript misleading. No high-affinity aptamer is presented in this study. If the docking truly presented a bound conformation of an aptamer to a protein, a sub-micromolar Kd would be expected, based on the number of interactions that they make.

(3) The binding energies estimated from calculations and those obtained from the gel-shift experiments are vastly different, as calculated from the Kd measurements, making them useless for comparison, except for estimating relative affinities.

Author response:

Reviewer #1 (Public review):

Summary:

In this study, the authors attempt to devise general rules for aptamer design based on structure and sequence features. The main system they are testing is an aptamer targeting a viral sequence.

Strengths:

The method combines a series of well-established protocols, including docking, MD, and a lot of system-specific knowledge, to design several new versions of the Ta aptamer with improved binding affinity.

We thank the reviewer for this accurate summary and for recognizing the strength of our integrated computational–experimental workflow in improving aptamer affinity. We will emphasize this contribution more clearly in the revised Introduction.

Weaknesses:

The approach requires a lot of existing knowledge and, impo rtantly, an already known aptamer, which presumably was found with SELEX. In addition, although the aptamer may have a stronger binding affinity, it is not clear if any of it has any additional useful properties such as stability, etc.

Thanks for these critical comments.

(1) On the reliance on a known aptamer: We agree that our CAAMO framework is designed as a post-SELEX optimization platform rather than a tool for de novo discovery. Its primary utility lies in rationally enhancing the affinity of existing aptamers that may not yet be sequence-optimal, thereby complementing experimental technologies such as SELEX. In the revised manuscript, we plan to clarify this point more explicitly in both the Introduction and Discussion sections, emphasizing that the propose CAAMO framework is intended to serve as a complementary strategy that accelerates the iterative optimization of lead aptamers.

(2) On stability and developability: We also appreciate the reviewer’s important reminder that affinity alone is not sufficient for therapeutic development. We acknowledge that the present study has focused mainly on affinity optimization, and properties such as nuclease resistance, structural stability, and overall developability were not evaluated. In the revised manuscript, we will add a dedicated section highlighting the critical importance of these characteristics and outlining them as key priorities for our future research efforts.

Reviewer #2 (Public review):

Summary:

This manuscript proposes a workflow for discovering and optimizing RNA aptamers, with application in the optimization of a SARS-CoV-2 RBD. The authors took a previously identified RNA aptamer, computationally docked it into one specific RBD structure, and searched for variants with higher predicted affinity. The variants were subsequently tested for RBD binding using gel retardation assays and competition with antibodies, and one was found to be a stronger binder by about three-fold than the founding aptamer. Overall, this would be an interesting study if it were performed with truly high-affinity aptamers, and specificity was shown for RBD or several RBD variants.

Strengths:

The computational workflow appears to mostly correctly find stronger binders, though not de novo binders.

We thank the reviewer for the clear summary and for acknowledging that our workflow effectively prioritizes stronger binders.

Weaknesses:

(1) Antibody competition assays are reported with RBD at 40 µM, aptamer at 5 µM, and a titration of antibody between 0 and 1.2 µg. This approach does not make sense. The antibody concentration should be reported in µM. An estimation of the concentration is 0-8 pmol (from 0-1.2 µg), but that's not a concentration, so it is unknown whether enough antibody molecules were present to saturate all RBD molecules, let alone whether they could have displaced all aptamers.

Thanks for your insightful comment. We have calculated that 0–1.2 µg antibody corresponds to a final concentration range of 0–1.6 µM (see Author response image 1). In practice, 1.2 µg was the maximum amount of commercial antibody that could be added under the conditions of our assay. In the revised manuscript, we plan to report all antibody quantities in molar concentrations in the Materials and Methods section for clarity and rigor.

Author response image 1.<br /> Estimation of antibody concentration. Assuming a molecular weight of 150 kDa, dissolving 1.2 µg of antibody in a 5 µL reaction volume results in a final concentration of 1.6 µM.<br />

As shown in Figure 5D of the main text, the purpose of the antibody–aptamer competition assay was not to achieve full saturation but rather to compare the relative competitive binding of the optimized aptamer (Ta^G34C) versus the parental aptamer (Ta). Molecular interactions at this scale represent a dynamic equilibrium of binding and dissociation. While the antibody concentration may not have been sufficient to saturate all available RBD molecules, the experimental results clearly reveal the competitive binding behavior that distinguishes the two aptamers. Specifically, two consistent trends emerged:

(1) Across all antibody concentrations, the free RNA band for Ta was stronger than that of Ta^G34C, while the RBD–RNA complex band of the latter was significantly stronger, indicating that Ta^G34Cbound more strongly to RBD.

(2) For Ta, increasing antibody concentration progressively reduced the RBD–RNA complex band, consistent with antibody displacing the aptamer. In contrast, for Ta^G34C, the RBD–RNA complex band remained largely unchanged across all tested antibody concentrations, suggesting that the antibody was insufficient to displace Ta^G34C from the complex.

Together, these observations support the conclusion that Ta^G34C exhibits markedly stronger binding to RBD than the parental Ta aptamer, in line with the predictions and objectives of our CAAMO optimization framework.

(2) These are not by any means high-affinity aptamers. The starting sequence has an estimated (not measured, since the titration is incomplete) KD of 110 µM. That's really the same as non-specific binding for an interaction between an RNA and a protein. This makes the title of the manuscript misleading. No high-affinity aptamer is presented in this study. If the docking truly presented a bound conformation of an aptamer to a protein, a sub-micromolar Kd would be expected, based on the number of interactions that they make.

In fact, our starting sequence (Ta) is a high-affinity aptamer, and then the optimized sequences (such as Ta^G34C) with enhanced affinity are undoubtedly also high-affinity aptamers. See descriptions below:

(1) Origin and prior characterization of Ta. The starting aptamer Ta (referred to as RBD-PB6-Ta in the original publication by Valero et al., PNAS 2021, doi:10.1073/pnas.2112942118) was selected through multiple positive rounds of SELEX against SARS-CoV-2 RBD, together with counter-selection steps to eliminate non-specific binders. In that study, Ta was reported to bind RBD with an IC₅₀ of ~200 nM as measured by biolayer interferometry (BLI), supporting its high affinity and specificity.

(2) Methodological differences between EMSA and BLI measurements. We acknowledge that the discrepancy between our obtained binding affinity (K_d = 110 µM) and the previously reported one (IC₅₀ ~ 200 nM) for the same Ta sequence arises primarily from methodological and experimental differences between EMSA and BLI. Namely, different experimental measurement methods can yield varied binding affinity values. While EMSA may have relatively low measurement precision, its relatively simple procedures were the primary reason for its selection in this study. Particularly, our framework (CAAMO) is designed not as a tool for absolute affinity determination, but as a post-SELEX optimization platform that prioritizes relative changes in binding affinity under a consistent experimental setup. Thus, the central aim of our work is to demonstrate that CAAMO can reliably identify variants, such as Ta^G34C, that bind more strongly than the parental sequence under identical assay conditions.

(3) Evidence of specific binding in our assays. We emphasize that the binding observed in our EMSA experiments reflects genuine aptamer–protein interactions. As shown in Figure 2G of the main text, a control RNA (Tc) exhibited no detectable binding to RBD, whereas Ta produced a clear binding curve, confirming that the interaction is specific rather than non-specific.

(3) The binding energies estimated from calculations and those obtained from the gel-shift experiments are vastly different, as calculated from the Kd measurements, making them useless for comparison, except for estimating relative affinities.

We thank the reviewer for raising this important point. CAAMO was developed as a post-SELEX optimization tool with the explicit goal of predicting relative affinity changes (ΔΔG) rather than absolute binding free energies (ΔG). Empirically, CAAMO correctly predicted the direction of affinity change for 5 out of 6 designed variants (e.g., ΔΔG < 0 indicates enhanced binding free energy relative to WT); such predictive power for relative ranking is highly valuable for prioritizing candidates for experimental testing. Our prior work on RNA–protein interactions likewise supports the reliability of relative affinity predictions (see: Nat Commun 2023, doi:10.1038/s41467-023-39410-8). In the revised manuscript we will explicitly state that the primary utility of CAAMO is to accurately predict affinity trends and to rank variants for follow-up, and we will moderate any statements that could be interpreted as claims about precise absolute ΔΔG values.

Confused on how this is known. Suspicion or established fact, where is the information coming from?

eLife Assessment

This study is a valuable contribution to the evidence base. However, the evidence provided is incomplete as the study results only partially support the study conclusions. Addressing the methodological and reporting issues raised by the peer reviewers and properly aligning the claim made for providing a tool for early warning with the study analysis/results would improve the study quality and usefulness of its findings.

Reviewer #1 (Public review):

This is my first review of this manuscript. The authors included previous reviews for a different journal with a length of 90 and 39 pages; I did not review this reply in my assessment of the paper itself. Influenza prediction is not my area of expertise.

A major concern is that the model is trained in the midst of the COVID-19 pandemic and its associated restrictions and validated on 2023 data. The situation before, during, and after COVID is fluid, and one may not be representative of the other. The situation in 2023 may also not have been normal and reflective of 2024 onward, both in terms of the amount of testing (and positives) and measures taken to prevent the spread of these types of infections. A further worry is that the retrospective prospective split occurred in October 2020, right in the first year of COVID, so it will be impossible to compare both cohorts to assess whether grouping them is sensible.

The outcome of interest is the number of confirmed influenza cases. This is not only a function of weather, but also of the amount of testing. The amount of testing is also a function of historical patterns. This poses the real risk that the model confirms historical opinions through increased testing in those higher-risk periods. Of course, the models could also be run to see how meteorological factors affect testing and the percentage of positive tests. The results only deal with the number of positive (only the overall number of tests is noted briefly), which means there is no way to assess how reasonable and/or variable these other measures are. This is especially concerning as there was massive testing for respiratory viruses during COVID in many places, possibly including China.

(1) Although the authors note a correlation between influenza and the weather factors. The authors do not discuss some of the high correlations between weather factors (e.g., solar radiation and UV index). Because of the many weather factors, those plots are hard to parse.

(2) The authors do not actually compare the results of both methods and what the LSTM adds.

Minor comments:

(3) The methods are long and meandering. They could be cleaned up and shortened. E.g., there is no need for 30 lines on PCR testing; the study area should come before the study design. The authors discuss similar elements in multiple places; this whole section can be shortened considerably without affecting the content.

(4) How reliable is the "Our Word in Data" website for subnational coverage of restrictions? Some of the authors are from Putian and should be able to confirm the accuracy for both studied areas.

(5) Figure 2A is hard to parse; it would make more sense to plot these as line plots (y=count, x=month).

Reviewer #2 (Public review):

Summary:

The study aimed to assess the associations between meteorological drivers and influenza is important although not new. The authors used only 6 years of surveillance data and deep learning models, combining distributed lag non-linear models (DLNM) with Bayesian-optimized LSTM neural networks for predictive modeling. The key interest in this area is to explore the subtropical locations, where influenza is less common and circulates year-round. The authors further claimed that such an association could be able to provide an early warning in the community. In this direction, the current manuscript has several scopes of improvements and clarification of the claims, as I list here.

Strengths:

Study design based on a prospective cohort to analyse the data for retrospective outcomes.

Weaknesses:

(1) The rationale of the study is not clearly stated.

(2) Several issues with methodological and data integration should be clarified.

(3) Validation of the models is not presented clearly.

(4) The claim for providing tools for 'early warning' was not validated by analysis and results.

Author response:

Reviewer # 1 (Public review):

A major concern is that the model is trained in the midst of the COVID-19 pandemic and its associated restrictions and validated on 2023 data. The situation before, during, and after COVID is fluid, and one may not be representative of the other. The situation in 2023 may also not have been normal and reflective of 2024 onward, both in terms of the amount of testing (and positives) and measures taken to prevent the spread of these types of infections. A further worry is that the retrospective prospective split occurred in October 2020, right in the first year of COVID, so it will be impossible to compare both cohorts to assess whether grouping them is sensible.

We fully concur with the reviewer that the COVID-19 pandemic represents a profound confounding factor that fundamentally impacts the interpretation and generalizability of our model. This is a critical point that deserves a more thorough treatment. In the revised manuscript, we will add a dedicated subsection in the Discussion to explicitly analyze the pandemic’s impact. We will reframe our model’s contribution not as a universally generalizable tool for a hypothetical “normal” future, but as a robust framework demonstrated to capture complex epidemiological dynamics under the extreme, non-stationary conditions of a real-world public health crisis. We will argue that its strong performance on the 2023 validation data, a unique post-NPI “rebound” year, specifically showcases its utility in modeling volatile periods.

The outcome of interest is the number of confirmed influenza cases. This is not only a function of weather, but also of the amount of testing. The amount of testing is also a function of historical patterns. This poses the real risk that the model confirms historical opinions through increased testing in those higher-risk periods. Of course, the models could also be run to see how meteorological factors affect testing and the percentage of positive tests. The results only deal with the number of positive (only the overall number of tests is noted briefly), which means there is no way to assess how reasonable and/or variable these other measures are. This is especially concerning as there was massive testing for respiratory viruses during COVID in many places, possibly including China.

The reviewer raises a crucial point regarding surveillance bias, which is inherent in studies using reported case data. We acknowledge this limitation and will address it more transparently.

(1) Clarification of Available Data: Our manuscript states that over the six-year period, a total of 20,488 ILI samples were tested, yielding 3,155 positive cases (line 471; Figure 1). We will make this denominator more prominent in the Methods section. However, the reviewer is correct that our models for Putian and the external validation for Sanming utilize the daily positive case counts as the outcome. The reality of our surveillance data source is that while we have the aggregate total of tests over six years, obtaining a reliable daily denominator of all respiratory virus tests conducted (not just for ILI patients as per the surveillance protocol) is not feasible. This is a common constraint in real-world public health surveillance systems.

(2) Justification and Discussion: We will add a detailed paragraph to the Limitations section to address this. We will justify our use of case counts as it is the most direct metric for assessing public health burden and planning resource allocation (e.g., hospital beds, antivirals). We will also explain that modeling the positivity rate presents its own challenges, as the ILI denominator is also subject to biases (e.g., shifts in healthcare-seeking behavior, co-circulation of other pathogens causing similar symptoms). We will thus frame our work as forecasting the direct surveillance signal that public health officials monitor daily.

Although the authors note a correlation between influenza and the weather factors. The authors do not discuss some of the high correlations between weather factors (e.g., solar radiation and UV index). Because of the many weather factors, those plots are hard to parse.

This is an excellent point. Our preliminary analysis (Supplementary Figure S2) indeed confirms a strong positive correlation between solar radiation and the UV index. Perhaps the reviewer overlooked the contents of the supplementary information document. We have included the figure for their review. Our original discussion did explicitly address this multicollinearity, summarized as follows: We acknowledge the high correlation between certain meteorological variables. We then explain that our two-stage modeling approach is designed to mitigate this issue. In the first stage, the DLNM models assess the impact of each variable individually, thus isolating their non-linear and lagged effects without being confounded by interactions. In the second stage, the LSTM network, by its nature, is a powerful non-linear function approximator that is robust to multicollinearity and can learn the complex, interactive relationships between all input features, including correlated ones.

Figure S2. Scatterplot matrix illustrating correlations between Influenza cases and meteorological factors. This comprehensive scatterplot matrix visualizes the relationships between influenza-like illness (ILI) cases, influenza A and B cases, and multiple meteorological variables, including average temperature, humidity, precipitation, wind speed, wind direction, solar radiation, and ultraviolet (UV) index. The figure is composed of three distinct sections that collectively provide an in-depth analysis of these relationships:

(1) Upper-right triangle: This section presents a Pearson correlation coefficient matrix, with color intensity reflecting the strength of correlations between the variables. Red cells represent positive correlations, while green cells represent negative correlations. The closer the coefficient is to 1 or -1, the darker the cell and the stronger the correlation, with statistically significant correlations marked by asterisks. This matrix allows for a rapid identification of notable relationships between influenza cases and meteorological factors.

(2) Lower-left triangle: This section contains scatterplots of pairwise comparisons between variables. These scatterplots facilitate the visual identification of potential linear or non-linear relationships, as well as any outliers or anomalies. This visualization is essential for evaluating the nature of interactions between meteorological factors and influenza cases.

(3) Diagonal: The diagonal displays the density distribution curves for each individual variable. These curves provide an overview of the distribution characteristics of each variable, revealing central tendencies, variance, and any skewness present in the data.

The authors do not actually compare the results of both methods and what the LSTM adds.

We thank the reviewer for this comment and realize we may not have signposted the comparison clearly enough. Our manuscript does present a direct comparison between the LSTM and ARIMA models in the Results section (lines 737-745) and Table 2, where performance metrics (MAE, RMSE, MAPE, SMAPE) for both models on the 2023 validation set are detailed, showing LSTM’s superior performance, particularly for Influenza A. Furthermore, Figure 6 (panels A and B) visualizes the LSTM’s predictions against observed values, and Supplementary Figure S3 does the same for the ARIMA model, allowing for a visual comparison of their fit.

To address the reviewer’s concern, in the revised manuscript, we will:

(1) Add a more explicit comparative statement in the Results section, directly contrasting the key metrics and highlighting the LSTM’s advantages in capturing peak activities.

(2) Consider combining the visualizations from Figure 6 and Supplementary Figure S3 into a single, more powerful comparative figure that shows the observed data, the LSTM predictions, and the ARIMA predictions on the same plot.

Meandering methods; reliability of “Our Word in Data”; Figure 2A is hard to parse.

We will address these points comprehensively.

(3) Methods: We will significantly streamline and restructure the Methods section. We also wish to provide context that the manuscript’s current structure reflects an effort to incorporate feedback from multiple rounds of peer review across different journals, which may have led to some repetition. We will perform a thorough edit to improve its conciseness and logical flow.

(4) Data Reliability: The reviewer raises a crucial and highly insightful question regarding the validity of using a national-level index to represent local public health interventions. This is a critical aspect of our model’s construction, and we are grateful for the opportunity to provide a more thorough justification.

We acknowledge that the ideal variable would be a daily, quantitative, city-level index of non-pharmaceutical interventions (NPIs). However, the practical reality of the data landscape in China is that such granular, publicly accessible databases for subnational regions do not exist. Given this constraint, our choice of the Our World in Data (OWID) national stringency index was the result of a careful consideration process, and we believe it serves as the best available proxy for our study context.

In the revised manuscript, we will significantly expand the Methods section to articulate our rationale, which is threefold:

National Policy Coherence: During the COVID-19 pandemic in mainland China, core NPIs, particularly mandatory face-covering policies in shared public spaces, were implemented with a high degree of national uniformity. While local governments had some autonomy, they operated within a centrally defined framework, ensuring a baseline level of policy consistency across the country.

Local Context Alignment: A key factor supporting the use of this national proxy is the specific epidemiological context of Putian during the study period. For the vast majority of the pandemic, Putian was classified as a low-risk area with only sporadic COVID-19 cases. Consequently, the city’s public health measures consistently aligned with the standard national guidelines. It did not experience prolonged or exceptionally strict local lockdowns that would cause a significant deviation from the national-level policy trends captured by the OWID index.

Validation by Local Public Health Experts: Most critically, and to directly address your suggestion, our co-authors from the Putian Center for Disease Control and Prevention have meticulously reviewed the OWID stringency index against their on-the-ground, institutional knowledge of the mandates that were in effect. They have confirmed that the categorical levels (0-4) and the temporal trends of the OWID index provide a faithful representation of the public health restrictions concerning face coverings as experienced by the population of Putian.

Therefore, we will revise our manuscript to make it clear that the use of the OWID index was not a choice of convenience, but a necessary and well-vetted decision. Given the unavailability of official local data, the OWID index, cross-validated by our local experts, represents the most rigorous and appropriate variable available to account for the profound impact of NPIs on influenza transmission in our model.

(5) Figure 2A: We agree completely and will replace the heatmap with a multi-line plot or a stacked area chart to better visualize the temporal dynamics of influenza subtypes.

We have preliminarily completed the redrawing of Figure 3A. The new and old versions are presented for your review to determine which figure is more suitable for this manuscript in terms of scientific accuracy and visual impact.

Reviewer #2 (Public review):

Weakness (1):

The rationale of the study is not clearly stated.

We appreciate the reviewer’s critique and acknowledge that the unique contribution of our study needs to be articulated more forcefully. Our introduction (lines 105-140) attempted to outline the limitations of existing studies, but we will revise it to be much sharper. The revised introduction will state unequivocally that our study’s rationale is to address a confluence of specific, unresolved gaps in the literature: 1) The persistent challenge of forecasting influenza in subtropical regions with their erratic seasonality; 2) The lack of studies that build subtype-specific models for Influenza A and B, which we show have distinct meteorological drivers; 3) The methodological gap in integrating the explanatory power of DLNM with the predictive power of a rigorously, Bayesian-optimized LSTM network; and 4) The unique opportunity to develop and test a model on data that encompasses the unprecedented disruption of the COVID-19 pandemic, a critical test of model robustness.

Weakness (2):

Several issues with methodological and data integration should be clarified.

We interpret this as a general statement, with the specific issues detailed in the reviewer’s subsequent points and the “Recommendations for the authors” section. We will meticulously address each of these specific points in our revision. For instance, as a demonstration of our commitment to clarification, we will provide a much more detailed justification for our choice of benchmark model (ARIMA), as detailed in our response to Recommendation #11.

Reviewer #2 (Recommendation  for the authors):

The authors should justify why the baseline model selection was made by comparing the LSTM model only with ARIMA? How the outcomes could be sensitive to other commonly used machine learning methods, such as Random Forest or XGBoost, etc, as a benchmark for their performance.

The reviewer raises a highly pertinent question regarding the selection of our benchmark model. A robust comparison is indeed essential for contextualizing the performance of our proposed LSTM network. Our choice to benchmark against the ARIMA model was a deliberate and principled decision, grounded in the specific literature of influenza forecasting at the intersection of climatology and epidemiology.

In the revised manuscript, we will expand our justification within the Methods section and reinforce it in the Discussion. Our rationale is as follows:

(1) ARIMA as the Established Standard: As we briefly noted in our original introduction (lines 110-113), the ARIMA model is arguably the most widely established and frequently cited statistical method for time-series forecasting of influenza incidence, including studies investigating meteorological drivers. It serves as the conventional benchmark against which novel methods in this specific domain are often evaluated. Therefore, demonstrating superiority over ARIMA is the most direct and scientifically relevant way to validate the incremental value of our deep learning approach.

(2) A Focused Scientific Hypothesis: Our primary hypothesis was that the LSTM network, with its inherent ability to capture complex non-linearities and long-term dependencies, could overcome the documented limitations of linear autoregressive models like ARIMA in the context of climate-influenza dynamics. Our study was designed specifically to test this hypothesis.

(3) Avoiding a “Bake-off” without a Clear Rationale: While other machine learning models like Random Forest or XGBoost are powerful, they are not established as the standard baseline in this particular niche of literature. Including them would shift the focus from a targeted comparison against the conventional standard to a broader, less focused “bake-off” of various algorithms. Such an exercise, while potentially interesting, would risk diluting the core message of our paper and would be undertaken without a clear, literature-driven hypothesis for why one of these specific tree-based models should be the next logical benchmark.

Therefore, we will argue in the revised manuscript that our focused comparison with ARIMA provides the clearest and most meaningful assessment of our model’s contribution to the existing body of work on climate-informed influenza forecasting. We will, however, explicitly acknowledge in the Discussion that future work could indeed benefit from a broader comparative analysis as the field continues to evolve and adopt a wider array of machine learning techniques.

Similarly, for some of the reviewer’s recommendations that do not require significant time and effort to implement, such as recommendation 7, we have also redrawn Figure 3 based on your feedback. It is provided for your review.

Figure 3 presents the time series of the cases. I wonder whether the data for these factors and outcomes are daily or aggregated by week/month? I suggest representing it in 9x1 format with a single x-axis to compare, instead of 3x3 format. Authors can refer similar plot in https://doi.org/ 10.1371/journal.pcbi.1012311 in Figure 1.

We are deeply grateful for the reviewer’s valuable suggestion and thoughtful provision of reference illustrations. Based on their input, we have redrawn Figure 3 and have included it for their review.

Weakness (3):

Validation of the models is not presented clearly.

We were concerned by this comment and conducted a thorough self-assessment of our manuscript. We believe we have performed a multi-faceted validation, but we have evidently failed to present it with sufficient clarity and structure. Our validation strategy, detailed across the Methods and Results sections, includes:

• Internal Out-of-Time Validation: Using 2023 data as a hold-out set to test the model trained on 2018-2022 data (lines 695-696, 705-710; Figure 6A, B).

• External Validation: Testing the trained model on an independent dataset from a different city, Sanming (lines 730-736; Figure 6I, J).

• Benchmark Model Comparison: Quantitatively comparing the LSTM’s performance against the standard ARIMA model using multiple error metrics (lines 737-745; Table 2).

• Interpretability Validation (Sanity Check): Using SHAP analysis to ensure the model’s predictions are driven by epidemiologically plausible factors (lines 746-755; Figure 6E-H).

To address the reviewer’s valid critique of our presentation, we will significantly restructure the relevant parts of the Results section. We will create explicit subheadings such as “Internal Validation,” “External Validation,” and “Comparative Performance against ARIMA Benchmark” to make our comprehensive validation process unambiguous and easy to follow.

Weakness (4):

The claim for providing tools for 'early warning' was not validated by analysis and results.

We agree with this assessment entirely. This aligns with the eLife Assessment and comments from Reviewer #1. Our primary revision will be to systematically recalibrate the manuscript's language. We will replace all instances of “early warning tool” with more accurate and modest phrasing, such as “high-performance forecasting framework” or “a foundational model for future warning systems.” We will ensure that our revised title, abstract, and conclusions precisely reflect what our study has delivered: a robust predictive model, not a field-ready public health intervention tool.

This sentence means that after picking the right tone and a way of writing for your document you should keep improving your writing to keep the reader interested.

eLife Assessment

This landmark manuscript comprehensively examines the roles of nine structural proteins in herpes simplex virus 1 (HSV-1) assembly and nuclear egress. By integrating cryo-light microscopy and soft X-ray tomography, the study presents an innovative approach to investigating viral assembly within cells. The research is thoroughly executed, yielding exceptional data that explain previously unknown functions expected to bear widespread influence. This work is of broad interest to virologists, cellular biologists, and structural biologists, offering a robust, contextually rich methodology for studying large protein complex assembly within the cellular environment, serving as an excellent starting point for high-resolution techniques.

Reviewer #1 (Public review):

Summary:

Nahas et al. investigated the roles of herpes simplex virus 1 (HSV-1) structural proteins using correlative cryo-light microscopy and soft X-ray tomography. The authors generated nine viral variants with deletions or mutations in genes encoding structural proteins. They employed a chemical fixation-free approach to study native-like events during viral assembly, enabling observation of a wider field of view compared to cryo-ET. The study effectively combined virology, cell biology, and structural biology to investigate the roles of viral proteins in virus assembly and budding.

Strengths:

(1) The study presented a novel approach to studying viral assembly in cellulo.

(2) The authors generated nine mutant viruses to investigate the roles of essential proteins in nuclear egress and cytoplasmic envelopment.

(3) The use of correlative imaging with cryoSIM and cryoSXT allowed for the study of viral assembly in a near-native state and in 3D.

(4) The study identified the roles of VP16, pUL16, pUL21, pUL34, and pUS3 in nuclear egress.

(5) The authors demonstrated that deletion of VP16, pUL11, gE, pUL51, or gK inhibits cytoplasmic envelopment.

(6) The manuscript is well-written, clearly describing findings, methods, and experimental design.

(7) The figures and data presentation are of good quality.

(8) The study effectively correlated light microscopy and X-ray tomography to follow virus assembly, providing a valuable approach for studying other viruses and cellular events.

(9) The research is a valuable starting point for investigating viral assembly using more sophisticated methods like cryo-ET with FIB-milling.

(10) The study proposes a detailed assembly mechanism and tracks the contributions of studied proteins to the assembly process.

(11) The study includes all necessary controls and tests for the influence of fluorescent proteins.

Weaknesses:

Overall, the manuscript does not have any major weaknesses, just a few minor comments, which were mostly solved in the revised version of the manuscript.

Comments on the latest version:

I reviewed the responses and the updated manuscript, and I am very pleased with how the authors have revised it. The manuscript was already strong, but with the addition of the summary table and the separated images, it is now excellent.

Reviewer #2 (Public review):

Summary:

For centuries, humans have been developing methods to see ever smaller objects, such as cells and their contents. This has included studies of viruses and their interactions with host cells during processes extending from virion structure to the complex interactions between viruses and their host cells: virion entry, virus replication and virion assembly, and release of newly constructed virions. Recent developments have enabled simultaneous application of fluorescence-based detection and intracellular localization of molecules of interest in the context of sub-micron resolution imaging of cellular structures by electron microscopy.

The submission by Nahas et al., extends the state-of-the-art for visualization of important aspects of herpesvirus (HSV-1 in this instance) virion morphogenesis, a complex process that involves virus genome replication, and capsid assembly and filling in the nucleus, transport of the nascent nucleocapsid and some associated tegument proteins through the inner and outer nuclear membranes to the cytoplasm, orderly association of several thousand mostly viral proteins with the capsid to form the virion's tegument, envelopment of the tegumented capsid at a virus-tweaked secretory vesicle or at the plasma membrane, and release of mature virions at the plasma membrane.

In this groundbreaking study, cells infected with HSV-1 mutants that express fluorescently tagged versions of capsid (eYFP-VP26) and tegument (gM-mCherry) proteins were visualized with 3D correlative structured illumination microscopy and X-ray tomography. The maturation and egress pathways thus illuminated were studied further in infections with fluorescently tagged viruses lacking one of nine viral proteins.

Strengths:

This outstanding paper meets the journal's definitions of Landmark, Fundamental, Important, Valuable, and Useful. The work is also Exceptional, Compelling, Convincing, and Solid. The work is a tour de force of classical and state-of-the-art molecular and cellular virology. Beautiful images accompanied by appropriate statistical analyses and excellent figures. The numerous complex issues addressed are explained in a clear and coordinated manner; the sum of what was learned is greater than the sum of the parts. Impacts go well beyond cytomegalovirus and the rest of the herpesviruses, to other viruses and cell biology in general.

Comments on the latest version:

This is a very nice paper. The authors responded affirmatively to the suggestions and questions of the reviewers.

Reviewer #3 (Public review):

Summary:

Kamal L. Nahas et al. demonstrated that pUL16, pUL21, pUL34, VP16, and pUS3 are involved in the egress of the capsids from the nucleous, since mutant viruses ΔpUL16, ΔpUL21, ΔUL34, ΔVP16, and ΔUS3 HSV-1 show nuclear egress attenuation determined by measuring the nuclear:cytoplasmic ratio of the capsids, the dfParental, or the mutants. Then, they showed that gM-mCherry+ endomembrane association and capsid clustering were different in pUL11, pUL51, gE, gK, and VP16 mutants. Furthermore, the 3D view of cytoplasmic budding events suggests an envelopment mechanism where capsid budding into spherical/ellipsoidal vesicles drives the envelopment.

Strengths:

The authors employed both structured illumination microscopy and cellular ultrastructure analysis to examine the same infected cells, using cryo-soft-X-ray tomography to capture images. This combination, set here for the first time, enabled the authors to obtain holistic data regarding a biological process, as a viral assembly. Using this approach, the researchers studied various stages of HSV-1 assembly. For this, they constructed a dual-fluorescently labelled recombinant virus, consisting of eYFP-tagged capsids and mCherry-tagged envelopes, allowing for the independent identification of both unenveloped and enveloped particles. They then constructed nine mutants, each targeting a single viral protein known to be involved in nuclear egress and envelopment in the cytoplasm, using this dual-fluorescent as the parental one. The experimental setting, both the microscopic and the virological, is robust and well-controlled. The manuscript is well-written, and the data generated is robust and consistent with previous observations made in the field.

I congratulate the authors. The work is robust, and I personally highlight the way they managed to include others' results merged among their own, providing a complete view of the story.

Comments on the latest version:

I reviewed the responses and the updated manuscript, and I agree with the reviewer's #1 words: "The manuscript was already strong, but with the addition of the summary table and the separated images, it is now excellent."

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

Nahas et al. investigated the roles of herpes simplex virus 1 (HSV-1) structural proteins using correlative cryo-light microscopy and soft X-ray tomography. The authors generated nine viral variants with deletions or mutations in genes encoding structural proteins. They employed a chemical fixation-free approach to study native-like events during viral assembly, enabling observation of a wider field of view compared to cryo-ET. The study effectively combined virology, cell biology, and structural biology to investigate the roles of viral proteins in virus assembly and budding.

Strengths:

(1) The study presented a novel approach to studying viral assembly in cellulo.

(2) The authors generated nine mutant viruses to investigate the roles of essential proteins in nuclear egress and cytoplasmic envelopment.

(3) The use of correlative imaging with cryoSIM and cryoSXT allowed for the study of viral assembly in a near-native state and in 3D.

(4) The study identified the roles of VP16, pUL16, pUL21, pUL34, and pUS3 in nuclear egress.

(5) The authors demonstrated that deletion of VP16, pUL11, gE, pUL51, or gK inhibits cytoplasmic envelopment.

(6) The manuscript is well-written, clearly describing findings, methods, and experimental design.

(7) The figures and data presentation are of good quality.

(8) The study effectively correlated light microscopy and X-ray tomography to follow virus assembly, providing a valuable approach for studying other viruses and cellular events.

(9) The research is a valuable starting point for investigating viral assembly using more sophisticated methods like cryo-ET with FIB-milling.

(10) The study proposes a detailed assembly mechanism and tracks the contributions of studied proteins to the assembly process.

(11) The study includes all necessary controls and tests for the influence of fluorescent proteins.

Weaknesses:

Overall, the manuscript does not have any major weaknesses, just a few minor comments:

(1) The gel quality in Figure 1 is inconsistent for different samples, with some bands not well resolved (e.g., for pUL11, GAPDH, or pUL20).

We thank the reviewer for their suggestion. We tried to resolve the bands several times, but unfortunately this was the best outcome we could achieve.

(2) The manuscript would benefit from a summary figure or table to concisely present the findings for each protein. It is a large body of manuscript, and a summary figure showing the discovered function would be great.

We thank the reviewer for their suggestion. We have created a summary table (Table 2).

(3) Figure 2 lacks clarity on the type of error bars used (range, standard error, or standard deviation). It says, however, range, and just checking if this is what the authors meant.

We thank the reviewer for double-checking, but it is meant to be range, as reported in the legend. We used range because there are only two data points for each time point, which are insufficient to calculate standard deviation or standard error.

(4) The manuscript could be improved by including details on how the plasma membrane boundary was estimated from the saturated gM-mCherry signal. An additional supplementary figure with the data showing the saturation used for the boundary definition would be helpful.

We appreciate the suggestion and have included an example of how saturated gM-mCherry signal was used to delineate the cytoplasm in Supp. Fig. 4A.

(5) Additional information or supplementary figures on the mask used to filter the YFP signal for Figure 4 would be helpful.

Thanks, we have adapted the text in the results section to clarify: “eYFP-VP26 signal was manually inspected to determine threshold values that filtered out background and included pixels containing individual or clustered puncta that represent capsids.”

(6) The figure legends could include information about which samples are used for comparison for significance calculations. As the colour of the brackets is different from the compared values (dUL34), it would be great to have this information in the figure legend.

Thanks, we have adapted Fig. 4B to make the colour of the brackets match the colour used for the ΔUL34 mutant, and we have included labels next to the brackets for clarity. We have applied similar adjustments to Fig. 5D & E and Supp. Fig. 4C.

(7) In Figure 5B, the association between YFP and mCherry signals is difficult to assess due to the abundance of mCherry signal; single-channel and combined images might improve visualization.

Thanks, we have provided split and combined channel views in Supp. Fig. 4B to improve visualization.

(8) In Figure 6D, staining for tubulin could help identify the cytoskeleton structures involved in the observed virus arrays.

We thank the reviewer for their suggestion, which we think would be interesting future work to build on the current study. Given the competitive nature of access to the cryoSIM and cryoSXT, CLXT, including staining for tubulin was outside the scope of additional experiments we were able to conduct at this time.

(9) It is unclear in Figure 6D if the microtubule-associated capsids are with the gM envelope or not, as the signal from mCherry is quite weak. It could be made clearer with the split signals to assess the presence of both viral components.

We have provided split channels to the figure to aid with visualization.

(10) The representation of voxel intensity in Figure 8 is somewhat confusing. Reversion of the voxel intensity representation to align brighter values with higher absorption, which would simplify interpretation.

We thank the reviewer for this suggestion. In contrast to fluorescence microscopy where high intensities reflect signal, low intensities represent signal (absorbance of X-rays) in cryoSXT. We respectfully decided not to reverse the values, as we believe that could cause more confusion. We have instead added a black-to-white gradient bar to illustrate that low voxel intensities correspond to dark signal in Fig 8.

(11) The visualization in panel I of Figure 8 might benefit from a more divergent colormap to better show the variation in X-ray absorbance.

We thank the reviewer for their suggestion. We experimented with a few different colour schemes but concluded that the current one produced the clearest results and was most accessible for color-blind viewers.

(12) Figure 9 would be enhanced by images showing the different virus sizes measured for the comparative study, which would help assess the size differences between different assembly stages.

We thank the reviewer for their suggestion and have included images to accompany the graph.

Overall, this is an excellent manuscript and an enjoyable read. It would be interesting to see this approach applied to the study of other viruses, providing valuable insights before progressing to high-resolution methods.

Reviewer #2 (Public review):

Summary:

For centuries, humans have been developing methods to see ever smaller objects, such as cells and their contents. This has included studies of viruses and their interactions with host cells during processes extending from virion structure to the complex interactions between viruses and their host cells: virion entry, virus replication and virion assembly, and release of newly constructed virions. Recent developments have enabled simultaneous application of fluorescence-based detection and intracellular localization of molecules of interest in the context of sub-micron resolution imaging of cellular structures by electron microscopy.

The submission by Nahas et al., extends the state-of-the-art for visualization of important aspects of herpesvirus (HSV-1 in this instance) virion morphogenesis, a complex process that involves virus genome replication, and capsid assembly and filling in the nucleus, transport of the nascent nucleocapsid and some associated tegument proteins through the inner and outer nuclear membranes to the cytoplasm, orderly association of several thousand mostly viral proteins with the capsid to form the virion's tegument, envelopment of the tegumented capsid at a virus-tweaked secretory vesicle or at the plasma membrane, and release of mature virions at the plasma membrane.

In this groundbreaking study, cells infected with HSV-1 mutants that express fluorescently tagged versions of capsid (eYFP-VP26) and tegument (gM-mCherry) proteins were visualized with 3D correlative structured illumination microscopy and X-ray tomography. The maturation and egress pathways thus illuminated were studied further in infections with fluorescently tagged viruses lacking one of nine viral proteins.

Strengths:

This outstanding paper meets the journal's definitions of Landmark, Fundamental, Important, Valuable, and Useful. The work is also Exceptional, Compelling, Convincing, and Solid. The work is a tour de force of classical and state-of-the-art molecular and cellular virology. Beautiful images accompanied by appropriate statistical analyses and excellent figures. The numerous complex issues addressed are explained in a clear and coordinated manner; the sum of what was learned is greater than the sum of the parts. Impacts go well beyond cytomegalovirus and the rest of the herpesviruses, to other viruses and cell biology in general.

Reviewer #3 (Public review):

Summary:

Kamal L. Nahas et al. demonstrated that pUL16, pUL21, pUL34, VP16, and pUS3 are involved in the egress of the capsids from the nucleous, since mutant viruses ΔpUL16, ΔpUL21, ΔUL34, ΔVP16, and ΔUS3 HSV-1 show nuclear egress attenuation determined by measuring the nuclear:cytoplasmic ratio of the capsids, the dfParental, or the mutants. Then, they showed that gM-mCherry+ endomembrane association and capsid clustering were different in pUL11, pUL51, gE, gK, and VP16 mutants. Furthermore, the 3D view of cytoplasmic budding events suggests an envelopment mechanism where capsid budding into spherical/ellipsoidal vesicles drives the envelopment.

Strengths:

The authors employed both structured illumination microscopy and cellular ultrastructure analysis to examine the same infected cells, using cryo-soft-X-ray tomography to capture images. This combination, set here for the first time, enabled the authors to obtain holistic data regarding a biological process, as a viral assembly. Using this approach, the researchers studied various stages of HSV-1 assembly. For this, they constructed a dual-fluorescently labelled recombinant virus, consisting of eYFP-tagged capsids and mCherry-tagged envelopes, allowing for the independent identification of both unenveloped and enveloped particles. They then constructed nine mutants, each targeting a single viral protein known to be involved in nuclear egress and envelopment in the cytoplasm, using this dual-fluorescent as the parental one. The experimental setting, both the microscopic and the virological, is robust and well-controlled. The manuscript is well-written, and the data generated is robust and consistent with previous observations made in the field.

Weaknesses:

It would be helpful to find out what role the targeted proteins play in nuclear egress or envelopment acquisition in a different orthoherpesvirus, like HSV-2. This would confirm the suitability of the technical approach set and would also act as a way to validate their mechanism at least in one additional herpesvirus beyond HSV-1. So, using the current manuscript as a starting point and for future studies, it would be advisable to focus on the protein functions of other viruses and compare them.

We appreciate the suggestion and agree that this would be a great starting point for future studies. At present, we do not have a panel of mutant viruses in HSV-2 or another orthoherpesvirus, and it would be significant work to generate them, so we consider this outside the scope of the current study.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

(1) There are enough uncommon abbreviations in the text to justify the inclusion of an abbreviation list.

We thank the reviewer for the suggestion, but we define all uncommon abbreviations at first mention and an abbreviations list is not part of eLife’s house style.

(2) The complex paragraph on p. 7 would be much easier to digest if broken into smaller chunks. Consider similar treatment for other lengthy landmark-free blocks of text, e.g., the one that begins on p. 14. Subheadings would help.

We thank the reviewer for this suggestion. We have divided large paragraphs into more easily digestible chunks throughout the manuscript, for example in the discussion where the previous monolithic 3rd paragraph has been divided into five shorter, focussed paragraphs.

(3) Table 1 needs units.

We thank the reviewer for noticing our omission and apologise for the oversight - the table has been updated accordingly.

Reviewer #3 (Recommendations for the authors):

(1) Toward the end of the manuscript, I missed some lines attempting to speculate on the origin/nature of the spherical/ellipsoidal vesicles providing the envelopment. Would it be possible to incorporate this in the Discussion section?

Thank you for noticing that omission. We have now included a few lines speculating that they may represent recycling endosomes, trans-Golgi network vesicles, or a hybrid compartment.

(2) I congratulate the authors. The work is robust, and I personally highlight the way they managed to include others' results merged with their own, providing a complete view of the story.

We thank the reviewer for their kind words.

Note to editors

In addition to these responses to the reviewer’s comments, we have also now included in the methods section details of the Tracking of Indels by Decomposition (TIDE) analysis we performed (data in Supplementary Figure 3) that was omitted by mistake from the original submission.

To protect culture, it's important to take the data out of the hands of big companies and have individuals instead create.

Strong statement, and important to know. We are privileged to know the language we do.

A correct way to use A.I., strengthening what's already known but protecting the culture behind it.

We're losing culture the more we use A.I.

A.I. isn't equally available for everyone, meaning we cannot say everyone is fairly represented in the development of our future.

Have you ever been met with a deceitful piece of misinformation on TikTok? If so, what was it, and how did it affect your view of the platform?

He chose the less traveled road meaning, this is a hard choice that not many people take in life; a path more risky and challenging yet, one that the writer chose to take. It is a choice that is not influenced by anyone else but, a choice made on your own. In other words, he is showing individualism in his choice.

He doubted the path he chose because he knows there is no turning back that this path he is on will lead to more and more, a series of events.

He apologizes because he can only choose one path and must leave the other behind.

The writer is giving us visuals. We can picture that both paths are covered in leaves and this also can confirm that the yellow wood does indicate the season of fall because in fall, the leaves fall off of the trees.

This tell the time of season such as yellow indicating fall because in the season of fall, the leaves turn yellow.

Original title of Woolf's Mrs Dalloway.

Do you believe the SIFT method is better than any other method? Why or why not?

This is a very nice warning. In any level of education, one sliver of mis info' will more often than not land you in big trouble down the line.

"What adaptive responses might aquatic microbes exhibit under increased stressors like warming and nutrient influx?" Some microbes might adapt by becoming more tolerant to higher saline levels in the water, which would cause a decline in the microbes that are less tolerant to saline. Also, some that are more tolerant to heat, might become more dominant, which would cause a decrease in the microbes that are less tolerant.

Discussion Question: "As microbiologists, what strategies could be adopted to anticipate, detect, or mitigate harmful microbial growth in warming aquatic systems?" We can do more comparitive studies on different bacteria in the same bodies of water, bacteria that are dependent on one another for survival. Comparing and contrasting their growth and adjustments to the climate can give us clues on what is happening in the water. And if one bacteria is more harmful than the other, maybe we can find a way to slow down its growth, or promote growth in the other bacteria.

https://www.sciencedirect.com/science/article/pii/S1470160X25002559

"In what ways do more frequent and intense precipitation events influence microbial contamination in aquatic systems, and why?" Higher frequency of more intense precipitation results in more runoff into aquatic systems, which in turn promotes greater chances of microbial contamination.

"How might rising water termperature, driven by climate change, alter microbial growth rates and community structures in aquatic environments?" Warmer water termperatures promote a favorable environment for harmful algal blooms. The warmer the water, and the longer it remains at an increased temperature, the longer the harmful algal blooms will last. Also with warmer water, the microbial growth rate could be even greater, if the exponential phase lasts longer.

Sometimes i forget what is the point of the article that is when i go read the tittle again to make sure to focus on the topic of it again.

Highlighting words I do not understand makes the text easier for me because I can always go back and look up the meaning of the words.

This has help me understand better while reading all of it at first.

DVD was apart of the cycle that is outdated technology, streaming right now is king of television broadcasting. And one day if a more mainstream or convenient way to stream shows is found then that will become the standard. DVD made reruns basically useless because why would you need reruns when you can just pop the DVD in and watch the show that you want to whenever you want.

This is true, the ability to start stop and rewind/ rewatch movies and shows whenever you want was a new feature that has been brought to T.V via the DVD model. It allows the viewer to be more in control and engage with the content however they want

This was basically bottling the idea of the flow theory captured in a DVD so you can rewatch it whenever you want.

The DVD model is becoming more and more outdated by the day but its principles still remain the same. Replay ability was why DVD blew up in the first place and also its applicability to this model.

I wasn't aware that this was a thing. I'm assuming it's saying that people who rented DVD's through Netflix were only emailed a disc inside an envelope and not the whole box set.

DVD box sets became a huge success because instead of waiting days for a new episode of a show to be broadcasted, people were able to put a DVD disc in a DVD player and had access to the whole season or show.

Something about this scene is almost terrifying. I like how he later describes his brother as a madman, and I feel like this works so well after the author really hammers home how essentially dead his brother was. Especially because he "comes back to life" at the mention of his brother's name.

Allen Barsky believes that social workers should avoid communicating online with groups that involve political affiliations or any type of political cause. This is a practice that I will abide by to avoid unethical conduct using technology. If I have colleagues on social media that are violating this code of conduct, I will remind them about the adequate measures we must take as social workers to prevent the unethical conduct of colleagues, including unethical conduct using technology.

People are just expected to allow A.I. to take over without a say in the game.

I feel like this is why it's important to know what is copy-righted online. People think they can take any image or writing, but most of it is protected by the owner. It should be taken more seriously.

I haven't heard this comparison before, but it does seem to work in the context. Amplifying A.I. by comparing it to something as terrible as colonization shows the consequences A.I. can bring.

eLife Assessment

The ratio of nuclei to cell volume is a well-controlled parameter in eukaryotic cells. This study now reports important findings that expand our understanding of the regulatory relationship between cell size and number of nuclei. The evidence supporting the conclusions is convincing obtained by applying appropriate and validated methodology in line with current state-of-the-art. The paper will be of broad interest for cell biologists and fungal biotechnologists seeking to understand mechanisms determining cell size and number of nuclei and why this knowledge might also be of importance for the production of enzymes and thus production strains not only of Aspergillus oryzae but also other industrially used fungi.

Reviewer #1 (Public review):

Filamentous fungi are established work horses in biotechnology with Aspergillus oryzae as a prominent example with a thousand-year of history. Still the cell biology and biochemical properties of the production strains is not well understood. The paper of the Takeshita group describes the change in nuclear numbers and correlate it to different production capacities. They used microfluidic devices to really correlate the production with nuclear numbers. In addition, they used microdissection to understand expression profile changes and found an increase of ribosomes. The analysis of two genes involved in cell volume control in S. pombe did not reveal conclusive answers to explain the phenomenon. It appears that it is a multi-trait phenotype. Finally, they identified SNPs in many industrial strains and tried to correlate them to the capability of increasing their nuclear numbers.

The methods used in the paper range from high quality cell biology, Raman spectroscopy to atomic force and electron microscopy and from laser microdissection to the use of microfluidic devices to study individual hyphae.

This is a very interesting, biotechnologically relevant paper with the application of excellent cell biology.

Comments on revised version:

The authors addressed all suggestions satisfactorily.

Reviewer #2 (Public review):

Summary:

In the study presented by Itani and colleagues it is shown that some strains of Aspergillus oryzae - especially those used industrially for the production of sake and soy sauce - develop hyphae with a significantly increased number of nuclei and cell volume over time. These thick hyphae are formed by branching from normal hyphae and grow faster and therefore dominate the colonies. The number of nuclei positively correlates with the thicker hyphae and also the amount of secreted enzymes. The addition of nutrients such as yeast extract or certain amino acids enhanced this effect. Genome and transcriptome analyses identified genes, including rseA, that are associated with the increased number of nuclei and enzyme production. The authors conclude from their data involvement of glycosyltransferases, calcium channels and the tor regulatory cascade in regulation of cell volume and number of nuclei. Thicker hyphae and an increased number of nuclei was also observed in high-production strains of other industrially used fungi such as Trichoderma reesei and Penicillium chrysogenum, leading to the hypothesis that the mentioned phenotypes are characteristic of production strains which is of significant interest for fungal biotechnology.

Strengths:

The study is very comprehensive and involves application of divers state-of-the-art cell biological, biochemical and genetic methods. Overall, the data are properly controlled and analyzed, and the figures and movies are of excellent quality.The results are particularly interesting with regard to the elucidation of molecular mechanisms that regulate the size of fungal hyphae and the number of nuclei. For this, the authors have discovered a very good model: (regular) strains with a low number of nuclei and strains with high number of nuclei. Also, the results can be expected to be of interest for the further optimization of industrially relevant filamentous fungi.

In the revision the authors addressed all my comments and as a result produced an even stronger study.

Reviewer #3 (Public review):

Summary:

The authors seek to determine the underlying traits that support the exceptional capacity of Aspergillus oryzae to secrete enzymes and heterologous proteins. To do so, they leverage the availability of multiple domesticated isolates of A. oryzae along with other Aspergillus species to perform comparative imaging and genomic analysis.

Strengths:

The strength of this study lies in the use of multifaceted approaches to identify significant differences in hyphal morphology that correlate with enzyme secretion, which is then followed by the use of genomics to identify candidate functions that underlie these differences.

Weaknesses:

The authors addressed all suggestions satisfactorily.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Recommendations for the authors): 

The authors addressed all suggestions satisfactorily. 

Reviewer #2 (Recommendations for the authors):

The authors have adequately dealt with the comments. 

Reviewer #3 (Recommendations for the authors):

(1) Line 157. Although the authors have added a statement acknowledging that addition of YE increased hyphal width and secretion in A. nidulans without increasing nuclear number, they have not indicated how this result might impact their model. It might just boil down to variation between the different Aspergilli, but it merits attention. 

(2) Line 341. To extend the argument, you might consider adding this citation (https://elifesciences.org/articles/76075), which provides evidence that nuclear size might scale with osmotic pressure based on the density of macromolecules in the nucleus vs. cytoplasm.

Thanks for the suggestion.

L341 This is likely related to the phenomenon in which a decrease in cell size is accompanied by a reduction in nuclear size (66).

(3) Line 343. Neurospora crass hyphal cells can exceed 100 nuclei... 

Changed.

Acquaintance

hi its emma

i can see this

YOOO

hello yall

This is... Beautiful. Love the E

i can see this

hi!

Hi

Hi it's Ciara

hi its working barrett

yes

Hello

what was the world like in 1750?

Romeo thinks dreams tells the truth

Lady Capulet says Juliet to fall in love with Paris

Nurse is happy about Juliet’s match with Paris, she think Paris is a good guy for her

Nurse says Juliet will be fourteen on the night of the mentioned holiday on the text ( Lammas ), and an earthquake happened eleven years ago that’s how nuts keeps track of her time

She and the majority of the american people in fact

eLife Assessment

This study presents a valuable finding regarding the role of Arp2/3 and the actin nucleators N-WASP and WAVE complexes in myoblast fusion. The data presented is convincing, and the work will be of interest to biologists studying skeletal muscle stem cell biology in the context of skeletal muscle regeneration.

Reviewer #1 (Public review):

Overall, the manuscript reveals the role for actin polymerization to drive fusion of myoblasts during adult muscle regeneration. This pathway regulates fusion in many contexts, but whether it was conserved in adult muscle regeneration remained unknown. Robust genetic tools and histological analyses were used to convincingly support the claims.

Reviewer #2 (Public review):

To fuse, differentiated muscle cells must rearrange their cytoskeleton and assemble actin-enriched cytoskeletal structures. These actin foci are proposed to generate mechanical forces necessary to drive close membrane apposition and the fusion pore formation. While the study of these actin-rich structures has been conducted mainly in drosophila and in vertebrate embryonic development, the present manuscript present clear evidence this mechanism is necessary for fusion of adult muscle stem cells in vivo, in mice. The data presented here clearly demonstrate that ARP2/3 and SCAR/WAVE complexes are required for differentiating satellite cells fusion into multinucleated myotubes, during skeletal muscle regeneration.

Reviewer #3 (Public review):

The authors have satisfactorily addressed my inquiries. However, I had to look quite hard to find where they responded to my final comment regarding the potential role of Arpc2 post-fusion during myofiber growth and/or maintenance, which I eventually located on page 7. I would appreciate it if the authors could state this point more explicitly, perhaps by adding a sentence such as "However, we cannot rule out the possibility that Arpc2 may also play a role in....." to improve clarity of communication.

While I understood from the original version that this issue falls beyond the immediate scope of the study, I believe it is important to adopt a more cautious and rigorous interpretative framework, especially given the widespread use of this experimental approach. In particular, when a gene could potentially have additional roles in myofibers, it may be helpful to explicitly acknowledge that possibility. Even if Arpc2 may not necessarily be one of them, such roles cannot be fully excluded without direct testing.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review): 

Overall, the manuscript reveals the role of actin polymerization to drive the fusion of myoblasts during adult muscle regeneration. This pathway regulates fusion in many contexts, but whether it was conserved in adult muscle regeneration remained unknown. Robust genetic tools and histological analyses were used to support the claims convincingly. 

We very much appreciate the positive comments from this Reviewer.

There are a few interpretations that could be adjusted. 

The beginning of the results about macrophages traversing ghost fibers after regeneration was a surprise given the context in the abstract and introduction. These results also lead to new questions about this biology that would need to be answered to substantiate the claims in this section. Also, it is unclear the precise new information learned here because it seems obvious that macrophages would need to extravasate the basement membrane to enter ghost fibers and macrophages are known to have this ability. Moreover, the model in Figure 4D has macrophages and BM but there is not even mention of this in the legend. The authors may wish to consider removing this topic from the manuscript. 

We appreciate this comment and acknowledge that the precise behavior of macrophages when they infiltrate and/or exit the ghost fibers during muscle regeneration is not the major focus of this study. However, we think that visualizing macrophages squeezing through tiny openings on the basement membrane to infiltrate and/or exit from the ghost fibers is valuable. Thus, we have moved the data from the original main Figure 2 to the new Figure S1. 

Regarding the model in Figure 4D, we have removed the macrophages because the depicted model represents a stage after the macrophages’ exit from the ghost fiber. 

Which Pax7CreER line was used? In the methods, the Jax number provided is the Gaka line but in the results, Lepper et al 2009 are cited, which is not the citation for the Gaka line. 

The Pax7^CreER line used in this study is the one generated in Lepper et al. 2009. We corrected this information in “Material and Methods” of the revised manuscript. 

Did the authors assess regeneration in the floxed mice that do not contain Cre as a control? Or is it known these alleles do not perturb the function of the targeted gene? 

We examined muscle regeneration in the floxed mice without Cre. As shown in Figure 1 below, none of the homozygous ArpC2^fl/fl, N-WASP^fl/fl, CYFIP1^fl/fl or N-WASP^fl/fl;CYFIP1^fl/fl alleles affected  muscle regeneration, indicating that these alleles do not perturb the function of the targeted gene.  

Author response image 1.

The muscle regeneration was normal in mice with only floxed target gene(s). Cross sections of TA muscles were stained with anti-Dystrophin and DAPI at dpi 14. n = 3 mice of each genotype, and > 80 ghost fibers in each mouse were examined. Mean ± s.d. values are shown in the dot-bar plot, and significance was determined by two-tailed student’s t-test. ns: not significant. Scale bar: 100 μm.

The authors comment: 'Interestingly, expression of the fusogenic proteins, MymK and MymX, was up-regulated in the TA muscle of these mice (Figure S4F), suggesting that fusogen overexpression is not able to rescue the SCM fusion defect resulted from defective branched actin polymerization.' It is unclear if fusogens are truly overexpressed because the analysis is performed at dpi 4 when the expression of fusogens may be decreased in control mice because they have already fused. Also, only two animals were analyzed and it is unclear if MymX is definitively increased. The authors should consider adjusting the interpretation to SCM fusion defect resulting from defective branched actin polymerization is unlikely to be caused by a lack of fusogen expression. 

We agree with the Reviewer that fusogen expression may simply persist till later time points in fusion mutants without being up-regulated. We have modified our interpretation according to the Reviewer’s suggestion. 

Regarding the western blots in the original Figure S4F, we now show one experiment from each genotype, and include the quantification of MymK and MymX protein levels from 3 animals in the revised manuscript (new Figure S5F-S5H). 

Reviewer #1 (Recommendations for the authors): 

(1) The ArpC2 cKO data could be presented in a clearer fashion. In the text, ArpC2 is discussed but in the figure, there are many other KOs presented and ArpC2 is the fourth one shown in the figure. The other KOs are discussed later. It may be worthwhile for the authors to rearrange the figures to make it easier for readers. 

Thank you for this suggestion. We have rearranged the genotypes in the figures accordingly and placed ArpC2 cKO first. 

The authors comment: 'Since SCM fusion is mostly completed at dpi 4.5 (Figure 1B) (Collins et al. 2024)'. This is not an accurate statement of the cited paper. While myofibers are formed by dpi 4.5 with centralized nuclei, there are additional fusion events through at least 21dpi. The authors should adjust their statement to better reflect the data in Collins et al 2024, which could include mentioning that primary fusions could be completed at dpi 4.5 and this is the process they are studying. 

We have adjusted our statement accordingly in the revised manuscript.

The authors comment: 'Consistent with this, the frequency distribution of SCM number per ghost fiber displayed a dramatic shift toward higher numbers in the ArpC2^cKO mice (Figure S5C). These results indicate that the actin cytoskeleton plays an essential role in SCM fusion as the fusogenic proteins. Should it read 'These results indicate that the actin cytoskeleton plays AS an essential role in SCM fusion as the fusogenic proteins'? 

Yes, and we adjusted this statement accordingly in the revised manuscript. 

Minor comments 

(1) In the results the authors state 'To induce genetic deletion of ArpC2 in satellites....'; 'satellites' is a term not typically used for satellite cells. 

Thanks for catching this. We changed “satellites” to satellite cells.

(2) In the next sentence, the satellite should be capitalized. 

Done.

(3) The cross-section area should be a 'cross-sectional area'. 

Changed.

Reviewer #2 (Public review):

To fuse, differentiated muscle cells must rearrange their cytoskeleton and assemble actinenriched cytoskeletal structures. These actin foci are proposed to generate mechanical forces necessary to drive close membrane apposition and fusion pore formation. 

While the study of these actin-rich structures has been conducted mainly in drosophila, the present manuscript presents clear evidence this mechanism is necessary for the fusion of adult muscle stem cells in vivo, in mice. 

We thank this Reviewer for the positive comment.

However, the authors need to tone down their interpretation of their findings and remember that genetic proof for cytoskeletal actin remodeling to allow muscle fusion in mice has already been provided by different labs (Vasyutina E, et al. 2009 PMID: 19443691; Gruenbaum-Cohen Y, et al., 2012 PMID: 22736793; Hamoud et al., 2014 PMID: 24567399). In the same line of thought, the authors write they "demonstrated a critical function of branched actin-propelled invasive protrusions in skeletal muscle regeneration". I believe this is not a premiere, since Randrianarison-Huetz V, et al., previously reported the existence of finger-like actin-based protrusions at fusion sites in mice myoblasts (PMID: 2926942) and Eigler T, et al., live-recorded said "fusogenic synapse" in mice myoblasts (PMID: 34932950). Hence, while the data presented here clearly demonstrate that ARP2/3 and SCAR/WAVE complexes are required for differentiating satellite cell fusion into multinucleated myotubes, this is an incremental story, and the authors should put their results in the context of previous literature. 

In this study, we focused on elucidating the mechanisms of myoblast fusion during skeletal muscle regeneration, which remained largely unknown. Thus, we respectfully disagree with this Reviewer that “this is an incremental story” for the following reasons – 

First, while we agree with this Reviewer that “genetic proof for cytoskeletal actin remodeling to allow muscle fusion in mice has already been provided by different labs”, most of the previous genetic studies, including ours (Lu et al. 2024), characterizing the roles of actin regulators (Elmo, Dock180, Rac, Cdc42, WASP, WIP, WAVE, Arp2/3) in mouse myoblast fusion were conducted during embryogenesis (Laurin et al. 2008; Vasyutina et al. 2009; Gruenbaum-Cohen et al. 2012; Tran et al. 2022; Lu et al. 2024), instead of during adult muscle regeneration, the latter of which is the focus of this study. 

Second, prior to this study, several groups tested the roles of SRF, CaMKII theta and gemma, Myo10, and Elmo, which affect actin cytoskeletal dynamics, in muscle regeneration. These studies have shown that knocking out SRF, CaMKII, Myo10, or Elmo caused defects in mouse muscle regeneration, based on measuring the cross-sectional diameters of regenerated myofibers only (Randrianarison-Huetz et al. 2018; Eigler et al. 2021; Hammers et al. 2021; Tran et al. 2022). However, none of these studies visualized myoblast fusion at the cellular and subcellular levels during muscle regeneration in vivo. For this reason, it remained unclear whether the muscle regeneration defects in these mutants were indeed due to defects in myoblast fusion, in particular, defects in the formation of invasive protrusions at the fusogenic synapse. Thus, the previous studies did not demonstrate a direct role for the actin cytoskeleton, as well as the underlying mechanisms, in myoblast fusion during muscle regeneration in vivo.

Third, regarding actin-propelled invasive protrusions at the fusogenic synapse, our previous study (Lu et al. 2024) revealed these structures by fluorescent live cell imaging and electron microscopy (EM) in cultured muscle cells, as well as EM studies in mouse embryonic limb muscle, firmly establishing a direct role for invasive protrusions in mouse myoblast fusion in cultured muscle cells and during embryonic development. Randrianarison-Huetz et al. (2018) reported the existence of finger-like actin-based protrusions at cell contact sites of cultured mouse myoblasts. It was unclear from their study, however, if these protrusions were at the actual fusion sites and if they were invasive (Randrianarison-Huetz et al. 2018). Eigler et al. (2021) reported protrusions at fusogenic synapse in cultured mouse myoblasts. It was unclear from their study, however, if the protrusions were actin-based and if they were invasive (Eigler et al. 2021). Neither Randrianarison-Huetz et al. (2018) nor Eigler et al. (2021) characterized protrusions in developing mouse embryos or regenerating adult muscle. 

Taken together, to our knowledge, this is the first study to characterize myoblast fusion at the cellular and subcellular level during mouse muscle regeneration. We demonstrate that branched actin polymerization promotes invasive protrusion formation and myoblast fusion during the regeneration process. We believe that this work has laid the foundation for additional mechanistic studies of myoblast fusion during skeletal muscle regeneration.

The citations in the original manuscript were primarily focused on previous in vivo studies of Arp2/3 and the actin nucleation-promoting factors (NPFs), N-WASP and WAVE (Richardson et al. 2007; Gruenbaum-Cohen et al. 2012), and of invasive protrusions mediating myoblast fusion in intact animals (Drosophila, zebrafish and mice) (Sens et al. 2010; Luo et al. 2022; Lu et al. 2024). We agree with this reviewer, however, that it would be beneficial to the readers if we provide a more comprehensive summary of previous literature, including studies of both intact animals and cultured cells, as well as studies of additional actin regulators upstream of the NPFs, such as small GTPases and their GEFs. Thus, we have significantly expanded our Introduction to include these studies and cited the corresponding literature in the revised manuscript.

Reviewer #2 (Recommendations for the authors): 

(1) I am concerned that the authors did not evaluate the efficiency of the target allele deletion efficiency following Pax7-CreER activation. The majority, if not all, of the published work focusing on this genetic strategy presents the knock-down efficiency using either genotyping PCR, immunolocalization, western-blot; etc... 

(2) Can the authors provide evidence that the N-WASP, CYFIP1, and ARPC2 proteins are depleted in TAM-treated tissue? Alternatively, can the author perform RT-qPCR on freshly isolated MuSCs to validate the absence of N-WASP, CYFIP1, and ARPC2 mRNA expression?

Thank you for these comments. We have assessed the target allele deletion efficiency with isolated satellite cells from TAM-injected mice in which Pax7-CreER is activated. Western blot analyses showed that the protein levels of N-WASP, CYFIP1, and ArpC2 significantly decreased in the satellite cells of knockout mice. Please see the new Figure S2.

Reviewer #3 (Public review): 

The manuscript by Lu et al. explores the role of the Arp2/3 complex and the actin nucleators NWASP and WAVE in myoblast fusion during muscle regeneration. The results are clear and compelling, effectively supporting the main claims of the study. However, the manuscript could benefit from a more detailed molecular and cellular analysis of the fusion synapse. Additionally, while the description of macrophage extravasation from ghost fibers is intriguing, it seems somewhat disconnected from the primary focus of the work. 

Despite this, the data are robust, and the major conclusions are well supported. Understanding muscle fusion mechanism is still a widely unexplored topic in the field and the authors make important progress in this domain. 

We appreciate the positive comments from this Reviewer.

We agree with this Reviewer and Reviewer #1 that the macrophage study is not the primary focus of the work. However, we think that visualizing macrophages squeezing through tiny openings on the basement membrane to infiltrate and/or exit from the ghost fibers is valuable. Thus, we have moved the data from the original main Figure 2 to the new Figure S1. 

I have a few suggestions that might strengthen the manuscript as outlined below.  

(1) Could the authors provide more detail on how they defined cells with "invasive protrusions" in Figure 4C? Membrane blebs are commonly observed in contacting cells, so it would be important to clarify the criteria used for counting this specific event. 

Thanks for this suggestion. We define invasive protrusions as finger-like protrusions projected by a cell into its fusion partner. Based on our previous studies (Sens et al. 2010; Luo et al. 2022; Lu et al. 2024), these invasive protrusions are narrow (with 100-250 nm diameters) and propelled by mechanically stiff actin bundles. In contrast, membrane blebs are spherical protrusions formed by the detachment of the plasma membrane from the underlying actin cytoskeleton. In general, the blebs are not as mechanically stiff as invasive protrusions and would not be able to project into neighboring cells. Thus, we do not think that the protrusions in Figure 4B are membrane blebs. We clarified the criteria in the text and figure legends of the revised manuscript.

(2) Along the same line, please clarify what each individual dot represents in Figure 4C. The authors mention quantifying approximately 83 SCMs from 20 fibers. I assume each dot corresponds to data from individual fibers, but if that's the case, does this imply that only around four SCMs were quantified per fiber? A more detailed explanation would be helpful. 

To quantitatively assess invasive protrusions in Ctrl and mutant mice, we analyzed 20 randomly selected ghost fibers per genotype. Within each ghost fiber, we examined randomly selected SCMs in a single cross section (a total of 83, 147 and 93 SCMs in Ctrl, ArpC2^cKO and MymX^cKO mice were examined, respectively). 

In Figure 4C, each dot was intended to represent the percentage of SCMs with invasive protrusions in a single cross section of a ghost fiber. However, we mistakenly inserted a wrong graph in the original Figure 4C. We sincerely apologize for this error and have replaced it with the correct graph in the new Figure 4C.

(3) Localizing ArpC2 at the invasive protrusions would be a strong addition to this study. Furthermore, have the authors examined the localization of Myomaker and Myomixer in ArpC2 mutant cells? This could provide insights into potential disruptions in the fusion machinery.

We have examined the localization of the Arp2/3 complex on the invasive protrusions in cultured SCMs and included the data in Figure 4A of the original manuscript. Specifically, we showed enrichment of mNeongreen-tagged Arp2, a subunit of the Arp2/3 complex, on the invasive protrusions at the fusogenic synapse of cultured SCMs (see the enlarged panels on the right; also see supplemental video 4). The small size of the invasive protrusions on SCMs prevented a detailed analysis of the precise Arp2 localization along the protrusions.  Please see our recently published paper (Lu et al. 2024) for the detailed localization and function of the Arp2/3 complex during invasive protrusion formation in cultured C2C12 cells. 

We have also attempted to localize the Arp2/3 complex in the regenerating muscle in vivo using an anti-ArpC2 antibody (Millipore, 07-227-I), which was used in many studies to visualize the Arp2/3 complex in cultured cells. Unfortunately, the antibody detected non-specific signals in the regenerating TA muscle of the ArpC2^cKO animals. Thus, it cannot be used to detect specific ArpC2 signals in muscle tissues. Besides the specificity issue of the antibody, it is technically challenging to visualize invasive protrusions with an F-actin probe at the fusogenic synapses of regenerating muscle by light microscopy, due to the high background of F-actin signaling within the muscle cells. 

Regarding the fusogens, we show that both are present in the TA muscle of the ArpC2^cKO animals by western blot (Figure S5F-S5H). Thus, the fusion defect in these animals is not due to the lack of fusogen expression. Since the focus of this study is on the role of the actin cytoskeleton in muscle regeneration, the subcellular localization of the fusogens was not investigated in the current study. 

(4) As a minor curiosity, can ArpC2 WT and mutant cells fuse with each other?

Our previous work in Drosophila embryos showed that Arp2/3-mediated branched actin polymerization is required in both the invading and receiving fusion partners (Sens et al. 2010).  To address this question in mouse muscle cells, we co-cultured GFP⁺ WT cells with mScarleti⁺ WT (or mScarleti⁺ ArpC2^cKO cells) in vitro and assessed their ability to fuse with one another. We found that ArpC2^cKO cells could barely fuse with WT cells (new Figure 3F and 3G), indicating that the Arp2/3-mediated branched actin polymerization is required in both fusion partners. This result is consistent with our findings in Drosophila embryos. 

(5) The authors report a strong reduction in CSA at 14 dpi and 28 dpi, attributing this defect primarily to failed myoblast fusion. Although this claim is supported by observations at early time points, I wonder whether the Arp2/3 complex might also play roles in myofibers after fusion. For instance, Arp2/3 could be required for the growth or maintenance of healthy myofibers, which could also contribute to the reduced CSA observed, since regenerated myofibers inherit the ArpC2 knockout from the stem cells. Could the authors address or exclude this possibility? This is rather a broader criticism of how things are being interpreted in general beyond this paper. 

This is an interesting question. It is possible that Arp2/3 may play a role in the growth or maintenance of healthy myofibers. However, the muscle injury and regeneration process may not be the best system to address this question because of the indispensable early step of myoblast fusion. Ideally, one may want to knockout Arp2/3 in myofibers of young healthy mice and observe fiber growth in the absence of muscle injury and compare that to the wild-type littermates. Since these experiments are out of the scope of this study, we revised our conclusion that the fusion defect in ArpC2^cKO mice should account, at least in part, for the strong reduction in CSA at 14 dpi and 28 dpi, without excluding additional possibilities such as Arp2/3’s potential role in the growth or maintenance of healthy myofibers.  

References:

Eigler T, Zarfati G, Amzallag E, Sinha S, Segev N, Zabary Y, Zaritsky A, Shakked A, Umansky KB, Schejter ED et al. 2021. ERK1/2 inhibition promotes robust myotube growth via CaMKII activation resulting in myoblast-to-myotube fusion. Dev Cell 56: 3349-3363 e3346.

Gruenbaum-Cohen Y, Harel I, Umansky KB, Tzahor E, Snapper SB, Shilo BZ, Schejter ED. 2012. The actin regulator N-WASp is required for muscle-cell fusion in mice. Proc Natl Acad Sci U S A 109: 11211-11216.

Hammers DW, Hart CC, Matheny MK, Heimsath EG, Lee YI, Hammer JA, 3rd, Cheney RE, Sweeney HL. 2021. Filopodia powered by class x myosin promote fusion of mammalian myoblasts. Elife 10.

Laurin M, Fradet N, Blangy A, Hall A, Vuori K, Cote JF. 2008. The atypical Rac activator Dock180 (Dock1) regulates myoblast fusion in vivo. Proc Natl Acad Sci U S A 105: 15446-15451.

Lu Y, Walji T, Ravaux B, Pandey P, Yang C, Li B, Luvsanjav D, Lam KH, Zhang R, Luo Z et al. 2024. Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion. Nat Cell Biol 26: 1860-1877.

Luo Z, Shi J, Pandey P, Ruan ZR, Sevdali M, Bu Y, Lu Y, Du S, Chen EH. 2022. The cellular architecture and molecular determinants of the zebrafish fusogenic synapse. Dev Cell 57: 1582-1597 e1586.

Randrianarison-Huetz V, Papaefthymiou A, Herledan G, Noviello C, Faradova U, Collard L, Pincini A, Schol E, Decaux JF, Maire P et al. 2018. Srf controls satellite cell fusion through the maintenance of actin architecture. J Cell Biol 217: 685-700.

Richardson BE, Beckett K, Nowak SJ, Baylies MK. 2007. SCAR/WAVE and Arp2/3 are crucial for cytoskeletal remodeling at the site of myoblast fusion. Development 134: 4357-4367.

Sens KL, Zhang S, Jin P, Duan R, Zhang G, Luo F, Parachini L, Chen EH. 2010. An invasive podosome-like structure promotes fusion pore formation during myoblast fusion. J Cell Biol 191: 1013-1027.

Tran V, Nahle S, Robert A, Desanlis I, Killoran R, Ehresmann S, Thibault MP, Barford D, Ravichandran KS, Sauvageau M et al. 2022. Biasing the conformation of ELMO2 reveals that myoblast fusion can be exploited to improve muscle regeneration. Nat Commun 13: 7077.

Vasyutina E, Martarelli B, Brakebusch C, Wende H, Birchmeier C. 2009. The small G-proteins Rac1 and Cdc42 are essential for myoblast fusion in the mouse. Proc Natl Acad Sci U S A 106: 8935-8940.

This is a perfect example of my last two annotations, the character has a main plot point but each week faces a new set of trivial new issues. In the end the main plot point is fixed but the episodes leading up to the culmination of the show does nothing to the actual purpose of the show.

Once again, how shows that used to air weekly work. They may give the illusion of providing a solution but it is a flimsy or weak one that can easily be broken and replaced with a new conflict for the characters to solve and the viewer to digest. It is a very easy way to get as many stories into the show as possible.

That's how shows like sitcoms or 90's shows that would air every week would work. The cast of characters would have to face a new issue weekly that would get "solved" but would be right back the next week with a different issue.

I know some people who don't watch certain shows they put on TV, but have it as background noise while they work on something else. Some people actually watch the series but others end up having a short attention span and pull out their phone or do something else while it continue playing on Tv.

I agree with this because in a cinema narration (movies), there is a beginning, middle, and end with one focus. TV narration (shows) doesn't always have a clear ending because the series can move different directions with plots so there are episodes and seasons to continue the stories.

1. Relaxing in a hot spring in the rainforest
2. Wandering across a land full of forms of water
3. Eating a hearty and nutritious dinner, then going to meet my friends Brains, Alan, Tin Tin and Slomo

This section of the NASW code of ethics raises important questions regarding power and structural inequality because it is not clear and concise about what is personal information that should not be shared. I would assume that the personal information includes personal email, personal number, home address, etc. I believe this code should go into more detail to deter a violation.

This ethical standard is applied in my field work daily while serving adults with disabilities who are seeking competitive employment. The vocational counselors and I work together to assist clients in their efforts to identify and clarify their employment goals. If the goal poses a potential risk to themselves or others, the counselor's and their guardians limit their right to self-determination. Safety comes first.

This small paragraph stood out to me because of the high statistical number of older adult who experience agism. This shows that agism is happening much more than we realize and not in good ways. The most common form is internalized which means that older people are even thinking of this of themselves.

This part of the data was surprise but not to me. It shows that women experienced more ageism than man have. I wonder why is this? Is this because many assume that men "do more" for them selfs and can be more stubborn. This also makes he wander if women are just more open to talk about it then men as well. I assume that men have a harder time being honst about agesim than possibly women.

This sentence is very interesting to me, because it says that only 7.2% of the group rated their mental health as fair/poor, but 32.3% say that they have depressive symptoms. This suggests that many participants experiencing depressive symptoms may not interpret or label their overall mental health as being “poor.” This tells me that the elderly population either does not want to discuss mental health issues, or is not properly informed about mental health.

I liked how this section was incorporated since there is not one specific reason for everyday ageism. I also like how it shows the difference in how the use of social media can change someone. This can help people caring for this population to understand factors that have gone into everyday ageism.

exactly what i was about to say and goes a little bit agains tmy rpbb research?

this is an intersting poitn I would like to look into further

interesting hypothesis; thinking the opposite in the context of RPBB; there is no refuge for the insects as temperatures increase and lower-elev habitat "creeps" into higher elevations

• striking first line, and very good hook
• I mean the obvious question is who the fuck is this boy and why is he being kept in just an obviously inhumane place?
• For some reason its really portrayed as idyllic
• he has no dialogue
• yikes bro, he was like raping kids or some shit?

why are the ships from America traveling to China? wouldn't America want to keep their merchant?

I think that the Guano islands act of 1856 is interesting because a part of it is the dried sea bird.

Thats fascinating that god wanted the United states to be expanded across the continent to help generate the popular support for projects. like why did they want to do that why not keep it small?

Luther believed true Christian has been lost and needed a complete return to biblical foundations, not just institutional adjustments. This marked a major turning point in European religious history. Luther went further than earlier reforms by calling not for reform but for a fundamental rebirth of Christianity.

This passage highlights how Akbar's policies of tolerance supported Hindu practices, while also explaining Hinduism's ancient roots, diversity, and unifying traditions compared to other world religions. While Hinduism has many sects and traditions, they tend to coexist under a share religious and cultural umbrella.

This passage shows that overtime, Sikhs built a strong warrior tradition, especially during the periods of persecution under the Mughal Empire. The Khalsa formalized this identify, making Sikhs both spiritual and martial leaders in India.

Despite reforms, the Ottoman Empire could not keep pace with industrialization, economic growth, and the military advances in Western Europe. Internal corruption, nationalist, and external pressures weakened the state.

I LOVE THIS and 100% AGREE! Inclusion not only benefits those with disabilities, it benefits all involved!

Teachers need to realize that a "one size fits all" approach is not the best practice when teaching kiddos with or without disabilities.

This right here is the very essence of what Special Education teachers and teachers in general should strive for!

While in some cases where a student with ASD who is low functioning may not benefit and could be more of a disruption to a general education classroom, the spectrum is so large that to just say that general statement for all kids with ASD is far from accurate or true. I know so many kids with ASD who do absolutely amazing in the general education class!

Inclusion needs to be modeled from a young age. A lot of times with children don't accept or include others it's because they've never been in that type of situation and proper inclusion and acceptance hasn't been modeled. Now, there are always going to be those select few kids that aren't kind or accepting, however this should not be the case for the majority or more than one or two students. If that's the case then I would look at the teacher and see what they could do better to promote inclusion better.

YES! I have seen this recently in the school I work in. We have general education teachers who have special needs students in their classroom and they are not trained at all in how to include them in the classroom. These teachers either don't think these children can do things other non-disabled students can, or the opposite, they expect the students to perform in other areas like non-disabled students. It's required of the case managers in our district to provide an "IEP At a Glance" to all teachers their SPED students would work with. Teachers are to read through, ask questions, and sign off stating they understand the document and will follow it in their classroom. I have seen so many general education teachers just quickly sign off on the At a Glance not really having looked through it. Part of that is lack of initiative, the other they haven't been trained enough to fully understand the seriousness and how to then implement this in the classroom.

This is hard for me to fathom. I think because I have worked in Special Education for several years now and have been studying Special Education and all of the laws and entities here in the United State with very clear guidelines and definitions, its hard to think that so many other countries don't have systems and guidelines and definitions in place.

Geographically speaking, I would be curious to see a comparison of what this looks like. For example, what that exclusivity looks like in the United States compared to non-Western societies.

I think this is one of the best statements and definitions the author makes. This definition really encompasses what inclusion is!

Mutidomestic means that we have access to to people culutre like sushi,mexican, and other foods We listen to the same music, shop like Kpop

• Flat World Theory saying that the world is more accesible with call, zoom, email
• employees can work every whwer
• tranporatation

Mostly everyone died.

The musicians were killed first, then the singers and spectators, then the other people as well. Many different people were killed in brutal ways.

On the first day everything went fine but the second day is when bad things started to happen.

He keeps saying they came to his house in Mexico because they are friends.

Cortes is claiming him and Montezuma are friends.

The captain only speaks Spanish.

Montezuma welcomed the Cortes.

Montezuma was the king.

This source was written by Aztec authors who described the description of the Tenochtilan.

This sounds chaotic. How can a story continue with no starting point and ending point? However, Murray describes later that this is the point. An adventure with so many paths to explore in a closed loop that we are comforted by it.

This is by far the clearest way to illustrate the idea of the rhizome story. It is a rather complex idea to comprehend and this makes it much easier to wrap your head around.

The improper use of the rhizomal structure--at it's base an attempt to elaborate on and determine narrative structure by the interactor's agency-- fails to maintain any level of coherence within itself to allow for comprehension of the work. They seem to take the lens of the most beautiful poem written scribbled on some bathroom wall. Perhaps more careful planning and development of technology for this will help (as it already has).

This is a particularly unique way to structure a story. In m opinion the most diverse and least used. Murray attempts to explain that the readers navigational choices through the use of hypertexts gives more hints to the story and thus more information to the reader on the plot leading to a much more complex mind involving experience.

I feel like this applies to "The boy in the book", in which we are also given multiple choices throughout the course of the story, but the effects of those choices are very limited. We get to choose where Nathan go and who he meets, but we do not get to decide what he ask or what he looks for when he gets to the places where we wants him to be (the Internet search is a major example of this.) The question then, should be to what degree should we get to make choices and should our choices affect the narrative for it to be considered true agency?

While this is true, it also means that there will be no opportunity for a satisfying ending, which can also be a draw for the interactor. The never-ending format of these stories means that the journey is the whole point of the work, but can it really be experienced as a whole if the journey has no destination?

Here the author is contrasting linear story telling and multithread story telling. This can show how a single version of an event may feel limiting, specifically in context of trauma. The multithreaded version can validate and express multiple perspectives and emotions which I think relates nicely to Hana Feels as we experienced multiple perspectives which I thought helped the story develop.

I think this statement is true and reflects what happened in the readings we have had so far. Having a wider understanding of the story makes it more engaging and helps the reader feel more connected to the story.

I believe this to be true for some digital stories but not all. There are many instances where the choices we make do not seem to make a difference in the story or we see no immediate consequence to our choice. Is this done on purpose? There must be a reason for this choice as it seems very unsatisfying to the reader.

When I read this sentence, it reminded me of human's desire for accomplishment. I think thats why adventure maze works well. Just like when you are playing a game of Escape room, you are actively trying to find a way out and each step and subtle clue you find makes you feel closer to the goal. Adventure maze uses this sense of accomplishment to bring powerfulness to the player.

This speaks to how, in Hana Feels, her own feelings about cutting herself feel like they're being treated as a real-life scenario rather than a story when playing the game and making decisions, since you are making choices that will impact her mental health and her possibly continuing self-harm.

This section highlights the benefits of using an interactive narrative for the plot of an adventure maze. No matter the case, Murray suggests that a digital environment suits these kinds of story's particularly well because they provide a wide arrange of choices from the reader to change the course of action and therefore the narrative. This gives the reader the freedom of multiple different paths and also the author the ability to change the course of the story. Overall, leading to a more immersive and subjective reality for the interactor.

This statement begins with a kind of dictionary definition of agency but I think it's the least interesting and apt way of thinking about how we participate and engage with digital works or even with digital space. In the interactive digital narratives we have read, "meaningful action" isn't always connected to a result or an outcome, but is instead part of a process of discovering or of uncovering ways of thinking or seeing.

Nigh all computations are based on an equal sign, and so all forms of interaction within games are forced to accept the logic of exchange in a sense of transaction rather than potlatch.These transactional actions allow the player to advance and so lead you to the ultimate transaction between the author where you broker your actions throughout the game for an ending you hope for.

I believe that this really highlights what choices are all about when you look at them from an outside perspective. Having the ability to understand different perspectives on why things happen and how choices can impact a situation makes people more understanding in social and life situations.

Core to this is, I believe, a sense of loss- a player who cannot lose anything (time, progress, efficiency, etc) is a player that has no challenge in facing threats. If there's no downside to taking a risk by facing the truth, but only an upside, why wouldn't every player take that choice? There must at least be the perception of a risk- such as a bad ending that can be restarted from the last point.

The first thing I think of when this comes to mind is the idea of the Lord of the Rings text adventure for MS-DOS that I played briefly as a kid, or most text adventures generally- they typically work off of a spacial, landmark-focused movement system, which really is more like the "paths" of a choose-your-own-adventure or digital narrative. It's the most "physical" version of this idea.

Is this maybe due to the context of the time- that computers are seen as solely productivity tools, or at best, entertainment boxes for mindless games?

Being inspired by seeing a film like Little Amelie, or The Character Of Rain

I like this term. Trouillot’s earlier discussion of Western models of linear time and how it became the assumed default hides the messiness, uncertainty, and multi-directionality of actual history. It also hides the fact that there is more than one way to look at time, the Western view excluded non-western ideas of time.

Who is the "real Columbus," as stated in the previous sentence, Columbus wears many hats. Historical figures are often battlegrounds for memory and meaning. The “real Columbus” is not a single, fixed person but a symbol whose identity shifts depending on who is telling the story and for what purpose.

What does it mean when a single historical figure like Columbus can symbolize both pride and oppression? How should educators or institutions navigate these conflicting narratives? In some places, Columbus is celebrated as a symbol of heritage, in others, he represents colonial violence and is resisted or reimagined in turn. This reflects Trouillot’s broader argument that history is not just about what happened, but how it is told and by whom. The same event or figure can be mobilized for competing purposes, depending on the narrative power of local actors and their specific relationship to the figure as well as colonialism, resistance, and identity.

To strip the event of its complexity and create a single, neat moment. In reality, this "discovery" was part of a long, messy process involving exploration, conquest, colonization, resistance, and genocide. But through dominant historical narratives, this process has been flattened into one symbolic moment, a flattening making it easier to celebrate, commemorate, or teach.

This reflects Trouillot’s broader idea of how power simplifies history, turning ongoing, contested events into fixed "facts" to serve the elite purpose (think national pride, colonial legacy). In recent years, there has been push back against this mythologized version of Columbus. It has been argued that what has been celebrated as a moment of “discovery” was in fact the beginning of centuries of violence and oppression. This could be an example of a challenge to the “single and simple moment” framing that Trouillot criticizes. History is not just about what happened, but about who gets to narrate what happened, and how those narratives are shaped by power. Are there any other examples that stick out where a complex, messy historical process has been flattened into a single, simple moment?

How can ideology override reality? in this context the alliance between the Church and state created a fiction of incompatibility between Christians, Muslims, and Jews—despite the fact that these groups often lived together, traded, and interacted peacefully in everyday life. This is a good example of Trouillot’s point about how power produces selective narratives/stories, here, the idea of a “pure” Christendom justifies violence and exclusion. By controlling the narrative, peaceful coexistence seems like betrayal, and war seems like salvation. In this way, ideology didn’t just describe the world, it reshaped it, silencing the messy reality in favor of a simplified and convenient myth.

I found Wallace’s message about conscious awareness really powerful. His point that we have a choice in how we perceive and interpret everyday situations like a frustrating trip to the grocery store made me reflect on how often I default to frustration instead of empathy. It’s a reminder that practicing awareness can shift our whole experience of the world, even in the most mundane moments.

No matter how much you invest in the marketing of the IFC5, diven the scope of the change, it is a revolution and NOT evolution.

Using STEP modules would do this equally well.

Key point. Very hard to set when rebuilding IFC from the ground zero.

If is is more rich and granular, how can it be simpler?

Yes, building information is rich. That's why IFC is a rich (and inherently complex) schema.

The original objection against STEP was, that is it too semanticaly rich. Is this not a contradiction?

STEP is also language independent. You can use any programming language to work with it.

So, there will be the mesh AND the parametric definition. You can do that in IFC already by using multiple geometry contexts.

IFC schema only describes what is needed. Any simplification will mean data loss.

This is great, if we only want to support 3D coordination.

Yes, we do need a schema language to make any sense of the data. That's what STEP/EXPRESS does very well.

Both authoring tools and viewers already do this on a large scale.

IFC has been using mapped geometries since ever. Nothing new or improved.

This is important! Triangulated mesh is great for some of the use cases, but not for all. Do we want to constraint the use of IFC just coordination? Why do we need any streaming capabilities in that case?

While it is possible to store one schema in many serialization formats, it decreases the reliability of the implementations. To claim IFC support, all the vendors will need to implement all the serialization formats with their quirks and workarounds. The final implementation matrix will make the IFC support very expensive and error prone, contrary to the goal of making it easier for startups and ISVs.

Major SW vendors will love to simplify to the point where the only suitable way to exchange their data is their proprietary format.

Here, USD is claimed to be flatter and simpler. Let's see later.

All very well defined in STEP.

These files will be much bigger than STEP files, at least twice. How does this help interoperability?

I've seen this picture a lot before

interesting way of portraying this

2

2

2

Keeping up with the homework

Is this the only empirical work? I thought there were others underway. Worth our digging into. Fwiw I can do an elicit.org query.

I'd say 'they argue' instead of 'emphasize'; the latter seems like a statement of absolute truth that we agree with.

The mastery of digital technologies today it requires increasingly attitudinal dispositions such as confidence, persistence, and the capacity to adapt in complex environments, beyond physical access to devices or procedural knowledge of software.

Should we adjust "the population of papers" to "the reference is" ? to be more explicit?

Version 6 of this preprint has been peer-reviewed and recommended by Peer Community in Neuroscience.<br /> See the peer reviews and the recommendation.

capitalize the first letters to match PDF: "Financial Statements (Unaudited)"