Suggests that the only reason they are visiting here is as tourists, which is what they are told later by the locals. Implies that she especially doesn't care about the past and sees this as a mere visit, whereas he originally came with the intention to reconnect with his old community (or so he thought.)

Mrs Jackson's unkind attitude towards their roots may also be influencing her husband's thought process, explaining why he was trying to deny his connection in the previous sentence.

Despite it clearly being a major part of his identity and heritage, Mr Jackson tries to deny or undermine his connection to this place. He likely shares some of the same sentiment about their friends as his wife, although to nowhere near the same extent.

Introduces the idea of the wife's focus on her image. She seems to be worried about what their friends in South Africa would think of them visiting somewhere run-down like this.

Personification of the shops that used to be lively, bustling markets and have been reduced to bland, uninspired storefronts. This is done to draw attention to the degradation of the town due to deindustrialisation and modernisation.

May also highlight how they are unwelcome here in their hometown since they have changed so much (i.e. the flat-faced shops represent the minds of the locals)

His sarcastic use of Scots here has a dual effect: it does show that he feels at home to some extent, but it may also just be him mocking his past self as a means of detaching himself from any sentimentality related to the area.

Suggests for a moment that he comes near to regretting his actions against black people, as he has some understanding that he is the one inflicting this suffering upon them now. This, unfortunately, doesn't end up coming to anything (▼)

Immediately asking him what he wants portrays the factor as quite cold and heartless as he clearly doesn't care about his clients.

Suggests that even the other workers here are unhappy with their lives, contributing to the idea that perhaps Glasgow was, in reality, a horrible place for Mr Jackson.

Shows Mr Jackson's highly racist mindset, particularly in saying "By God" when referring to them as this suggests that he is appalled to even be compared to a black person.

This line is also ironic as although he understands that he was "treated like a black," he doesn't seem to understand that people like him (Apartheid in South Africa) are the very reason for this.

Again characterises Mr Jackson as hypocritical as now that the tables have turned, he wishes to put others in the same exact position he despised being in.

The fact that he thinks of this Scottish slang as something sickening like "bile" suggests that he feels resentment for his country of birth, which has been amplified by this trip.

This is a plausible explanation, but it also highlights how the factor prioritises his jobs. He seems to think that people of a lower social status deserve to wait longer than his richer clientele.

Suggests that the factor is only motivated by money and has no sense of charity for others.

The colour black is used here to represent the gloom and misery of poverty. The symbolism of the umbrella is presumably similar, relating to the rain, and by implication, feelings of depression or melancholy.

Hyperbole to emphasise just how unkind the factor was (at least in Mr Jackson's eyes) - suggests that he is so mean and stingy that he is unwilling to even speak.

Portrays the factor as particularly ungenerous, as he sounds very unwilling to respond to those who are less fortunate than him and cannot provide him with financial gain.

The narrator explains Mr Jackson's thought process and how he clearly holds grudges against those who wronged him. We get the implication that he has quite violent ideas in mind as to what he would do in revenge.

Links back to when the Jacksons were still the inferior ones, living off of limited money, whereas now they have acquired a large sum of wealth and are flaunting that to everyone who is in the same situation that they were.

The "discoloured doors" imply that the place has fallen into ruin, likely since people have seldom visited it, hence why it has remained padlocked.

The fact that he only "wondered vaguely" suggests that he doesn't really care about her, but is merely curious. This likely reflects their unfriendly, uncaring attitudes towards others.

Shows Mrs Jackson to be overly avoidant of dirt as she doesn't want to contaminate her luxuries with this horrible place. She is both physically and metaphorically distancing herself from the place she used to live.

More evidence of his wealth and sense of safety (or stupidity) since he isn't afraid to show this, even in a dangerous area.

Having achieved a position of power and wealth, Mr Jackson now feels the urge to boast to others, but understands that there is no longer anyone here to boast to. This ultimately characterises him as quite arrogant and egotistical.

Emphasises the change that the area has seen, but that Mr Jackson cannot seem to accept. Nothing is as he recalls as the place has grown and the places he remembers have been replaced.

Suggests that the quality of the new houses has worsened, or at least lost any of its personality and soul. They are no longer like homes and are mere buildings, hinting at the negative effects of modernisation on society.

Deindustrialisation was a major issue in Glasgow during this time period (roughly the 1960s), and refers to the socio-economic change that results from the removal of industrial activity in a region.

By destroying old buildings and decommissioning factories, many people were made homeless and unemployed, which led to a downfall in the economy and a rise in violent crime.

Despite clearly having vivid memories of her old life, Mrs Jackson only responds to her husband with short replies that seem to lack intrigue, reinforcing the idea that she is trying to forget about that whole period.

Tells us that Mrs Jamieson was subjected to serious domestic abuse, and was afraid to come out about it. The fact that this was noticed by Mrs Jackson (and presumably the others), but nothing was done to intervene lets us know that this isn't anything rare.

Gives a sense of how unpleasant and aggressive a place this is by conveying the magnitude of his fury and abuse. It is also suggested that this was quite a regular occurrence, making the whole thing sound very distressing.

The simile here suggests that he feels proud of taking part in these fights, linking to the ideas of gang violence and social division.

Creates a sense of danger due to the imagery of sharpness and precision, which could be emphasising the dangerous nature of life in impoverished urban areas.

Alternatively, this can be viewed as conveying the sense of neatness, which could highlight Mrs Jackson's contempt for her old neighbours.

Mockery of the Jamiesons, which links on to the idea of him being blue from the bruises he received while fighting.

The additional remark that it was likely more than one highlights the violent nature of Glasgow, even back then. This directly contradicts what Mr Jackson seems to remember, thinking of the place as quite safe and peaceful.

Here, the mention of the colour blue emphasises the religious divide since the blueness was presumably not present due to his religious beliefs; rather because of the beating he received.

Introduces the religious divide that was present in their neighbourhood as the wife's thoughts of the Protestants are clearly of spite, hence why she (the narrator; her thoughts) remarks "of course" in reference to them.

The fact that they "lived above them" could also be, to some extent, symbolic of the social hierarchy, suggesting that the Jacksons felt as if they were inferior to the others at the time.

Again shows their differing attitudes. He is proud of his roots and remembers the neighbours somewhat fondly, while she seems to have tried to forget about them now that she has achieved (what she views as) success in life

The choice to use the word 'distasteful' here emphasises the wife's dislike of her neighbours, with her opinions potentially exaggerated by her memories.

The third person narrative reveals the wife's thoughts and prejudiced opinions towards her old neighbours, whom she looks down upon, as is proven by the phrase "at that level."

A Glaswegian slang term derived from "you bastard", used frequently by violent gangs in the 1960s. Indicates the hostility of the environment, which Mr Jackson is seemingly unaware of.

Illustrates the poverty present within the neighbourhood (and much of urban Glasgow during this period) by suggesting that there is a lot of physical ruination and that repairs are few and far between.

Characterises Mrs Jackson as quite old, fatigued, and even fragile. The metaphor of being "like a lacy bag" portrays her as quite delicate, not only physically, but mentally too as she is unwilling to recall her past.

A veldt is a large, open grassland situated in southern parts of Africa, typically blanketed by numerous small shrubs. The term originates from the Afrikaans word for 'field', therefore linking the story to South Africa.

Suggests that he is excited to be back here, but not solely for nostalgic reasons; also because he is arrogantly proud of his newfound wealth and wants to brag to those who he knew.

Although her husband is quite eager to revisit his old home, Mrs Jackson feels immediately unsafe upon entering the area, telling him to "lock the car" as a safety precaution.

Suggests that he is quite pretentious as he feels the need to flaunt his wealth by means of wearing expensive jewellery to an impoverished neighbourhood.

Makes him come across as quite strong, powerful, and imposing, yet this contradicts what we later see of him, which parallels the way that his view of his old life has been distorted with time.

Contrasts the Jackson's wealth, represented by their "black polished car", with the poverty of the local area with its "brown tenement[s]."

Joe here, in Bellevue, WA!

Without putting in your opinion, analyze the strengths and weaknesses of each source, or pros and cons for your audience.

Pick the sources that offer the widest range of opinions.

This line encapsulates a key insight into the shift from rigid, rule-based methods to a more flexible, data-driven machine learning approach. It raises a question about the subjectivity involved in labeling data: How do we decide which matches are genuinely significant, and how might these decisions influence the algorithm's learning process?

Tell us more! What did Martens do before and after this time?

For information only. And, for clarification, "White" as stated here does not refer to their skin color, but rather that they were White Party sympathizers.

Tell us more! What other impact did Colby have on international relations?

Fascinating! Please give us a biographical sketch of each of the African Americans named here. What did they do before and after after leaving the university?

Tell us more! What happened to Bullitt after his work was scuttled [failed]?

Tell us more! How long did this last? What are the names, occupations, and accusations against people who were arrested, deported, executed?

Information only. Adds more context to why Russia distrusts(ed) the West.

Information only.

What were Wilson's 14 points, and how many of them were accepted/adopted?

For information only.

This line struck me because it challenges the conventional view of data cleaning as merely a process of correction. Instead, it suggests that the act of cleaning can create a new layer of information—a distinct data model that carries its own implications for analysis and interpretation. It raises a question about the epistemological role of these transformations: When we ‘clean’ data, are we simply removing errors, or are we actively reshaping the knowledge contained within the dataset?

it is a great resource from the Academy of Pediatrics but with many young children having tablets and or phones. How can we as caregivers and educators can help limit their exposure to difficult or disturbing media. It can be easily hidden even with filters such as "youtube kids" or child account settings.

The study on deciphering bad handwriting through Mechanical Turk reminds me of the broader role of crowdsourcing in machine learning. One notable example is the ImageNet dataset, which was built using crowdsourced labeling of images.

The example of Fold-It is fascinating because it highlights how human intuition and problem-solving skills can outperform computers in certain complex tasks. It reminds me of how CAPTCHA tests often rely on human pattern recognition to verify users.

The mention of YouTube’s aggressive copyright enforcement reminds me of the ongoing debate about the Digital Millennium Copyright Act (DMCA). The system often disproportionately affects small content creators, who may have their videos removed or demonetized due to false claims, even when their use of copyrighted material falls under fair use. One relevant source is the Electronic Frontier Foundation (EFF), which has criticized the DMCA’s takedown system for being overly automated and biased in favor of large media corporations. Their article “Takedown Hall of Shame” provides real-world examples of wrongful takedowns and how they harm online expression.

The chapter touches on the complex issue of content moderation on social media platforms and the legal concerns they face with allowing certain content to remain posted. One thing that particularly stands out to me is the balance social media companies must strike between freedom of expression and legal responsibility. For instance, the fact that sites like 8kun are trying to avoid scrutiny by moving to countries with looser regulations (like Russia) raises some deep concerns about accountability and the role of tech companies in preventing harmful content.

The section on Automated Moderators (bots) made me think about the limitations of AI in content moderation. While bots can quickly filter out spam and flag harmful content, they often struggle with nuance, sarcasm, or cultural context. I’ve seen many cases where platforms like YouTube or Instagram mistakenly flag harmless content while allowing harmful material to slip through. This raises the question: should platforms rely more on AI moderation, or should they invest in more human moderators to review flagged content? While AI can help with efficiency, it still seems far from replacing human judgment completely.

This statement is an appeal to kairos, because it creates a sense of urgency by pointing out the opportunity for change. By connecting this issue to a future event, she is emphasizing that it would be a time to influence policy decisions about these types of issues. By her saying "later this year" and "appoints a...commissioner who will prioritize equity" makes you feel a sense of urgency and like you can do something to change what is happening.

In this statement, the author makes an appeal to pathos. By putting the situation into perspective, stating that some people have a hard time affording gas and food, makes you feel bad or sympathetic of those people, who now have to worry about paying for expensive gas/electric.

This sentence would support ethos because it is using rational explaining, telling you what the PSC is supposed to acting in favor of the people. It is an effective use of ethos because it aligns with the widely accepted principle that the government agencies should protect the people they serve.

this is such an important topic and interesting article

hyperplasia (increase in number) + hypertrophy (increase in size)

This is basically the guideline/goals on asthma treatment procedures, we identify these factors in order to aid the patient.

Nicely put: this is the big issue. Our goal is to nip the inflammation in the bud before it can become chronic.

GET YOUR INFLUENZA SHOTS PEOPLE! It can be a nice introduction to your body without getting the actual disease.

Not a death sentence, but you have to make sure to manage the symptoms and the flare ups.

As opposed to COPD

Brand name only solutions being covered by insurance, nebulizers (the compressor and the associated tubing), masks to ease in the medication... the costs add up.

Also important to note that there is an intersectional element to this concept. Poor people, people who live in cities (think pollution, a lack of ventilation, pests) , minorities, all of this can continually increase chances of being afflicted with asthma. Thus, if a patient falls into the intersections of asthma, it would be wise to screen with proactiveness, before symptoms exacerbate.

REMEMBER REVERSIBLE!! It is not irreversible narrowing like with chronic asthma. The airways will become mucus filled and fibrotic, fibrosis is never reversible, you lose that elasticity and ease of breathing in and out with chronic asthma.

Keaton’s ability to push the boundaries of film as a medium is highlighted here

how visual gags and slapstick were integrated into narrative structures, especially in the work of Chaplin and Keaton. This connection between physical comedy and storytelling is a key aspect of silent film’s appeal.

It highlights the changing audience tastes and perceptions, emphasizing how silent comedies, once hysterically funny, now seem different due to different aesthetic societal dynamics

This shows how today’s audiences find silent film comedy old-fashioned and hard to relate to. It highlights how humor changes over time.

Believes the bank is acting in the interest of foreigners since a large part of the stocks are held by foreigners and the wealthy class

I think that through school as well as our experiences within the professional field, the more comfortable we will all become with learning how to walk the line of providing enough information vs. too much. It is a skill that is learned, no matter how much you read up on it.

Alt text for image to the right: 2023 Miata interior

This aligns with other research on failure training. Because you get diminishing returns when getting closer to muscular failure, it is probably a good idea to get close to but not up to failure to maximize muscle hypertrophy.

Without a agreed upon definition you add a confounding variable.

This is a very in dept research question(s). A lot of articles don't include its own section for the research question.

It sounds crazy but it is true. The more efficient and to the point you are with communication and executing plans the better off you are in the corporate world. The more I have worked at my internship with the limited resources and the high-expected results you have to learn to be scrappy.

Alt text for image above: Dashboard of the 2017 Mazda CX-5 Grand Touring trim level.

>Dynamic: Undergo change throughout the story. > Static: Remain unchanged and often serve specific narrative or thematic purposes.

The techniques an author uses to create and develop characters.

This can help improve an argument so the discussion has multiply perspectives.

The 2024 Mazda CX-50

She is not like other people because of the unique cultural experience of being of color in contrast with the people in her environment up to this point, but we hear how she is still like everybody else as she shares the universal experience of being human like any other person.

The Y symbolizing his connection to Harlem, her African American culture and life. Despite being part of a predominantly white educational institution, he remains tied to his culture down Harlem.

Setting: time and place

They are two separate things don't get these confused.

I can't imagine what a change it was to receive news the moment it happened versus finding out from papers or different publications. This reminds me of the infamous "Where were you when 9/11 happened?" as everyone found out at around the same time because the news was broadcast instantly across the country. It's so strange to think about the fact that news would break late, and then even then, some people found out sooner than others based on how effectively the publication spread. I was thinking about this before as well, when Marconi used to wireless telegraph to announce the winner of a sporting event right as it happened.

It's interesting to hear about this perception of masculinity, especially when comparing it to the modern understanding of masculinity. I don't think that most people today would describe technological proficiency as 'masculine', but it just shows how values have shifted over time. I also believe their talking more about masculinity in reference to the scientific world, but still.

It's so interesting to see how popular media can influence the ideologies and values of an entire population. These viewpoints existed prior to the creation of this media, but it's crazy how broad the impact is.

I think celebrating individual achievement can be beneficial, and this exemplifies one of the reasons why. People can look up to someone accredited with a great achievement, putting a face to an invention that someone admires. It's easier to have one person as a role model, rather than a team of experts.

It's interesting reading this because I feel like nowadays, you are essentially forced to spend a lot of time on screens / online. I feel like we have gone backward in the sense that people crave face-to-face interaction and miss times when we were "tethered" to earthbound discourse.

This specific quote just shows that the grass isn’t always greener on the other side. To migrate there is always disadvantages and doesn’t get easier, but brave enough not knowing what’s to come

eLife Assessment

This valuable paper reports machine learning-based image analysis pipelines for the automated segmentation of micronuclei and the detection and sorting of micronuclei-containing cells. These are powerful new tools for researchers who study micronuclei and their physiologic consequences. The analysis of the new tools and their benchmarking is rigorous and convincing; applications and remaining limitations are well explained in the paper.

Reviewer #1 (Public review):

DiPeso et al. develop two tools to i) classify micronucleated (MN) cells, which they call VCS MN, and ii) segment micronuclei and nuclei with MNFinder. They then use these tools to identify transcriptional changes in MN cells.

The strengths of this study are:

- Developing highly specialized tools to speed up the analysis of specific cellular phenomena such as MN formation and rupture is likely valuable to the community and neglected by developers of more generalist methods.

- A lot of work and ideas have gone into this manuscript. It is clearly a valuable contribution.

- Combining automated analysis, single-cell labeling, and cell sorting is an exciting approach to enrich for phenotypes of interest, which the authors demonstrate here.

The authors addressed my original concerns related to the first version of this manuscript.

Reviewer #2 (Public review):

Summary:

Micronuclei are aberrant nuclear structures frequently seen following the missegregation of chromosomes. The authors present two image analysis methods, one robust and another rapid, to identify micronuclei (MN) bearing cells. To analyse their software efficacy, the authors study images of cells treated with MPS1 inhibitor to induce chromosome missegregation. Next, the authors use RNA-seq to assess the outcomes of their MN-identifying methods: they do not observe a transcriptomic signature specific to MN but find changes that correlate with aneuploidy status. Overall, this work offers new tools to identify MN-presenting cells, and it sets the stage with clear benchmarks for further software development.

Strengths:

Currently, there are no robust MN classifiers with a clear quantification of their efficiency across cell lines (mIoU score). The software presented here tries to address this gap. GitHub material (images, ground truth labels, tools, protocols, etc.) provided is a great asset to computational biologists. The method has been tested in more than one cell line. This method can help integrate cell biology and 'omics' data, making it suitable for multimodal studies.

Weaknesses:

Although the classifier outperforms available tools for MN segmentation by providing mIoU, it's not yet at a point where it can be reliably applied to functional genomics assays where we expect a range of phenotypic penetrance in most cell lines (e.g., misshapen, multinucleated, and lagging DNA in addition to micronucleated cells). The discussion considers the nature and proportion of MN in RPE1 cells, and how the classifier is well-suited for RPE1 that predominantly display MN structures. Whether the classifier can rigorously assign MN-presenting cells amidst drastic nuclear aberrancies following a spindle checkpoint loss needs to be tested in the future.

Reviewer #3 (Public review):

Summary:

The authors develop automated methods to visually identify micronuclei (MN) and MN-containing cells. The authors then use these methods to isolate MN-containing RPE-1 cells post-photoactivation and analyze transcriptional changes in cells with and without micronuclei. The authors find that RPE-1 cells with MN have similar transcriptomic changes as aneuploid cells and that MN rupture does not lead to vast changes in the transcriptome.

Strengths:

The authors develop a method that allows for automating measurements and analysis of micronuclei. This has been something that the field has been missing for a long time. Using such a method has the potential to greatly enhance the field's ability to analyze micronuclei and understand the downstream consequences. The authors also develop a method to identify cells with micronuclei in real-time, mark them using photoconversion, and then isolate them via cell sorting, which could change the way we isolate and study MN-containing cells, and the scale at which we do it. The authors use this method to look at the transcriptome. This method is very powerful as it can allow for the separation of a heterogenous population and subsequent analysis with a much higher sample number than previously possible.

Weaknesses:

The major weakness of this paper is the transcriptomic analysis of MN. There is in general large variance between replicates in experiments looking at cells with ruptured versus intact micronuclei. This limits our ability to assess if lack of changes are due to truly not having changes between these populations or experimental limitations. More transcriptomic analysis will be necessary to fully understand the downstream consequences of MN rupture.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

DiPeso et al. develop two tools to (i) classify micronucleated (MN) cells, which they call VCS MN, and (ii) segment micronuclei and nuclei with MMFinder. They then use these tools to identify transcriptional changes in MN cells.

The strengths of this study are:

(1) Developing highly specialized tools to speed up the analysis of specific cellular phenomena such as MN formation and rupture is likely valuable to the community and neglected by developers of more generalist methods.

(2) A lot of work and ideas have gone into this manuscript. It is clearly a valuable contribution.

(3) Combining automated analysis, single-cell labeling, and cell sorting is an exciting approach to enrich phenotypes of interest, which the authors demonstrate here.

Weaknesses:

(1) Images and ground truth labels are not shared for others to develop potentially better analysis methods.

We regret this omission and thank the reviewer for pointing it out. Both the images and ground truth labels for VCS MN and MNFinder are now available on the lab’s github page and described in the README.txt files. VCS MN: https://github.com/hatch-lab/fast-mn. MNFinder: https://github.com/hatch-lab/mnfinder.

(2) Evaluations of the methods are often not fully explained in the text.

The text has been extensively updated to include a full description of the methods and choices made to develop the VCS MN and MNFinder image segmentation modules.

(3) To my mind, the various metrics used to evaluate VCS MN reveal it not to be terribly reliable. Recall and PPV hover in the 70-80% range except for the PPV for MN+. It is what it is - but do the authors think one has to spend time manually correcting the output or do they suggest one uses it as is?

VCS MN attempts to balance precision and recall with speed to reduce the fraction of MN changing state from intact to ruptured during a single cell cycle during a live-cell isolation experiment. In addition, we chose to prioritize inclusion of small MN adjacent to the nucleus in our positive calls. This meant that there were more false positives (lower PPV) than obtained by other methods but allowed us to include this highly biologically relevant class of MN in our MN+ population. Thus, for a comprehensive understanding of the consequences of MN formation and rupture, we recommend using the finder as is. However, for other visual cell sorting applications where a small number of highly pure MN positive and negative cells is preferred, such as clonal outgrowth or metastasis assays, we would recommend using the slower, but more precise, MNFinder to get a higher precision at a cost of temporal resolution. In addition, MNFinder, with its higher flexibility and object coverage, is recommended for all fixed cell analyses.

Reviewer #2 (Public review):

Summary:

Micronuclei are aberrant nuclear structures frequently seen following the missegregation of chromosomes. The authors present two image analysis methods, one robust and another rapid, to identify micronuclei (MN) bearing cells. The authors induce chromosome missegregation using an MPS1 inhibitor to check their software outcomes. In missegregation-induced cells, the authors do not distinguish cells that have MN from those that have MN with additional segregation defects. The authors use RNAseq to assess the outcomes of their MN-identifying methods: they do not observe a transcriptomic signature specific to MN but find changes that correlate with aneuploidy status. Overall, this work offers new tools to identify MN-presenting cells, and it sets the stage with clear benchmarks for further software development.

Strengths:

Currently, there are no robust MN classifiers with a clear quantification of their efficiency across cell lines (mIoU score). The software presented here tries to address this gap. GitHub material (tools, protocols, etc) provided is a great asset to naive and experienced computational biologists. The method has been tested in more than one cell line. This method can help integrate cell biology and 'omics' studies.

Weaknesses:

Although the classifier outperforms available tools for MN segmentation by providing mIOU, it's not yet at a point where it can be reliably applied to functional genomics assays where we expect a range of phenotypic penetrance.

We agree that the MNFinder module has limitations with regards to the degree of nuclear atypia and cell density that can be tolerated. Based on the recall and PPV values and their consistency across the majority conditions analyzed, we believe that MNFinder can provide reliable results for MN frequency, integrity, shape, and label characteristics in a functional genomics assay in many commonly used adherent cell lines. We also added a discussion of caveats for these analyses, including the facts that highly lobulated nuclei will have higher false positive rates and that high cell confluency may require additional markers to ensure highly accurate assignment of MN to nuclei.

Spindle checkpoint loss (e.g., MPS1 inhibition) is expected to cause a variety of nuclear atypia: misshapen, multinucleated, and micronucleated cells. It may be difficult to obtain a pure MN population following MPS1 inhibitor treatment, as many cells are likely to present MN among multinucleated or misshapen nuclear compartments. Given this situation, the transcriptomic impact of MN is unlikely to be retrieved using this experimental design, but this does not negate the significance of the work. The discussion will have to consider the nature, origin, and proportion of MN/rupture-only states - for example, lagging chromatids and unaligned chromosomes can result in different states of micronuclei and also distinct cell fates.

We appreciate the reviewer’s comments and now quantify the frequency of other nuclear atypias and MN chromosome content in RPE1 cells after 24 h Mps1 inhibition (Fig. S1). In summary, we find only small increases in nuclear atypia, including multinucleate cells, misshapen nuclei, and chromatin bridges, compared to the large increase in MN formation. This contrasts with what is observed when mitosis is delayed using nocodazole or CENPE inhibitors where nuclear atypia is much more frequent. Importantly, after Mps1 inhibition, RPE1 cells with MN were only slightly more likely to have a misshapen nucleus compared to cells without MN (Fig. S1C).

Interestingly, this analysis showed that the VCS MN pipeline, which uses the Deep Retina segmenter to identify nuclei, has a strong bias against lobulated nuclei and frequently fails to find them (Fig. S2B). Therefore, the cell populations analyzed by RNAseq were largely depleted of highly misshapen nuclei and differences in nuclear atypia frequency between MN+ and MN- cells in the starting population were lost (Fig. S9A, compare to Fig. S1C). This strongly suggests that the transcript changes we observed reflect differences in MN frequency and aneuploidy rather than differences in nuclei morphology.

We agree with the reviewer that MN rupture frequency and formation, and downstream effects on cell proliferation and DNA damage, are sensitive to the source of the missegregated chromatin. In the revised manuscript we make clear that we chose Mps1 inhibition because it is strongly biased towards whole chromosome MN (Fig. S1E), limiting signal from DNA damage products, including chromosome fragments and chromatin bridges. This provides a base line to disambiguate the consequences of micronucleation and DNA damage in more complex chromosome missegregation processes, such as DNA replication disruption and irradiation. 

Reviewer #3 (Public review):

Summary:

The authors develop a method to visually analyze micronuclei using automated methods. The authors then use these methods to isolate MN post-photoactivation and analyze transcriptional changes in cells with and without micronuclei of RPE-1 cells. The authors observe in RPE-1 cells that MN-containing cells show similar transcriptomic changes as aneuploidy, and that MN rupture does not lead to vast changes in the transcriptome.

Strengths:

The authors develop a method that allows for automating measurements and analysis of micronuclei. This has been something that the field has been missing for a long time. Using such a method has the potential to advance micronuclei biology. The authors also develop a method to identify cells with micronuclei in real time and mark them using photoconversion and then isolate them via FACS. The authors use this method to study the transcriptome. This method is very powerful as it allows for the sorting of a heterogenous population and subsequent analysis with a much higher sample number than could be previously done.

Weaknesses:

The major weakness of this paper is that the results from the RNA-seq analysis are difficult to interpret as very few changes are found to begin with between cells with MN and cells without. The authors have to use a 1.5-fold cut-off to detect any changes in general. This is most likely due to the sequencing read depth used by the authors. Moreover, there are large variances between replicates in experiments looking at cells with ruptured versus intact micronuclei. This limits our ability to assess if the lack of changes is due to truly not having changes between these populations or experimental limitations. Moreover, the authors use RPE-1 cells which lack cGAS, which may contribute to the lack of changes observed. Thus, it is possible that these results are not consistent with what would occur in primary tissues or just in general in cells with a proficient cGAS/STING pathway.

We agree with the reviewer’s assessment of the limitations of our RNA-Seq analysis. After additional analysis, we propose an alternative explanation for the lower expression changes we observe in the MN+ and Mps1 inhibitor RNA-Seq experiments. In summary, we find that VCS MN has a strong bias against highly lobulated nuclei that depletes this class of cells from both the bulk analysis and the micronucleated cell populations (Fig. S9A). Based on this result, we propose that our analysis reduces the contribution of nuclear atypia to these transcriptional changes and that nuclear morphology changes are likely a signaling trigger associated with aneuploidy.

We believe that this finding strengthens our overall conclusion that MN formation and rupture do not cause transcriptional changes, as suppressing the signaling associated with nuclei atypia should increase sensitivity to changes from the MN. However, we cannot completely rule out that MN formation or rupture cause a broad low-level change in transcription that is obscured by other signals in the dataset.

As to cGAS signaling, several follow up papers and even the initial studies from the Greenburg lab show that MN rupture does not activate cGAS and does not cause cGAS/STING-dependent signaling in the first cell cycle (see citations and discussion in text). Therefore, we expect the absence of cGAS in RPE1 cells will have no effect in the first cell cycle, but could alter the transcriptional profile after mitosis. Although analysis of RPE1  cGAS+ cells or primary cells in these experiments will be required to definitively address this point, we believe that our interpretation of our RNAseq results is sufficiently backed up by the literature to warrant our conclusion that MN formation and rupture do not induce a transcriptional response in the first cell cycle.

Reviewer #1 (Recommendations for the authors):

I do not recommend additional experimental or computational work. Instead, I just recommend adapting the claims of the manuscript to what has been done. I am just asking for further clarification and minor rewriting.

(1) The manuscript is written like a molecular biology paper with sparse explanations of the authors' reasoning, especially in the development of their algorithms. I was often lost as to why they did things in one way or another.

The revised manuscript has thorough explanations and additional data and graphics defining how and why the VCS MN and MNFinder modules were developed. We hope that this clears up many of the questions the reviewer had and appreciate their guidance on making it more readable for scientists from different backgrounds.

(2) Evaluations of their method are often not fully explained, for example:

"On average, 75% of nuclei per field were correctly segmented and cropped."

"MN segments were then assigned to 'parent' nuclei by proximity, which correctly associated 97% of MN."

Were there ground truth images and labels created? How many? For example, I don't know how the authors could even establish a ground-truth for associating MNs to nuclei if MNs happened to be almost equidistant between two nuclei in their images.

I suggest a separate subsection early in the Results section where the underlying imaging data + labels are presented.

We added new sections to the text and figures at the beginning of the VCS MN and MNFinder subsections (Fig. S2 and Fig. S5) with specific information about how ground truth images and labels were generated for both modules and how these were broken up for training, validation, and testing.

We also added information and images to explain how ground truth MN/nucleus associations were derived. In summary, we took advantage of the fact that 2xDendra-NLS is present at low levels in the cytoplasm to identify cell boundaries. This combined with a subconfluent cell population allowed us to unambiguously group MN and nuclei for 98% of MN, we estimate. These identifications were used to generate ground truth labels and analyze how well proximity defines MN/nuclei groups (Fig.s S1 and S2).

(3) Overall, I find the sections long and more subtitles would help me better navigate the manuscript.

Where possible, we have added subtitles.

(4) Everything following "To train the model, H2B channel images were passed to a Deep Retina neural net ..." is fully automated, it seems to me. Thus, there seems to be no human intervention to correct the output before it is used to train the neural network. Therefore, I do not understand why a neural network was trained at all if the pipeline for creating ground truth labels worked fully automatically. At least, the explanations are insufficient.

We apologize for the initial lack of clarity in the text and included additional details in the revision. We used the Deep Retina segmenter to crop the raw images to areas around individual nuclei to accelerate ground truth labeling of MN. A trained user went through each nucleus crop and manually labeled pixels belonging to MN to generate the ground truth dataset for training, validation, and imaging in VCS MN (Fig. S2A).

(5) To my mind, the various metrics used to evaluate VCS MN reveal it not to be terribly reliable. Recall and PPV hover in the 70-80% range except for the PPV for MN+. It is what it is - but do the authors think one has to spend time manually correcting the output or do they suggest one uses it as is? I understand that for bulk transcriptomics, enrichment may be sufficient but for many other questions, where the wrong cell type could contaminate the population, it is not.

Remarks in the Results section on what the various accuracies mean for different applications would be good (so one does not need to wait for the Discussion section).

One of the strengths of the visual cell sorting system is that any image analysis pipeline can be used with it. We used VCS MN for the transcriptomics experiment, but for other applications a user could run visual cell sorting in conjunction with MNFinder for increased purity while maintaining a reasonable recall or use a pre-existing MN segmentation program that gives 100% purity but captures only a specific subgroup of micronucleated cells (e.g. PIQUE). 

To maintain readability, especially with the expansion of the results sections, we kept the discussion of how we envision using visual cell sorting for other MN-based applications in the discussion section.

(6) I am confused about what "cell" is referring to in much of the manuscript. Is it the nucleus + MNs only? Is it the whole cell, which one would ordinarily think it is? If so, are there additional widefield images, where one can discern cell boundaries? I found the section "MNFinder accurately ..." very hard to read and digest for this reason and other ambiguous wording. I suggest the authors take a fresh look at their manuscript and see whether the text can be improved for clarity. I did not find it an easy read overall, especially the computational part.

After re-examining how “cell” was used, we updated the text to limit its use to the MNFinder arm tasked with identifying MN-nucleus associations where the convex hull defined by these objects is used to determine the “cell” boundary. In all other cases we have replaced cell with “nucleus” because, as the reviewer points out, that is what is being analyzed and converted. We hope this is clearer.

(7) Post-FACS PPVs are not that great (Figure 3c). It depends on the question one wants to answer whether ~70% PPV is good enough. Again, would be good to comment on.

We added discussion of this result to the revision. In summary, a likely reason for the reduced PPV is that, although we maintain the cells in buffer with a Cdk1 inhibitor, we know that some proportion of the cells go through mitosis post-sorting. Since MN are frequently reincorporated into the nucleus after mitosis (Hatch et al, 2013; Zhang et al., 2015), we expect this to reduce the MN+ population. Thus, we expect that the PPV in the RNAseq population is higher than what we can measure by analyzing post-sorted cells that have been plated and analyzed later.

(8) I am thoroughly confused as to why the authors claim that their system works in the "absence of genetic perturbations" and why they emphasize the fact that their cells are non-transformed: They still needed a fluorescent label and they induce MNs with a chemical Mps1 inhibitor. (The latter is not a genetic manipulation, of course, but they still need to enrich MNs somehow. That is, their method has not been tested on a cell population in which MNs occur naturally, presumably at a very low rate, unless I missed something.) A more careful description of the benefits of their method would be good.

We apologize for the confusion on these points and hope this is clarified in the revision. We were comparing our system, which can be made using transient transfection, if desired, to current tools that disambiguate aneuploidy and MN formation by deleting parts of chromosomes or engineering double strand breaks with CRISPR to generate single chromosome-specific missegregation events. Most of these systems require transformed cancer cells to obtain high levels of recombination. In contrast, visual cell sorting can isolate micronucleated cells from any cell line that can exogenously express a protein, including primary cells and non-transformed cells like RPE1s.

Other minor points:

(1) The authors should not refer to "H2B channels" but to "H2B-emiRFP703 channels". It may seem obvious to the authors but for someone reading the manuscript for the very first time, it was not. I was not sure whether there were additional imaging modalities used for H2B/nucleus/chromatin detection before I went back and read that only fluorescence images of H2B-emiRFP703 were used. To put it another way, the authors are detecting fluorescence, not histones -- unless I misunderstood something.

To address this point, we altered the text to read “H2B-emiRFP703” when discussing images of this construct. For MNFinder some images were of cells expressing H2B-GFP, which has also been clarified.

(2) If the level of zoom on my screen is such that I can comfortably read the text, I cannot see much in the figure panels. The features that I should be able to see are the size of a title. The image panels should be magnified.

In the revision, the images are appended to the end at full resolution to overcome this difficulty. Thank you for your forbearance.

Reviewer #2 (Recommendations for the authors):

The methods are adequately explained. The Results text narrating experiments and data analysis is clear. Interpretation of a few results could be clarified and strengthened as explained below.

(1) RNAseq experiments are a good proof of principle. To strengthen their interpretation in Figures 4 and 6, I would recommend the authors cite published work on checkpoint/MPS1 loss-induced chromosome missegregation (PMID: 18545697, PMID: 33837239, PMC9559752) and consider in their discussion the 'origin' and 'proportion' of micronucleated cells and irregularly shaped nuclei expected in RPE1 lines. This will help interpret Figure 6 findings on aneuploidy signature accurately. Not being able to see an MN-specific signature could be due to the way the biological specimen is presented with a mixture of cells with 'MN only' or 'rupture' or 'MN along with misshapen nuclei'. These features may all link to aneuploidy rather than 'MN' specifically.

We appreciate the reviewer’s suggestion and added a new analysis of nuclear atypia after Mps1 inhibition in RPE1 cells to Fig. S1. Overall, we found that Mps1 inhibition significantly, but modestly, increased the proportion of misshapen nuclei and chromatin bridges. Multinucleate cells were so rare that instead of giving them their own category we included them in “misshapen nuclei.” These results are consistent with images of Msp1i treated RPE1 cells from He et al. 2019 and Santaguida et al. 2017 and distinct from the stronger changes in nuclear morphology observed after delaying mitosis by nocodazole or CENPE inhibition.

We also found that the Deep Retina segmenter used to identify nuclei in VCS MN had a significant bias against highly lobulated nuclei (Fig. S2B) that led to misshapen nuclei being largely excluded from the RNAseq analyses. As a result we found no enrichment of misshapen nuclei, chromatin bridges, or dead/mitotic nuclear morphologies in MN+ compared to MN- nuclei in our RNASeq experiments (Fig. S9A).

(2) As the authors clarify in the response letter, one round of ML is unlikely to result in fully robust software; additional rounds of ML with other markers will make the work robust. It will be useful to indicate other ML image analysis tools that have improved through such reiterations. They could use reviews on challenges and opportunities using ML approaches to support their statement. Also in the introduction, I would recommend labelling as 'rapid' instead of 'rapid and precise' method.

We updated the text to reference review articles that discuss the benefit of additional training for increasing ML accuracy and changed the text to “rapid.”

(3) The lack of live-cell studies does not allow the authors to distinguish the origin of MN (lagging chromatids or unaligned chromosomes). As explained in 1, considering these aspects in discussion would strengthen their interpretation. Live-cell studies can help reduce the dependencies on proximity maps (Figure S2).

The revised text includes new references and data (Fig. S1E) demonstrating that Mps1 inhibition strongly biases towards whole chromosome missegregation and that MN are most likely to contain a single centromere positive chromosome rather than chromatin fragments or multiple chromosomes.

(4) Mean Intersection over Union (mIOU) is a good measure to compare outcomes against ground truth. However, the mIOU is relatively low (Figure 2D) for HeLa-based functional genomics applications. It will help to discuss mIOU for other classifiers (non-MN classifiers) so that they can be used as a benchmark (this is important since the authors state in their response that they are the first to benchmark an MN classifier). There are publications for mitochondria, cell cortex, spindle, nuclei, etc. where IOU has been discussed.

We added references to classifiers for other small cellular structures. We also evaluated major sources of error in MNFinder found that false negatives are enriched in very small MN (3 to 9 pixels, or about 0.4 µm² – 3 µm², Fig. S6B). A similar result was obtained for VCS MN (Fig. S3B). Because small changes in the number of pixels identified in small objects can have outsized effects on mIoU scores, we suspect that this is exerting downward pressure on the mIoU value. Based on the PPV and recall values we identified, we believe that MNFinder is robust enough to use for functional genomics and screening applications with reasonable sample sizes.

(5) Figure 5 figure legend title is an overinterpretation. MN and rupture-initiated transcriptional changes could not be isolated with this technique where several other missegregation phenotypes are buried (see point 1 above).

We decided to keep the figure title legend based on our analysis of known missegregation phenotypes in Fig. S1 and S9 showing that there is no difference in major classes of nuclear atypia between MN+ and MN- populations in this analysis. Although we cannot rule out that other correlated changes exist, we believe that the title represents the most parsimonious interpretation.

Minor comments

(1) The sentence in the introduction needs clarification and reference. "However, these interventions cause diverse "off-target" nuclear and cellular changes, including chromatin bridges, aneuploidy, and DNA damage." Off-target may not be the correct description since inhibiting MPS1 is expected to cause a variety of problems based on its role as a master kinase in multiple steps of the chromosome segregation process. Consider one of the references in point 1 for a detailed live-cell view of MPS1 inhibitor outcomes.

We have changed “off-target” to “additional” for clarity.

(2) In Figure 3 or S3, did the authors notice any association between the cell cycle phase and MN or rupture presence? Is this possible to consider based on FACS outcomes or nuclear shapes?

Previous work by our lab and others have shown that MN rupture frequency increases during the cell cycle (Hatch et al., 2013; Joo et al., 2023). Whether this is stochastic or regulated by the cell cycle may depend on what chromosome is in the MN (Mammel et al., 2021) and likely the cell line. Unfortunately, the H2B-emiRFP703 fluorescence in our population is too variable to identify cell cycle stage from FACS or nuclear fluorescence analysis.

(3) Figure 5 - Please explain "MA plot".

An MA plot, or log fold-change (M) versus average (A) gene expression, is a way to visualize differently expressed genes between two conditions in an RNASeq experiment and is used as an alternative to volcano plots. We chose them for our paper because most of the expression changes we observed were small and of similar significance and the MA plot spreads out the data compared to a volcano plot and allowed a better visualization of trends across the population.

(4) Page 7: "our results strongly suggest that protein expression changes in MN+ and rupture+ cells are driven mainly by increased aneuploidy rather than cellular sensing of MN formation and rupture.". This is an overstatement considering the mIOU limits of the software tool and the non-exclusive nature of MN in their samples.

We agree that we cannot rule out that an unknown masking effect is inhibiting our ability to observe small broad changes in transcription after MN formation or rupture. However, we believe we have minimized the most likely sources of masking effects, including nuclear atypia and large scale aneuploidy differences, and thus our interpretation is the most likely one.

Reviewer #3 (Recommendations for the authors):

Overall, the authors need to explain their methods better, define some technical terms used, and more thoroughly explain the parameters and rationale used when implementing these two protocols for identifying micronuclei; primarily as this is geared toward a more general audience that does not necessarily work with machine learning algorithms.

(1) A clearer description in the methods as to how accuracy was calculated. Were micronuclei counted by hand or another method to assess accuracy?

We significantly expanded the section on how the machine learning models were trained and tested, including how sensitivity and specificity metrics were calculated, in both the results and the methods sections. The code used to compare ground truth labels to computed masks is also now included in the MNFinder module available on the lab github page. 

(2) Define positive predictive value.

The text now says “the positive predictive value (PPV, the proportion of true positives, i.e. specificity) and recall (the proportion of MN found by the classifier, i.e. sensitivity)…”.

(3) Why is it a problem to use the VCS MN at higher magnifications where undersegmentation occurs? What do the authors mean by diminished performance (what metrics are they using for this?).

We have included a representative image and calculated mIoU and recall for 40x magnification images analyzed by MNFinder after rescaling in Fig. 2A. In summary, VCS MN only correctly labeled a few pixels in the MN, which was sufficient to call the adjacent nucleus “MN+” but not sufficient for other applications, such as quantifying MN area. In addition, VCS MN did much worse at identifying all the MN in 40x images with a recall, or sensitivity, metric of 0.36. We are not sure why. Developing MNFinder provided a module that was well suited to quantify MN characteristics in fixed cell images, an important use case in MN biology.

(4) The authors should compare MN that are analyzed and not analyzed using these methods and define parameters. Is there a size limitation? Closeness to the main nucleus?

We added two new figures defining what contributes to module error for both VCS MN (Fig. S3) and MNFinder (Fig. S6). For VCS MN, false negatives are enriched in very large or very small MN and tend to be dimmer and farther from the nucleus than true positives. False positives are largely misclassification of small dim objects in the image as MN. For MNFinder, the most missed class of MN are very small ones (3-9 px in area) and the majority of false positives are misclassifications of elongated nuclear blebs as MN.

(5) Are there parameters in how confluent an image must be to correctly define that the micronucleus belongs to the correct cell? The authors discussed that this was calculated based on predicted distance. However, many factors might affect proper calling on MN. And the authors should test this by staining for a cytosolic marker and calculating accuracy.

We updated the text with more information about how the cytoplasm was defined using leaky 2x-Dendra2-NLS signal to analyze the accuracy of MN/nucleus associations (Fig. S2G-H). In addition, we quantified cell confluency and distance to the first and second nearest neighbor for each MN in our training and testing image datasets. We found that, as anticipated, cells were imaged at subconfluent concentrations with most fields having a confluency around 30% cell coverage (Fig. S2E) and that the average difference in distance between the closest nucleus to an MN and the next closest nucleus was 3.3 fold (Fig. S2F). We edited the discussion section to state that the ability of MN/nuclear proximity to predict associations at high cell confluencies would have to be experimentally validated.

(6) The authors measure the ratio of Dendra2(Red) v. Dendra2 (Green) in Figure 3B to demonstrate that photoconversion is stable. This measurement, to me, is confusing, as in the end, the authors need to show that they have a robust conversion signal and are able to isolate these data. The authors should directly demonstrate that the Red signal remains by analyzing the percent of the Red signal compared to time point 0 for individual cells.

We found a bulk analysis to be more powerful than trying to reidentify individual cells due to how much RPE1 cells move during the 4 and 8 hours between image acquisitions. In addition, we sort on the ratio between red and green fluorescence per cell, rather than the absolute fluorescence, to compensate for variation in 2xDendra-NLS protein expression between cells. Therefore, demonstrating that distinct ratios remained present throughout the time course is the most relevant to the downstream analysis.

To address the reviewer’s concern, we replotted the data in Fig. 3B to highlight changes over time in the raw levels of red and green Dendra fluorescence (Fig. S7D). As expected, we see an overall decrease in red fluorescence intensity, and complementary increase in green fluorescence intensity, over 8 hours, likely due to protein turnover. We also observe an increase in the number of nuclei lacking red fluorescence. This is expected since the well was only partially converted and we expect significant numbers of unconverted cells to move into the field between the first image and the 8 hour image.

(7) The authors isolate and subsequently use RNA-sequencing to identify changes between Mps1i and DMSO-treated cells. One concern is that even with the less stringent cut-off of 1.5 fold there is a very small change between DMSO and MPS1i treated cells, with only 63 genes changing, none of which were affected above a 2-fold change. The authors should carefully address this, including why their dataset sees changes in many more pathways than in the He et al. and Santaguida et al. studies. Is this due to just having a decreased cut-off?

The reviewer correctly points out that we observed an overall reduction in the strength of gene expression changes between our dataset of DMSO versus Mps1i treated RPE1 cells compared to similar studies. We suggest a couple reasons for this. One is that the log₂ fold changes observed in the other studies are not huge and vary between 2.5 and -3.8 for He et al., 3.3 and -2.3 for Santaguida et al., and -0.8 and 1.6 for our study. This variability is within a reasonable range for different experimental conditions and library prep protocols. A second is that our protocol minimizes a potential source of transcriptional change – nuclear lobulation – that is present in the other datasets.

For the pathway analysis we did not use a fold-change cut-off for any data set, instead opting to include all the genes found to be significantly different between control and Mps1i treated cells for all three studies. Our read-depth was higher than that of the two published experiments, which could contribute to an increased DEG number. However, we hypothesize that our identification of a broader number of altered pathways most likely arises from increased sensitivity due to the loss of covering signal from transcriptional changes associated with increased nuclear atypia. Additional visual cell sorting experiments sorting on misshapen nuclei instead of MN would allow us to determine the accuracy of this hypothesis.

(8) Moreover, clustering (in Figure 5E) of the replicates is a bit worrisome as the variances are large and therefore it is unclear if, with such large variance and low screening depth, one can really make such a strong conclusion that there are no changes. The authors should prove that their conclusion that rupture does not lead to large transcriptional changes, is not due to the limitations of their experimental design.

We agree with the reviewers that additional rounds of RNAseq would improve the accuracy of our transcriptomic analysis and could uncover additional DEGs. However, we believe the overall conclusion to be correct based on the results of our attempt to validate changes in gene expression by immunofluorescence. We analyzed two of the most highly upregulated genes in the ruptured MN dataset, ATF3 and EGR1. Although we saw a statistically significant increase in ATF3 intensity between cells without MN and those with ruptured MN, the fold change was so small compared to our positive control (100x less) that we believe it is it is more consistent with a small increase in the probability of aneuploidy rather than a specific signature of MN rupture.

(9) The authors also need to address the fact that they are using RPE-1 cells more clearly and that the lack of effect in transcriptional changes may be simply due to the loss of cGAS-STING pathway (Mackenzie et al., 2017; Harding et al., 2017; etc.).

As we discuss above in the public comments section, the literature is clear that MN do not activate cGAS in the first cell cycle after their formation, even upon rupture. Therefore, we do not expect any changes in our results when applied to cGAS-competent cells. However, this expectation needs to be experimentally validated, which we plan to address in upcoming work.

ayahuasca as a serpent originating

eLife Assessment

This valuable study introduces a new method for detecting RNA modification. Since it does not rely on chemical modification of RNA, which often results in RNA degradation and therefore loss of RNA molecules, it complements other approaches for detecting RNA modification, and it might be of particular interest for sites where modifications are found in only a minority of interrogated molecules. The information provided is incomplete, however, to allow for comparison with other methods, since there is uncertainty regarding false positive and false negative rates.

Reviewer #2 (Public review):

The fledgling field of epitranscriptomics has encountered various technical roadblocks with implications as to the validity of early epitranscriptomics mapping data. As a prime example, the low specificity of (supposedly) modification-specific antibodies for the enrichment of modified RNAs, has been ignored for quite some time and is only now recognized for its dismal reproducibility (between different labs), which necessitates the development of alternative methods for modification detection. Furthermore, early attempts to map individual epitranscriptomes using sequencing-based techniques are largely characterized by the deliberate avoidance of orthogonal approaches aimed at confirming the existence of RNA modifications that have been originally identified.

Improved methodology, the inclusion of various controls, and better mapping algorithms as well as the application of robust statistics for the identification of false-positive RNA modification calls have allowed revisiting original (seminal) publications whose early mapping data allowed making hyperbolic claims about the number, localization and importance of RNA modifications, especially in mRNA. Besides the existence of m6A in mRNA, the detectable incidence of RNA modifications in mRNAs has drastically dropped.

As for m5C, the subject of the manuscript submitted by Zhou et al., its identification in mRNA goes back to Squires et al., 2012 reporting on >10.000 sites in mRNA of a human cancer cell line, followed by intermittent findings reporting on pretty much every number between 0 to > 100.000 m5C sites in different human cell-derived mRNA transcriptomes. The reason for such discrepancy is most likely of a technical nature. Importantly, all studies reporting on actual transcript numbers that were m5C-modified relied on RNA bisulfite sequencing, an NGS-based method, that can discriminate between methylated and non-methylated Cs after chemical deamination of C but not m5C. RNA bisulfite sequencing has a notoriously high background due to deamination artifacts, which occur largely due to incomplete denaturation of double-stranded regions (denaturing-resistant) of RNA molecules. Furthermore, m5C sites in mRNAs have now been mapped to regions that have not only sequence identity but also structural features of tRNAs. Various studies revealed that the highly conserved m5C RNA methyltransferases NSUN2 and NSUN6 do not only accept tRNAs but also other RNAs (including mRNAs) as methylation substrates, which in combination account for most of the RNA bisulfite-mapped m5C sites in human mRNA transcriptomes. Is m5C in mRNA only a result of the Star activity of tRNA or rRNA modification enzymes, or is their low stoichiometry biologically relevant?

In light of the short-comings of existing tools to robustly determine m5C in transcriptomes, other methods, like DRAM-seq, aiming to map m5C independently of ex situ RNA treatment with chemicals, are needed to arrive at a more solid "ground state", from which it will be possible to state and test various hypotheses as to the biological function of m5C, especially in lowly abundant RNAs such as mRNA.

Importantly, the identification of >10.000 sites containing m5C increases through DRAM-Seq, increases the number of potential m5C marks in human cancer cells from a couple of 100 (after rigorous post-hoc analysis of RNA bisulfite sequencing data) by orders of magnitude. This begs the question, whether or not the application of these editing tools results in editing artefacts overstating the number of actual m5C sites in the human cancer transcriptome.

Author response:

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

Reviewer #2:

(1) The use of two m⁵C reader proteins is likely a reason for the high number of edits introduced by the DRAM-Seq method. Both ALYREF and YBX1 are ubiquitous proteins with multiple roles in RNA metabolism including splicing and mRNA export. It is reasonable to assume that both ALYREF and YBX1 bind to many mRNAs that do not contain m⁵C.

To substantiate the author's claim that ALYREF or YBX1 binds m⁵C-modified RNAs to an extent that would allow distinguishing its binding to non-modified RNAs from binding to m⁵C-modified RNAs, it would be recommended to provide data on the affinity of these, supposedly proven, m⁵C readers to non-modified versus m⁵C-modified RNAs. To do so, this reviewer suggests performing experiments as described in Slama et al., 2020 (doi: 10.1016/j.ymeth.2018.10.020). However, using dot blots like in so many published studies to show modification of a specific antibody or protein binding, is insufficient as an argument because no antibody, nor protein, encounters nanograms to micrograms of a specific RNA identity in a cell. This issue remains a major caveat in all studies using so-called RNA modification reader proteins as bait for detecting RNA modifications in epitranscriptomics research. It becomes a pertinent problem if used as a platform for base editing similar to the work presented in this manuscript.

The authors have tried to address the point made by this reviewer. However, rather than performing an experiment with recombinant ALYREF-fusions and m⁵C-modified to unmodified RNA oligos for testing the enrichment factor of ALYREF in vitro, the authors resorted to citing two manuscripts. One manuscript is cited by everybody when it comes to ALYREF as m⁵C reader, however none of the experiments have been repeated by another laboratory. The other manuscript is reporting on YBX1 binding to m⁵C-containing RNA and mentions PAR-CLiP experiments with ALYREF, the details of which are nowhere to be found in doi: 10.1038/s41556-019-0361-y.<br /> Furthermore, the authors have added RNA pull-down assays that should substitute for the requested experiments. Interestingly, Figure S1E shows that ALYREF binds equally well to unmodified and m⁵C-modified RNA oligos, which contradicts doi:10.1038/cr.2017.55, and supports the conclusion that wild-type ALYREF is not specific m⁵C binder. The necessity of including always an overexpression of ALYREF-mut in parallel DRAM experiments, makes the developed method better controlled but not easy to handle (expression differences of the plasmid-driven proteins etc.)

Thank you for pointing this out. First, we would like to correct our previous response: the binding ability of ALYREF to m⁵C-modified RNA was initially reported in doi: 10.1038/cr.2017.55, (and not in doi: 10.1038/s41556-019-0361-y), where it was observed through PAR-CLIP analysis that the K171 mutation weakens its binding affinity to m⁵C -modified RNA.

Our previous experimental approach was not optimal: the protein concentration in the INPUT group was too high, leading to overexposure in the experimental group. Additionally, we did not conduct a quantitative analysis of the results at that time. In response to your suggestion, we performed RNA pull-down experiments with YBX1 and ALYREF, rather than with the pan-DRAM protein, to better validate and reproduce the previously reported findings. Our quantitative analysis revealed that both ALYREF and YBX1 exhibit a stronger affinity for m⁵C -modified RNAs. Furthermore, mutating the key amino acids involved in m⁵C recognition significantly reduced the binding affinity of both readers. These results align with previous studies (doi: 10.1038/cr.2017.55 and doi: 10.1038/s41556-019-0361-y), confirming that ALYREF and YBX1 are specific readers of m⁵C -modified RNAs. However, our detection system has certain limitations. Despite mutating the critical amino acids, both readers retained a weak binding affinity for m⁵C, suggesting that while the mutation helps reduce false positives, it is still challenging to precisely map the distribution of m⁵C modifications. To address this, we plan to further investigate the protein structure and function to obtain a more accurate m⁵C sequencing of the transcriptome in future studies. Accordingly, we have updated our results and conclusions in lines 294-299 and discuss these limitations in lines 109-114.

In addition, while the m⁵C assay can be performed using only the DRAM system alone, comparing it with the DRAM^mutC control enhances the accuracy of m⁵C region detection. To minimize the variations in transfection efficiency across experimental groups, it is recommended to use the same batch of transfections. This approach not only ensures more consistent results but also improve the standardization of the DRAM assay, as discussed in the section added on line 308-312.

(2) Using sodium arsenite treatment of cells as a means to change the m⁵C status of transcripts through the downregulation of the two major m⁵C writer proteins NSUN2 and NSUN6 is problematic and the conclusions from these experiments are not warranted. Sodium arsenite is a chemical that poisons every protein containing thiol groups. Not only do NSUN proteins contain cysteines but also the base editor fusion proteins. Arsenite will inactivate these proteins, hence the editing frequency will drop, as observed in the experiments shown in Figure 5, which the authors explain with fewer m⁵C sites to be detected by the fusion proteins.

The authors have not addressed the point made by this reviewer. Instead the authors state that they have not addressed that possibility. They claim that they have revised the results section, but this reviewer can only see the point raised in the conclusions. An experiment would have been to purify base editors via the HA tag and then perform some kind of binding/editing assay in vitro before and after arsenite treatment of cells.

We appreciate the reviewer’s insightful comment. We fully agree with the concern raised. In the original manuscript, our intention was to use sodium arsenite treatment to downregulate NSUN mediated m⁵C levels and subsequently decrease DRAM editing efficiency, with the aim of monitoring m⁵C dynamics through the DRAM system. However, as the reviewer pointed out, sodium arsenite may inactivate both NSUN proteins and the base editor fusion proteins, and any such inactivation would likely result in a reduced DRAM editing. This confounds the interpretation of our experimental data.

As demonstrated in Appendix A, western blot analysis confirmed that sodium arsenite indeed decreased the expression of fusion proteins. In addition, we attempted in vitro fusion protein purification using multiple fusion tags (HIS, GST, HA, MBP) for DRAM fusion protein expression, but unfortunately, we were unable to obtain purified proteins. However, using the Promega TNT T7 Rapid Coupled In Vitro Transcription/Translation Kit, we successfully purified the DRAM protein (Appendix B). Despite this success, subsequent in vitro deamination experiments did not yield the expected mutation results (Appendix C), indicating that further optimization is required. This issue is further discussed in line 314-315.

Taken together, the above evidence supports that the experiment of sodium arsenite treatment was confusing and we determined to remove the corresponding results from the main text of the revised manuscript.

Author response image 1.

(3) The authors should move high-confidence editing site data contained in Supplementary Tables 2 and 3 into one of the main Figures to substantiate what is discussed in Figure 4A. However, the data needs to be visualized in another way then excel format. Furthermore, Supplementary Table 2 does not contain a description of the columns, while Supplementary Table 3 contains a single row with letters and numbers.

The authors have not addressed the point made by this reviewer. Figure 3F shows the screening process for DRAM-seq assays and principles for screening high-confidence genes rather than the data contained in Supplementary Tables 2 and 3 of the former version of this manuscript.

Thank you for your valuable suggestion. We have visualized the data from Supplementary Tables 2 and 3 in Figure 4A as a circlize diagram (described in lines 213-216), illustrating the distribution of mutation sites detected by the DRAM system across each chromosome. Additionally, to improve the presentation and clarity of the data, we have revised Supplementary Tables 2 and 3 by adding column descriptions, merging the DRAM-ABE and DRAM-CBE sites, and including overlapping m⁵C genes from previous datasets.

Alt text for image below (for the image of the garage): Seacoast Mazda Service bays.

Alt text for image below (For the image of the silver and red car): Seacoast Mazda dealership lobby.

This is tricky because most of the time there is an entire team responsible for scientific development. I think it is natural to want to benefit from something that you worked hard on, even if the focus of the BAAS was scientific advancement rather than individual achievement. It's a shame that it is usually one person regarded for the work of many, but I don't think that this means that people should not receive credit for their work. Although, i can definitely see why the BAAS was not to fond of Marconi, as he went against what they stood for.

This seems to be the unique approach to presenting the technology that gave him his notoriety. Marconi was a flashy fellow, and it helped his legacy.

I feel like Marconi was a demonstration of how the public usually needs an individual to attribute an invention to. Even though it sounds like they often credited the team of people, it's easier to remember an individual.

It's interesting that despite the inventions that make the inventor 'great' being created in the inventor's lifetime, it's not until after the inventor's time has passed that they are hailed as such.

When comparing the Mazda MX-5 trim levels, you’ll notice everything from small, subtle differences to major upgrades—Mazda does it all with the Club.

Originally established in 1673 as a French trading outpost Chandernagore was a key french colony that was taken over by the british in 1794 but later returned to france in 1816. https://chandernagoremunicipalcorporation.in/About?id=About%20Us-History%20Of%20CMC#:~:text=Chandannagar%20was%20established%20as%20a,appointed%20governor%20of%20the%20city.

This war was triggered by the Death of King Charles VI. France opposed Maria Theresa's claim to the throne which was unsuccessful and later set the stage for the seven year war. https://www.britannica.com/biography/Maria-Theresa

goood

I talk about this in my current conclusion, but can now add citations

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ALL EXAMPLES OF TEST ANXIETY, CONNECTS TO PREVIOUS SOURCES

never thought about it this way, another good quote and example

easy quote to paraphrase

in other words, parents attitude torwards education is hugely important, and generally schooling can't do anything to change that.

explanation, and good quote to use.

standardized testing is good at testing groups of the same people.

Great idea.

HUGE GAP TO CLEAR FOR NON-NATIVE ENGLISH SPEAKERS

most important?

I agree with that statement. Identifying the core problem you're trying to solve is crucial because it ensures you're addressing something that truly matters to your target audience

How the polemers target to the focal tissue？

This can also be the literary device of symbolism of transformation of someone's mindset and consciousness.

Visual imagination to give an image of hope for the future.

The end rhyme with satisfied.

She critiques the unchecked power of men in marriage and society, drawing a parallel between male authority and the tyranny the colonies were resisting from Britain.

Abigail is making an early feminist argument, urging John to consider women's rights as the new government is being formed.

I had never thought about how what clothing affects any given scene within TGG the fact that the interactions change between Gatsby and Daisy based on how they dress. the difference in how the pink suits and military uniform affected the mood of the scene and there respective outcome.

This is something I have learned from this article. Creating a plan with a family is not something I have done before. This is piece of information I wish I had long ago. In my experience when relaying information to parents they just listen and move on.

This to me would feel like conflict and I would naturally shy away from it. This is a practice I want/need to start trying because I have good intentions and it would be a benefit to the child's growth and development.

Relationship building is ongoing work. This is something I practice but something I could try in different ways with parents that I find hard to reach. As first impressions can mean a lot when caring for someones child, even meeting them for the first time is a crucial time in the start of that parent/ teacher relationship.

eLife Assessment

This important study shows how genetic variation is associated with fecundity following a period of reproductive diapause in female Drosophila. The work identifies the olfactory system as central to successful diapause with associated changes in longevity and fecundity. While the methods used are convincing, a limitation of the study, as of any other laboratory-based investigation is the challenge of demonstrating how well measures for fitness related to diapause and its recovery correlates with realities encountered during development in the wild.

Reviewer #1 (Public review):

Summary:

The paper begins with phenotyping the DGRP for post-diapause fecundity, which is used to map genes and variants associated with fecundity. There are overlaps with genes mapped in other studies and also functional enrichment of pathways including most surprisingly neuronal pathways. This somewhat explains the strong overlap with traits such as olfactory behaviors and circadian rhythm. The authors then go on to test genes by knocking them down effectively at 10 degrees. Two genes, Dip-gamma and sbb are identified as significantly associated with post-diapause fecundity, which they also find the effects to be specific to neurons. They further show that the neurons in the antenna but not arista are required for the effects of Dip-gamma and sbb. They show that removing antenna has a diapause specific lifespan extending effect, which is quite interesting. Finally, ionotropic receptor neurons are shown to be required for the diapause associated effects.

Strengths:

Overall I find the experiments rigorously done and interpretations sound. I have no further suggestions except an ANOVA to estimate heritability of the post-diapause fecundity trait, which is routinely done in the DGRP and offers a global parameter regarding how reliable phenotyping is.

Weaknesses:

A minor point is I cannot find how many DGRP lines are used.

Author response:

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

Reviewer #1 (Public Review): 

Summary: 

The paper begins with phenotyping the DGRP for post-diapause fecundity, which is used to map genes and variants associated with fecundity. There are overlaps with genes mapped in other studies and also functional enrichment of pathways including most surprisingly neuronal pathways. This somewhat explains the strong overlap with traits such as olfactory behaviors and circadian rhythm. The authors then go on to test genes by knocking them down effectively at 10 degrees. Two genes, Dip-gamma and sbb, are identified as significantly associated with post-diapause fecundity, and they also find the effects to be specific to neurons. They further show that the neurons in the antenna but not the arista are required for the effects of Dip-gamma and sbb. They show that removing the antenna has a diapause-specific lifespan-extending effect, which is quite interesting. Finally, ionotropic receptor neurons are shown to be required for the diapause-associated effects. 

Strengths and Weaknesses: 

Overall I find the experiments rigorously done and interpretations sound. I have no further suggestions except an ANOVA to estimate the heritability of the post-diapause fecundity trait, which is routinely done in the DGRP and offers a global parameter regarding how reliable phenotyping is. 

We added to the Methods: “We performed a one-way ANOVA to get the mean squares for between-group and withingroup variances and calculated broad-sense heritability using the formula: H² = MS_G - MS_E / MS_G + (k-1) MS_E where MS_G - Mean square between groups and MS_G - Mean square within groups and k - Number of individuals per group. Using this formula, the broad-sense heritability for normalized post-diapause fecundity was found to be 0.51.” 

We added to the Results: “The broad-sense heritability for normalized post-diapause fecundity was found to be 0.51 (see Methods).”

A minor point is I cannot find how many DGRP lines are used. 

Response: We screened 193 lines and have added that to the Results. 

Reviewer #2 (Public Review):

Summary

In this study, Easwaran and Montell investigated the molecular, cellular, and genetic basis of adult reproductive diapause in Drosophila using the Drosophila Genetic Reference Panel (DGRP). Their GWAS revealed genes associated with variation in post-diapause fecundity across the DGRP and performed RNAi screens on these candidate genes. They also analyzed the functional implications of these genes, highlighting the role of genes involved in neural and germline development. In addition, in conjunction with other GWAS results, they noted the importance of the olfactory system within the nervous system, which was supported by genetic experiments. Overall, their solid research uncovered new aspects of adult diapause regulation and provided a useful reference for future studies in this field.

Strengths:

The authors used whole-genome sequenced DGRP to identify genes and regulatory mechanisms involved in adult diapause. The first Drosophila GWAS of diapause successfully uncovered many QTL underlying post-diapause fecundity variations across DGRP lines. Gene network analysis and comparative GWAS led them to reveal a key role for the olfactory system in diapause lifespan extension and post-diapause fecundity.

Comments on revised version:

While the authors have addressed many of the minor concerns raised by the reviewers, they have not fully resolved some of the key criticisms. Notably, two reviewers highlighted significant concerns regarding the phenotype and assay of post-diapause fecundity, which are critical to the study. The authors acknowledged that this assay could be confounded by the 'cold temperature endurance phenotype,' potentially altering the interpretation of their results.

However, they responded by stating that it is not obvious how to separate these effects experimentally. This leaves the analysis in this research ambiguous, as also noted by Reviewer #3.

We should have clarified earlier that we actually chose to measure post-diapause fecundity in order to minimize any impact of ‘cold temperature endurance.” In fact, we chose post-diapause fecundity as the appropriate measure of successful diapause for both technical and conceptual reasons. Conceptually, the benefit of diapause is to perpetuate the species. It seems obvious to us that post-diapause fecundity is more relevant to species propagation than other measures of diapause such as how many egg chambers contain yolk or how many eggs are laid. Technically, we chose 5-week diapause and recovery based on pilot studies that showed that nearly all DGRP lines showed excellent survival at 5 weeks in diapause conditions. Therefore, our experimental design minimized as much as possible any effect of cold temperature endurance - in the sense of the ability to survive at 10°C - on our phenotype. 

We apologize for not clarifying that point earlier and have added this text to the Results: “We chose 5 weeks based on pilot studies that showed that nearly all DGRP lines showed excellent survival at 5 weeks in diapause conditions while exhibiting sufficient variation in post-diapause fecundity to carry out GWAS. Beyond 5 weeks, fecundity was low and there was insufficient variation to conduct a GWAS.”

Additionally, I raised concerns about the validity of prioritizing genes with multiple associated variants. Although the authors agreed with this point, they did not revise the manuscript accordingly. The statement that 'Genes with multiple SNPs are good candidates for influencing diapause traits' is not a valid argument within the context of population and quantitative genetics.

We apologize for neglecting to revise the manuscript accordingly. We have revised Supplemental Table: S4 and ranked the genes by p-value.

It’s interesting how the term "spam" originated from a Monty Python sketch, where the word was repeated annoyingly—much like how actual spam messages flood inboxes today. The fact that spamming remains economically viable because of low costs to senders, while the burden falls on the public and service providers, highlights an ethical issue.

Hao’s article provides a deep dive into how Facebook’s internal incentives and algorithmic tweaks inadvertently fueled the spread of misinformation. By examining the platform’s structural issues, the piece highlights the ethical challenges that arise when engagement metrics drive content amplification, often at the cost of truth and public trust. This analysis is a crucial resource for understanding the broader implications of tech design on societal discourse, prompting us to question how platforms can be re-engineered to prioritize accurate information over sensationalism.

This idea of moderation as a key ethical principle makes a lot of sense, especially in how it applies to real life. Whether in decision-making, relationships, or even personal habits, extremes tend to cause instability, while balance leads to sustainability. It’s interesting to think about how this concept shows up across different cultures and philosophies, reinforcing the idea that moderation is not just a moral principle but a practical one for living well.

This idea is both provocative and timely, as it challenges the conventional power structures behind content moderation on social media. It suggests that if marginalized communities had an equal voice in shaping the rules, moderation practices might better protect against systemic bias and ensure fairer representation of diverse perspectives. This prompts us to reconsider who truly benefits from current moderation policies and how they might evolve to foster a more inclusive digital public sphere.

Janie wants to meet someone to be married with

The "Guinean culture and traditions" must be italicized for proper APA format referencing.

To correct: Cultural Insight Website. (n.d). Guinean culture and traditions. Retrieved from www.culturalinsight.com.

Here he shows how beautiful good memories are and how even though he lost people no one can take away those good memories

The lilac that he loves making an appearance in this poem during this terrible battle represents that this pain will stop as Spring returns each year

Here he paints a depressing picture. The fact he said suffer'd many times shows how depressing this battle was. There is no beauty just suffering

This shows how he enjoys the birds song as he paints a relaxing picture. Perhaps the bird reminds him that Spring is coming

Here he describes the beauty and changes of the day and night

Here he describes the beauty of these places which represent how happy things were before this passing

Here he paints a very dark and depressing picture of the coffin. It is not as colorful as the other parts of this poem hinting that this makes him sad. The fact that he gives the lilac to the coffin represents maybe Spring ending as before he talked about how Spring and that flower always returning but now, he gives that flower to the coffin. Also, the fact he loves that flower, and it is dead makes this part even sadder to imagine.

You can tell that Spring is a season he enjoys based on how beautifully he describes this area in this season. He describes it very colorfully which clues at the fact this season and place makes him happy.

Here Whitman is showing the beauty of this flower he loves in his writing. Not only does he describe how it looks but he describes the strong scent it smells of which romancizes it further.

Here it romantizes night. It shows the beauty of it's darkness and paints the dark and moody vibes of night.

This is romanizing Spring and showing it's beauty. It also shows how Spring comes back and is something to look forward too.

Author Response:

Reviewer #1 (Public Review):

[...] Strengths: This study utilized multiple in vitro approaches, such as proteomics, siRNA, and overexpression, to demonstrate that PCBP2 is an intrinsic factor of BMSC aging.

Weaknesses:

This study did not perform in vivo experiments.

Response: We will continue to conduct animal experiments in subsequent studies.

Reviewer #2 (Public Review):

[...] Weaknesses: It is unclear if PCBP2 can also function as an intrinsic factor for BMSC cells in female individuals. More work may be needed to further dissect the mechanism of how PCBP2 impacts FGF2 expression. Could PCBP2 impact the FGF2 expression independent of ROS?

Response: Thank you very much for your valuable comments, which is also the focus of our follow-up work. We will sort out the data and publish the relevant research results as soon as possible.

Additional context that would help readers interpret or understand the significance of the work: In the current work, the authors studied the aging process of BMSC cells, which are related to osteoporosis. Aging processes also impact many other cell types and their function, such as in muscle, skin, and the brain.

Response: Thank you very much for your valuable comments, we will continue to improve the writing logic of the article to make the article more understandable.

yes!! spending time and deciding carefully

eLife Assessment

This useful manuscript reports mechanisms behind the increase in fecundity in response to sub-lethal doses of pesticides in the crop pest, the brown plant hopper. The authors hypothesize that the pesticide works by inducing the JH titer, which through the JH signaling pathway induces egg development. Evidence for this is, however, incomplete.

Reviewer #1 (Public review):

Summary:

Gao et al. has demonstrated that the the pesticide emamectin benzoate (EB) treatment of brown plathopper (BPH) leads to increased egg laying in the insect, which is a common agricultural pest. The authors hypothesize that EB upregulates JH titer resulting in increased fecundity.

Strengths:

The finding that a class of pesticide increases fecundity of brown planthopper is interesting.

Weaknesses:

(1) EB is an allosteric modulator of GluCl. That means it EB physically interacts with GluCl initiating a structural change in the cannel protein. Yet the authors here central hypothesis is about how EB can upregulate the mRNA of GluCl. I do not know whether there is any evidence that an allosteric modulator can function as a transcriptional activator for the same receptor protein. The basic premise of the paper sounds counterintuitive. This is a structural problem and should be addressed by the authors by giving sufficient evidence about such demonstrated mechanisms before.<br /> (2) I am surprised to see a 4th instar larval application or treatment with EB results in upregulation of JH in the adult stages. Complicating the results further is the observation that a 4th instar EB application results in an immediate decrease in JH titer. There is a high possibility that this late JH titer increase is an indirect effect.<br /> (3) The writing quality of the paper needs improvement. Particularly with respect to describing processes, and abbreviations. In several instances authors have not adequately described the processes they have introduced, thus confusing the readers.<br /> (4) In the section 'EB promotes ovarian development' the authors have shown that EB treatment results in increased detention of eggs which contradicts their own results which show that EB promotes egg laying. Again, this is a serious contradiction that nullifies their hypothesis.<br /> (5) Furthermore, the results suggest that oogenesis is not affected by EB application. The authors should devote a section to discussing how they are observing increased egg numbers in EB-treated insects while not impacting Oogenesis.<br /> (6) Met is the receptor of JH and to my understanding, remains mostly constant in terms of its mRNA or protein levels throughout various developmental periods in many different insects. Therefore, the presence of JH becomes the major driving factor for physiological events and not the presence of the receptor Met. Here the authors have demonstrated an increase in Met mRNA as a result of EB treatment. Their central hypothesis is that EB increases JH titer to result in enhanced fecundity. JH action will not result in the activation of Met. Although not contradictory to the hypothesis, the increase in mRNA content of Met is contrary to the findings of the JH field thus far.<br /> (7) As pointed out before, it is hard to rationalize how a 4th instar exposure to EB can result in upregulation of key genes involved in JH synthesis at the adult stage. The authors must consider providing a plausible explanation and discussion in this regard.<br /> (8) I have strong reservations against such an irrational hypothesis that Met (the receptor for JH) and JH-Met target gene Kr-h1 regulates JH titer (Line 311, Fig 3 supplemental 2D). This would be the first report of such an event on the JH field and therefore must be analysed to depth before it may go to publication. I strongly suggest the authors remove such claims from the manuscript without substantiating it.<br /> (9) Kr-h1 is JH/Met target gene. The authors demonstrate that silencing of Kr-h1 results in inhibition of FAMeT, which is a gene involved in JH synthesis. The feedback loop in JH synthesis is unreported. Authors must go ahead with a mechanistic detail of Kr-h1 mediated JH upregulation before this can be concluded. Mere qPCR experiments are not sufficient to substantiate a claim that is completely contrary to the current understanding of JH signalling pathway.<br /> (10) Authors have performed knockdowns of JHAMT, Met and Kr-h1 to demonstrate the effect of these factors on fecundity n BPH. Additionally, they have performed rescue experiments with EB application on these knockdown insects (Figure 3K-M). This I believe is a very flawed experiment. The authors demonstrate EB works through JHAMT in upregulating JH titer. In the absence of JHAMT, EB application is not expected to rescue the phenotype. But authors have reported a complete rescue here. In the absence of Met, the receptor of JH, either EB or JH is not expected to rescue the phenotype. But a complete rescue has been reported. These two experimental results contradict their own hypothesis.<br /> (11) A significant section of the paper deals with how EB upregulates JH titer. JH is a hormone synthesized in the Corpora Allata. Yet the authors have chosen to use the whole body for all of their experiments. Changes in the whole body for mRNA of those enzymes involved in JH synthesis does may not reflect on the situation in Corpora Allata. Although working with corpora Allata is challenging, discarding the abdomen and thorax region and working with the head and neck region of the insect is easily doable. Results from such sampling is always more convincing when it comes to JH synthesis studies.<br /> (12) The phenomenon reported was specific for BPH and not found in other insects. This limits the implications of the study.<br /> (13) Overall, the molecular experiments are very poorly designed and can at best be termed superficial. There are several contradictions within the paper and no discussion or explanation has been provided for that.

Comments on revisions:

(1) The onus of making the revisions understandable to the reviewers lies with the authors. In its current form, how the authors have approached the review is hard to follow, in my opinion. Although the authors have taken a lot of effort in answering the questions posed by reviewers, parallel changes in the manuscript are not clearly mentioned. In many cases, the authors have acknowledged the criticism in response to the reviewer, but have not changed their narrative, particularly in the results section.<br /> (2) In the response to reviewers, the authors have mentioned line numbers in the main text where changes were made. But very frequently, those lines do not refer to the changes or mention just a subsection of changes done. The problem is throughout the document making it very difficult to follow the revision and contributing to the point mentioned above.<br /> (3) The authors need to infer the performed experiments rationally without over interpretation. Currently, many of the claims that the authors are making are unsubstantiated. As a result of the first review process, the authors have acknowledged the discrepancies, but they have failed to alter their interpretations accordingly.<br /> (4) I would like to point to the fact that there are significant experimental modifications added to the manuscript. The decision from the first cycle of review was given on 8th Nov 2024. The authors re-submitted the manuscript on 20th Nov 2024. It just beats my understanding, how so many experiments can be done in such a short time. The rush in resubmission is evident in the writing quality as well. Which I think is now poorer than the original version.<br /> (5) The writing quality is still extremely poor.

Author response:

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

eLife assessment

This useful manuscript reports mechanisms behind the increase in fecundity in response to sub-lethal doses of pesticides in the crop pest, the brown plant hopper. The authors hypothesize that the pesticide works by inducing the JH titer, which through the JH signaling pathway induces egg development. Evidence for this is, however, inadequate.

We greatly appreciate your valuable comments and constructive suggestions for our work. All in all, the manuscript has been carefully edited and improved following your suggestions. We also provide more evidence to support our statements by conducting new experiments. First, we found that also EB treatment of adult females can stimulate egg-laying. Second, EB treatment in female adults increases the number of mature eggs in the ovary and ovarioles. Third, EB treatment in females enhances the expression of the kr-h1 gene in the whole body of BPH. Finally, EB treatment in female adults increases the JHIII titer, but has no impact on the 20E titer.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Gao et al. have demonstrated that the pesticide emamectin benzoate (EB) treatment of brown planthopper (BPH) leads to increased egg-laying in the insect, which is a common agricultural pest. The authors hypothesize that EB upregulates JH titer resulting in increased fecundity.

Strengths:

The finding that a class of pesticide increases the fecundity of brown planthopper is interesting.

We greatly appreciate your positive comments on our work.

Weaknesses:

(1) EB is an allosteric modulator of GluCl. That means EB physically interacts with GluCl initiating a structural change in the cannel protein. Yet the authors' central hypothesis here is about how EB can upregulate the mRNA of GluCl. I do not know whether there is any evidence that an allosteric modulator can function as a transcriptional activator for the same receptor protein. The basic premise of the paper sounds counterintuitive. This is a structural problem and should be addressed by the authors by giving sufficient evidence about such demonstrated mechanisms before.

Thank you for your question. As the reviewer points out, EB physically interacts with its target protein GluCl and thus affects its downstream signaling pathway. In the manuscript, we reported that EB-treated brown planthoppers display increased expression of GluCl in the adult stage (Fig. 5A). Actually, there are many studies showing that insects treated with insecticides can increase the expression of target genes. For example, the relative expression level of the ryanodine receptor gene of the rice stem borer, Chilo suppressalis was increased 10-fold after treatment with chlorantraniliprole, an insecticide which targets the ryanodine receptor (Peng et al., 2017). Besides this, in Drosophila, starvation (and low insulin) elevates the transcription level of the sNPF and tachykinin receptors (Ko et al., 2015; Root et al., 2011). In brown planthoppers, reduction in mRNA and protein expression of a nicotinic acetylcholine receptor α8 subunit is associated with resistance to imidacloprid (Zhang et al., 2015). RNA interference knockdown of α8 gene decreased the sensitivity of N. lugens to imidacloprid (Zhang et al., 2015). Hence, expression of receptor genes can be regulated by diverse factors including insecticide treatment. In our case, we found that EB can upregulate its target gene GluCl. However, we did not claim that EB functions as transcriptional activator for GluCl, and we still do not know why EB treatment changes the expression of GluCl in the brown planthopper. Considering our experiments are lasting several days, it might be an indirect (or secondary) effect caused by other factors, which change the expression of GluCl gene upon EB action of the channel. One reason is maybe that the allosteric interaction with GluCl by EB makes it dysfunctional and the cellular response is to upregulate the channel/receptor to compensate. We have inserted text on lines 738 - 757 to explain these possibilities.

(2) I am surprised to see a 4th instar larval application or treatment with EB results in the upregulation of JH in the adult stages. Complicating the results further is the observation that a 4th instar EB application results in an immediate decrease in JH titer. There is a high possibility that this late JH titer increase is an indirect effect.

Thank you for your question. Treatment with low doses or sublethal doses of insecticides might have a strong and complex impact on insects (Gandara et al., 2024; Gong et al., 2022; Li et al., 2023; Martelli et al., 2022). We kept the 4th instar of brown planthoppers feeding on EB for four days. They will develop to 5th instar after four days treatment, which is the final nymphal stage of BPH. Since the brown planthopper is a hemimetabolous insect, we cannot rule out the possibility that an indirect effect of treatment with EB results in the upregulation of JH in the adult stages. In this new revised manuscript, we investigated the impact of EB treatment in the adult stage. We found that female adults treated with EB also laid more eggs than controls (Figure 1-figure supplement 1A). The following experiments were performed in adults to address how EB treated stimulates egg-laying in adult brown planthopper.

(1) We found that EB treatment in adults increases the number of mature eggs in ovary (new Figure 2-figure supplement 1). We add this results in lines 234 – 238 and 281-285.

(2) We measured the JH titer after the female adults had been treated with EB. We found that EB can also increase the JH titer but has no impact on the 20E titer in the female adult (Figure 3-S3A and B). We add this results in lines 351 – 356 and 281-285.

(3) EB treatment in adults increases the gene expression of JHAMT and Kr-h1 (Figure 3-S3C and D). We add this results in lines 378 – 379, lines 387-390 and lines 457-462.

(3) The writing quality of the paper needs improvement. Particularly with respect to describing processes and abbreviations. In several instances the authors have not adequately described the processes they have introduced, thus confusing readers.

Thank you for your suggestion. We have thoroughly revised the paper to improve clarity.

(4) In the section 'EB promotes ovarian development' the authors have shown that EB treatment results in increased detention of eggs which contradicts their own results which show that EB promotes egg laying. Again, this is a serious contradiction that nullifies their hypothesis.

Thank you for pointing this out. We revised the figure 2B to show number of mature eggs in the ovary. The number of mature eggs in ovaries of females that fed on EB was higher than in control females. We also show that BPH fed with EB laid more eggs than controls. Thus, our results suggest that EB promotes ovary maturation (and egg production) and also increases egg laying (Figure 1 and Table S1). Thus, we found that EB treatment can increase both the production of eggs and increase egg laying. We add this results in lines 234 – 238.

(5) Furthermore, the results suggest that oogenesis is not affected by EB application. The authors should devote a section to discussing how they are observing increased egg numbers in EB-treated insects while not impacting Oogenesis.

Thank you for your suggestions, and apologies for the lack of clarity in our initial explanation. First, we found that EB treatment led to an increase in the number of eggs laid by female brown planthoppers (Figure 1). Through dissection experiments, we observed that EB-treated females had more mature eggs in their ovaries (Figure 2A and B), indicating that the increased egg-laying was due to a larger production of mature eggs in the ovaries after EB treatment. This is now explained on lines 229-238.

Additionally, since there is no systematic description of oogenesis in the brown planthopper, we were the first to observe the oogenesis process in this species using immunohistochemistry and laser confocal microscopy. Based on the developmental characteristics, we defined the different stages of oogenesis (Figure 2C, Figure 2-figure supplement 2). We did not observe any significant effect of EB treatment on the various stages of oogenesis, indicating that EB treatment does not impair normal egg development (Figure 2D). Instead, the increase in vitellogenin accelerates the production of mature eggs. This is now explained on lines 243-262.

During the maturation process, eggs require uptake of vitellogenin, and an increase in vitellogenin (Vg) content can accelerate egg maturation, producing more mature eggs. Our molecular data suggest that EB treatment leads to an upregulation of vg expression. Based on these findings, we conclude that the increase in egg-laying caused by EB treatment is due to the upregulation of vg (Figure 3I), which raises vitellogenin content, promoting the uptake of vitellogenin by maturing eggs and resulting in the production of more mature eggs. We have revised the text on lines 389-395 to clarify this point.

(6) Met is the receptor of JH and to my understanding, remains mostly constant in terms of its mRNA or protein levels throughout various developmental periods in many different insects. Therefore, the presence of JH becomes the major driving factor for physiological events and not the presence of the receptor Met. Here the authors have demonstrated an increase in Met mRNA as a result of EB treatment. Their central hypothesis is that EB increases JH titer to result in enhanced fecundity. JH action will not result in the activation of Met. Although not contradictory to the hypothesis, the increase in mRNA content of Met is contrary to the findings of the JH field thus far.

Thank you for your comment. Our results showed that EB treatment can mildly increase (about 2-fold) expression of the Met gene in brown planthoppers (Figure 3G). And our data indicated that Met and FAMeT expression levels were not influenced so much by EB compared with kr-h1 and vg (Figure 3H and I). We agree that JH action will not result in the increase of Met. However, we cannot rule out the possibility of other factors (indirect effects), induced by EB treatment that increase the mRNA expression level of Met. One recent paper reported that downregulation of transcription factor CncC will increase met expression in beetles (see Figure 6A in this reference) (Jiang et al., 2023). Many studies have reported that insecticide treatment will activate the CncC gene signaling pathway, which regulates detoxification gene expression (Amezian et al., 2023; Fu et al., 2024; Hu et al., 2021). Hence, it is possible that EB might influence the CncC gene pathway which then induces met expression. This EB effect on met upregulation may be similar to the upregulation of GluCl and some other secondary effects. We have discussed this on lines 725-738.

(7) As pointed out before, it is hard to rationalize how a 4th instar exposure to EB can result in the upregulation of key genes involved in JH synthesis at the adult stage. The authors must consider providing a plausible explanation and discussion in this regard.

Thank you for your comments. It must be mentioned that although we exposed the BPH to EB at 4th instar, we make the insect feed on the EB-treated rice plants for four days. After that, the insect will develop into 5^th instar, the final nymphal stage of brown planthopper. Since brown planthoppers do not have a pupal stage, this might cause the EB presented to the insects last a longer time even in the adult stage. Besides this, we found that EB treatment will increase the weight of adult females (Figure 1-figure supplement 3E and F), which indicates that EB might increase food intake in BPHs that might produce more insulin peptide. Insulin might increase the JH synthesis at the adult stage. In our revised study we also investigate EB impairment in adult BPHs. We found that, similar to the nymphal stage, EB treatment in adult BPHs also increases the egg laying. Furthermore, the JH titer was increased after treatment of BPH with EB in adults. Besides this, GluCl and kr-h1 genes were also up-regulated after EB treatment in the adult stage. We have discussed this on lines 739-746.

(8) I have strong reservations against such an irrational hypothesis that Met (the receptor for JH) and JH-Met target gene Kr-h1 regulate JH titer (Line 311, Fig 3 supplemental 2D). This would be the first report of such an event on the JH field and therefore must be analysed in depth. I strongly suggest the authors remove such claims from the manuscript without substantiating it.

Thank you for your suggestions and comments. We have changed our claims in this revised MS. We found that EB treatment can enhance Kr-h1 expression. We have no evidence to support that JH can induce met expression. We have rewritten the manuscript to avoid confusion (see text on lines 725-735).

(9) Kr-h1 is JH/Met target gene. The authors demonstrate that silencing of Kr-h1 results in inhibition of FAMeT, which is a gene involved in JH synthesis. A feedback loop in JH synthesis is unreported. It is the view of this reviewer that the authors must go ahead with a mechanistic detail of Kr-h1 mediated JH upregulation before this can be concluded. Mere qPCR experiments are not sufficient to substantiate a claim that is completely contrary to the current understanding of the JH signalling pathway.

Thank you for your suggestions and comments. We agree that only qPCR experiments are not enough to provide this kind of claim. More evidences need to be provided to support this. We have revised the MS to avoid confusion (see text on lines 725-735).

(10) The authors have performed knockdowns of JHAMT, Met, and Kr-h1 to demonstrate the effect of these factors on fecundity in BPH. Additionally, they have performed rescue experiments with EB application on these knockdown insects (Figure 3K-M). This, I believe, is a very flawed experiment. The authors demonstrate EB works through JHAMT in upregulating JH titer. In the absence of JHAMT, EB application is not expected to rescue the phenotype. But the authors have reported a complete rescue here. In the absence of Met, the receptor of JH, either EB or JH is not expected to rescue the phenotype. But a complete rescue has been reported. These two experimental results contradict their own hypothesis.

Thank you for your comments. We thought that this rescue is possible since knockdown of the genes is incomplete when using dsRNA injection (and residual gene expression allows for EB action). It is not a total knockout and actually, these genes still have a low level of expression in the dsRNA-injected insects. Since EB can upregulate the expression of JHAMT, Met, and Kr-h1, it is reasonable that EB treatment can rescue the down-regulation effects of these three genes and make fecundity completely rescued. We have clarified this on lines 411-413).

(11) A significant section of the paper deals with how EB upregulates JH titer. JH is a hormone synthesized in the Corpora Allata. Yet the authors have chosen to use the whole body for all of their experiment. Changes in the whole body for mRNA of those enzymes involved in JH synthesis may not reflect the situation in Corpora Allata. Although working with Corpora Allata is challenging, discarding the abdomen and thorax region and working with the head and neck region of the insect is easily doable. Results from such sampling are always more convincing when it comes to JH synthesis studies.

Thank you for your suggestions. Because the head is very difficult to separate from the thorax region in brown planthoppers as you can see in Author response image 1. We are now trying to answer how EB regulates JH synthesis using Drosophila as a model.

Author response image 1.

The brown planthopper

(12) The phenomenon reported was specific to BPH and not found in other insects. This limits the implications of the study.

Thank you for your comments. The brown planthopper is a serious insect pest on rice in Asia. Our findings can guide the use of this insecticide in the field. Besides this, our findings indicated that EB, which targets GluCl can impair the JH titer. Our findings added new implications for how a neuronal system influences the JH signaling pathway. We will further investigate how EB influences JH in the future and will use Drosophila as a model to study the molecular mechanisms.

(13) Overall, the molecular experiments are very poorly designed and can at best be termed superficial. There are several contradictions within the paper and no discussion or explanation has been provided for that.

Thank you for your comments. We have revised the paper according to your suggestions and added further explanation of our results in the discussion parts and hope the conclusions are better supported in the new version. We have discussed this on lines 725-746 and 778-799.

Reviewer #2 (Public Review):

The brown plant hopper (BPH) is a notorious crop pest and pesticides are the most widespread means of controlling its population. This manuscript shows that in response to sublethal doses of the pesticide (EB), BPH females show enhanced fecundity. This is in keeping with field reports of population resurgence post-pesticide treatment. The authors work out the mechanism behind this increase in fecundity. They show that in response to EB exposure, the expression of its target receptor, GluCl, increases. This, they show, results in an increase in the expression of genes that regulate the synthesis of juvenile hormone (JH) and JH itself, which, in turn, results in enhanced egg-production and egg-laying. Interestingly, these effects of EB exposure are species-specific, as the authors report that other species of plant hoppers either don't show enhanced fecundity or show reduced fecundity. As the authors point out, it is unclear how an increase in GluCl levels could result in increased JH regulatory genes.

We greatly appreciate your valuable comments and constructive suggestion to our work. We will try to figure out how EB interacts with its molecular target GluCl and then increases JH regulatory genes in the future work using Drosophila as models.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Overall, the molecular experiments are very poorly designed and can at best be termed superficial. There are several contradictions within the paper and no discussion or explanation has been provided for that.

The authors should consider a thorough revision.

Thank you for your comments. We have thoroughly revised the paper according to your suggestions and added further experiments and explanations of our results in the discussion parts.

Reviewer #2 (Recommendations For The Authors):

It would help the reader to have more schematics along with the figures. The final figure is helpful, but knowing the JH pathway, and where it acts would help with the interpretations as one reads the manuscript and the figures. The pathways represented in 4N or 5J are helpful but could be improved upon for better presentation.

It would be nice to have some discussion on how the authors think EB exposure results in an increase in GluCl expression, and how that in turn affects the expression of so many genes.

Thank you for your comments. We have thoroughly revised the paper according to your suggestions and added further experiments and explanations of how we think EB exposure results in an increase in JH titer and other genes in the discussion parts. We have added the test on lines 753-761.

References

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How devilishly hard it must be to consolidate an island! All those independence-minded islanders means that as a conqueror in charge of a coalition, you can't do your job too well or else you'll start to look like the enemy.

No idea why, but this section is elegant, romantic, beautiful, and final to me. Yet, not for the poet. For the poet her journey has just begun. Or the Valkyrie's journey has just begun.

It feels ancient. Like it was written during the time of Thor, Oden, and Asgard. Maybe Greek or Roman mythology perhaps?

This sentence felt like a quick deep breath taken by the poet. The reason is the violent illusion that the beginning and end sentences portray in this piece. I totally agree with Abbie that it sounds like the middle of war. Not just a war, but a very violent war at that!

Then why is it not the default?

eLife Assessment

This valuable study confirms the association between the human leukocyte antigen (HLA)-II region and tuberculosis (TB) susceptibility in genetically admixed South African populations, specifically identifying a near-genome-wide significant association in the HLA-DPB1 gene, which originates from KhoeSan ancestry. The evidence supporting the association between the HLA-II region and TB susceptibility is solid, and the work will be of interest to those studying the genetic basis of tuberculosis susceptibility/infection resistance.

Reviewer #1 (Public review):

Summary:

The authors aimed to confirm the association between the human leukocyte antigen (HLA)-II region and tuberculosis (TB) susceptibility within admixed African populations. Building upon previous findings from the International Tuberculosis Host Genetics Consortium (ITHGC), this study sought to address the limitations of small sample size and the inclusion of admixed samples by employing the Local Ancestry Allelic Adjusted (LAAA) model, as well as identify TB susceptibility loci in an admixed South African cohort.

Strengths:

The major strengths of this study include the use of multiple TB case-control datasets from diverse South African populations and ADMIXTURE for global ancestry inference.

Weaknesses:

The major weakness of this study include insufficient significant novel discoveries and reliance on cross-validation. The use of existing models did not add value to this study.

Appraisal:<br /> The authors achieved their aims. However, the results still needed to be further validated in the future.

Impact:<br /> The innovative use of the LAAA model and the comprehensive dataset in this study may make contributions to the field of genetic epidemiology.

Reviewer #2 (Public review):

Summary:

This manuscript is about using different analytical approaches to allow ancestry adjustments to GWAS analyses amongst admixed populations. This work is a follow-on from the recently published ITHGC multi-population GWAS (https://doi.org/10.7554/eLife.84394), with the focus on the admixed South African populations. Ancestry adjustment models detected a peak of SNPs in the class II HLA DPB1, distinct from the class II HLA DQA1 loci signficant in the ITHGC analysis.

Strengths:

Excellent demonstration of GWAS analytical pipelines in highly admixed populations. Particularly the utility of ancestry adjustment to improve study power to detect novel associations. Further confirmation of the importance of the HLA class II locus in genetic susceptibility to TB.

Weaknesses:

Limited novelty compared to the group's previous existing publications and the body of work linking HLA class II alleles with TB susceptibility in South Africa or other African populations. This work includes only ~100 new cases and controls from what has already been published. High resolution HLA typing has detected significant signals in both the DQA1 and DPB1 regions identified by the larger ITHGC and in this GWAS analysis respectively (Chihab L et al. HLA. 2023 Feb; 101(2): 124-137).<br /> Despite the availability of strong methods for imputing HLA from GWAS data (Karnes J et Plos One 2017), the authors did not confirm with HLA typing the importance of their SNP peak in the class II region. This would have supported the importance of this ancestry adjustment versus prior ITHGC analysis.<br /> The populations consider active TB and healthy controls (from high-burden presumed exposed communities) and do not provide QFT or other data to identify latent TB infection.

Important methodological points for clarification and for readers to be aware of when reading this paper:

(1) One of the reasons cited for the lack of African ancestry-specific associations or suggestive peaks in the ITHGC study was the small African sample size. The current association test includes a larger African cohort and yields a near-genome-wide significant threshold in the HLA-DPB1 gene originating from the KhoeSan ancestry. Investigation is needed as to whether the increase in power is due to increased African samples and not necessarily the use of the LAAA model as stated on lines 295 and 296?

Authors response - The Manhattan plot in Figure 3 includes the results for all four models: the traditional GWAS model (GAO), the admixture mapping model (LAO), the ancestry plus allelic (APA) model and the LAAA model. In this figure, it is evident that only the LAAA model identified the association peak on chromosome 6, which lends support the argument that the increase in power is due to the use of the LAAA model and not solely due to the increase in sample size.<br /> Reviewer comment - This data supports the authors conclusions that increase power is related to the LAAA model application rather than simply increase sample size.

(2) In line 256, the number of SNPs included in the LAAA analysis was 784,557 autosomal markers; the number of SNPs after quality control of the imputed dataset was 7,510,051 SNPs (line 142). It is not clear how or why ~90% of the SNPs were removed. This needs clarification.

Authors response:<br /> In our manuscript (line 194), we mention that "...variants with minor allele frequency (MAF) < 1% were removed to improve the stability of the association tests." A large proportion of imputed variants fell below this MAF threshold and were subsequently excluded from this analysis.

Reviewers additional comment: The authors should specify the number of SNPs in the dataset before imputation and indicate what proportion of the 784,557 remaining SNPs were imputed. Providing this information might help the reader better understand the rationale behind the imputation process.

(3) The authors have used the significance threshold estimated by the STEAM p-value < 2.5x10-6 in the LAAA analysis. Grinde et al. (2019 implemented their significance threshold estimation approach tailored to admixture mapping (local ancestry (LA) model), where there is a reduction in testing burden. The authors should justify why this threshold would apply to the LAAA model (a joint genotype and ancestry approach).

Authors response: We describe in the methods (line 189 onwards) that the LAAA model is an extension of the APA model. Since the APA model itself simultaneously performs the null global ancestry only model and the local ancestry model (utilised in admixture mapping), we thus considered the use of a threshold tailored to admixture mapping appropriate for the LAAA model.

Reviewers additional comment: While the LAAA model is an extension of the APA model, the authors describe the LAAA test as 'models the combination of the minor allele and the ancestry of the minor allele at a specific locus, along with the effect of this interaction,' thus a joint allele and ancestry effects model. Grinde et al. (2019) proposed the significance threshold estimation approach, STEAM, specifically for the LA approach, which tests for ancestry effects alone and benefits from the reduced testing burden. However, it remains unclear why the authors found it appropriate to apply STEAM to the LAAA model, a joint test for both allele and ancestry effects, which does not benefit from the same reduction in testing burden.

(4) Batch effect screening and correction (line 174) is a quality control check. This section is discussed after global and local ancestry inferences in the methods. Was this QC step conducted after the inferencing? If so, the authors should justify how the removed SNPs due to the batch effect did not affect the global and local ancestry inferences or should order the methods section correctly to avoid confusion.

Authors response: The batch effect correction method utilised a pseudo-case-control comparison which included global ancestry proportions. Thus, batch effect correction was conducted after ancestry inference. We excluded 36 627 SNPs that were believed to have been affected by the batch effect. We have amended line 186 to include the exact number of SNPs excluded due to batch effect.<br /> The ancestry inference by RFMix utilised the entire merged dataset of 7 510 051 SNPs. Thus, the SNPs removed due to the batch effect make up a very small proportion of the SNPs used to conduct global and local ancestry inferences (less than 0.5%). As a result, we do not believe that the removed SNPs would have significantly affected the global and local ancestry inferences. However, we did conduct global ancestry inference with RFMix on each separate dataset as a sanity check. In the Author response tables 1 and 2, we show the average global ancestry proportions inferred for each separate dataset, the average global ancestry proportions across all datasets and the average global ancestry proportions inferred using the merged dataset. The SAC and Xhosa cohorts are shown in two separate tables due to the different number of contributing ancestral populations to each cohort. The differences between the combined average global ancestry proportions across the separate cohorts does not differ significantly to the global ancestry proportions inferred using the merged dataset.

This is an excellent response and should remain accessible to readers to clarify this issue.

Author response:

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

Reviewer #1 (Public review): 

Summary: 

The authors aimed to confirm the association between the human leukocyte antigen (HLA)-II region and tuberculosis (TB) susceptibility within admixed African populations. Building upon previous findings from the International Tuberculosis Host Genetics Consortium (ITHGC), this study sought to address the limitations of small sample size and the inclusion of admixed samples by employing the Local Ancestry Allelic Adjusted (LAAA) model, as well as identify TB susceptibility loci in an admixed South African cohort. 

Strengths: 

The major strengths of this study include the use of six TB case-control datasets collected over 30 years from diverse South African populations and ADMIXTURE for global ancestry inference. The former represents comprehensive dataset used in this study and the later ensures accurate determination of ancestral contributions. In addition, the identified association in the HLA-DPB1 gene shows near-genomewide significance, enhancing the credibility of the findings. 

Weaknesses: 

The major weakness of this study includes insufficient significant discoveries and reliance on crossvalidation. This study only identified one variant significantly associated with TB status, located in an intergenic region with an unclear link to TB susceptibility. Despite identifying multiple lead SNPs, no other variants reached the genome-wide significance threshold, limiting the overall impact of the findings. The absence of an independent validation cohort, with the study relying solely on crossvalidation, is also a major limitation. This approach restricts the ability to independently confirm the findings and evaluate their robustness across different population samples. 

Appraisal: 

The authors successfully achieved their aims of confirming the association between the HLA-II region and TB susceptibility in admixed African populations. However, the limited number of significant discoveries, reliance on cross-validation, and insufficient discussion of model performance and SNP significance weaken the overall strength of the findings. Despite these limitations, the results support the conclusion that considering local ancestry is crucial in genetic studies of admixed populations. 

Impact:  

The innovative use of the LAAA model and the comprehensive dataset in this study make substantial contributions to the field of genetic epidemiology. 

Reviewer #2 (Public review): 

Summary: 

This manuscript is about using different analytical approaches to allow ancestry adjustments to GWAS analyses amongst admixed populations. This work is a follow-on from the recently published ITHGC multi-population GWAS (https://doi.org/10.7554/eLife.84394), with a focus on the admixed South African populations. Ancestry adjustment models detected a peak of SNPs in the class II HLA DPB1, distinct from the class II HLA DQA1 loci significant in the ITHGC analysis. 

Strengths: 

Excellent demonstration of GWAS analytical pipelines in highly admixed populations. Further confirmation of the importance of the HLA class II locus in genetic susceptibility to TB. 

Weaknesses: 

Limited novelty compared to the group's previous existing publications and the body of work linking HLA class II alleles with TB susceptibility in South Africa or other African populations. This work includes only ~100 new cases and controls from what has already been published. High-resolution HLA typing has detected significant signals in both the DQA1 and DPB1 regions identified by the larger ITHGC and in this GWAS analysis respectively (Chihab L et al. HLA. 2023 Feb; 101(2): 124-137). Despite the availability of strong methods for imputing HLA from GWAS data (Karnes J et Plos One 2017), the authors did not confirm with HLA typing the importance of their SNP peak in the class II region. This would have supported the importance of this ancestry adjustment versus prior ITHGC analysis. 

The populations consider active TB and healthy controls (from high-burden presumed exposed communities) and do not provide QFT or other data to identify latent TB infection. 

Important methodological points for clarification and for readers to be aware of when reading this paper: 

(1) One of the reasons cited for the lack of African ancestry-specific associations or suggestive peaks in the ITHGC study was the small African sample size. The current association test includes a larger African cohort and yields a near-genome-wide significant threshold in the HLA-DPB1 gene originating from the KhoeSan ancestry. The investigation is needed as to whether the increase in power is due to increased African samples and not necessarily the use of the LAAA model as stated on lines 295 and 296? 

Thank you for your comment. The Manhattan plot in Figure 3 includes the results for all four models: the traditional GWAS model (GAO), the admixture mapping model (LAO), the ancestry plus allelic (APA) model and the LAAA model. In this figure, it is evident that only the LAAA model identified the association peak on chromosome 6, which lends support the argument that the increase in power is due to the use of the LAAA model and not solely due to the increase in sample size. 

(2) In line 256, the number of SNPs included in the LAAA analysis was 784,557 autosomal markers; the number of SNPs after quality control of the imputed dataset was 7,510,051 SNPs (line 142). It is not clear how or why ~90% of the SNPs were removed. This needs clarification. 

Thank you for your recommendation. In our manuscript (line 194), we mention that “…variants with minor allele frequency (MAF) < 1% were removed to improve the stability of the association tests.” A large proportion of imputed variants fell below this MAF threshold, and were subsequently excluded from this analysis. Below, we show the number of imputed variants across MAF bins for one of our datasets [RSA(A)] to substantiate this claim:  

Author response image 1.

(3) The authors have used the significance threshold estimated by the STEAM p-value < 2.5x10^-6 in the LAAA analysis. Grinde et al. (2019 implemented their significance threshold estimation approach tailored to admixture mapping (local ancestry (LA) model), where there is a reduction in testing burden. The authors should justify why this threshold would apply to the LAAA model (a joint genotype and ancestry approach). 

Thank you for your recommendation. We describe in the methods (line 189 onwards) that the LAAA model is an extension of the APA model. Since the APA model itself simultaneously performs the null global ancestry only model and the local ancestry model (utilised in admixture mapping), we thus considered the use of a threshold tailored to admixture mapping appropriate for the LAAA model.  

(4) Batch effect screening and correction (line 174) is a quality control check. This section is discussed after global and local ancestry inferences in the methods. Was this QC step conducted after the inferencing? If so, the authors should justify how the removed SNPs due to the batch effect did not affect the global and local ancestry inferences or should order the methods section correctly to avoid confusion. 

Thank you for your comments. The batch effect correction method utilised a pseudo-case-control comparison which included global ancestry proportions. Thus, batch effect correction was conducted after ancestry inference. We excluded 36 627 SNPs that were believed to have been affected by the batch effect. We have amended line 186 to include the exact number of SNPs excluded due to batch effect. 

The ancestry inference by RFMix utilised the entire merged dataset of 7 510 051 SNPs. Thus, the SNPs removed due to the batch effect make up a very small proportion of the SNPs used to conduct global and local ancestry inferences (less than 0.5%). As a result, we do not believe that the removed SNPs would have significantly affected the global and local ancestry inferences. However, we did conduct global ancestry inference with RFMix on each separate dataset as a sanity check. In the tables below, we show the average global ancestry proportions inferred for each separate dataset, the average global ancestry proportions across all datasets and the average global ancestry proportions inferred using the merged dataset. The SAC and Xhosa cohorts are shown in two separate tables due to the different number of contributing ancestral populations to each cohort. The differences between the combined average global ancestry proportions across the separate cohorts does not differ significantly to the global ancestry proportions inferred using the merged dataset. 

Author response table 1.

Comparison of global ancestry proportions across the separate SAC datasets and the merged cohort.

Author response table 2.

Comparison of global ancestry proportions in the Xhosa dataset and the merged cohort. 

Reviewer #1 (Recommendations for the authors): 

Suggestions for Improved or Additional Experiments, Data, or Analyses:   

(1) It might be beneficial to consider splitting the data into separate discovery and validation cohorts rather than relying solely on cross-validation. This approach could provide a stronger basis for independently confirming the findings. 

Thank you for your suggestion. However, we are hesitant to divide our already modest dataset (n=1544) into separate discovery and validation cohorts, as this would reduce the statistical power to detect significant associations.

(2) Clearly stating the process of cross-validation in the methods section and reporting relevant validation statistics, such as accuracy, sensitivity, specificity, and area under the curve (AUC), would provide a more comprehensive assessment of the model's performance.  

Thank you for your recommendation. We would like to highlight this article, “GWAS in the southern African context” (1), which evaluated the performance of the LAAA model compared to other models in three- and five-way admixed populations. Given the thorough evaluation of the model’s performance in that study, we did not find it necessary to reassess its performance in this manuscript.   

(3) Analysing racial cohorts separately to see if you can replicate previous results and find significant markers in combined non-African populations that are not evident in African-only samples might be useful. 

Thank you for your suggestion. We would like to respectfully note that race is a social construct, and its use as a proxy for genetic ancestry can be problematic (2). In our study, we rather rely on genetic ancestry inferred using ancestry inference software to provide a more accurate representation of our cohort's genetic diversity. Additionally, our cohort consists mostly of a highly admixed population group, with some individuals exhibiting ancestral contributions from up to five different global populations. Therefore, it is not possible to categorize our samples into distinct “Africanonly” or “non-African” groups.

(4) It might be worthwhile to consider using polygenic risk scores (PRS) to combine multiple genetic influences. This approach could help in identifying cumulative genetic effects that are not apparent when examining individual SNPs.  

Thank you for your recommendation. While constructing a polygenic risk score (PRS) is beyond the scope of the current study, but an ongoing interest in our group, we recognize its potential value and will consider incorporating this approach in future research endeavours or a separate publication. A recent publication by Majara et al showed that that PRS accuracy is low for all traits and varies across ancestrally and ethnically diverse South African groups (3).

Recommendations for Improving the Writing and Presentation: 

Including a more thorough discussion of the methodological limitations, such as the challenges of studying admixed populations and the potential limitations of the LAAA model, would provide a more balanced perspective. 

Thank you for your suggestion. To provide a more balanced perspective, we included the limitations of our study in the discussion, from line 429 to like 451.

Minor Corrections to the Text and Figures: 

Including all relevant statistics would improve clarity. For example, providing confidence intervals for the odds ratios and discussing any observed trends or outliers would be beneficial. 

Thank you for your recommendation. We have added 95% confidence intervals to all odds ratios reported in Table 3. However, beyond the association peak identified in the HL-II region associated with the phenotype, we do not observe any other trends or outliers in or LAAA analysis.  

Reviewer #2 (Recommendations for the authors): 

Points for improvement: 

(1) Related to the different datasets and inclusions in previous publications, it would also be good to better understand the different numbers of cases and controls included across the previous and current analyses, or discussion thereof. For instance, the RSA(M) dataset includes 555/440 cases/controls for this analysis and only 410/405 cases/controls in the ITHGC analysis. Other discrepancies are noted across the other published datasets compared to those included in this analysis, and these always need to be detailed in a supplement or similar to better understand if this could have introduced bias or was in fact correct based on the additional ancestry-related restriction applied.  

Thank you for your comments. Table 1 of our manuscript lists number of individuals in the RSA(M) dataset, including related individuals. As described in line 131, related individuals were subsequently excluded during quality control: “Individual datasets were screened for relatedness using KING software (Manichaikul et al., 2010) and individuals up to second degree relatedness were removed.” The ITHGC only reported the number of unrelated individuals included their analyses, which would account for the discrepancies in the reported number of cases and controls.  

(2) The imbalance between cases and controls in this analysis is quite striking, and it is unusual to have the imbalance favour cases over controls. This contrasts with the ITHGC, where there are substantially more controls. There is no comment on how this could potentially impact this analysis. 

Thank you for your comment. We have included a note on our case-control imbalance in the discussion:

“While many studies discuss methods for addressing case-control imbalances with more controls than cases (which can inflate type 1 error rates (Zhou et al. 2018; Dai et al. 2021; Öztornaci et al. 2023), few address the implications of a large case-to-control ratio like ours (952 cases to 592 controls). To assess the impact of this imbalance, we used the Michigan genetic association study (GAS) power calculator (Skol et al. 2006). Under an additive disease model with an estimated prevalence of 0.15, a disease allele frequency of 0.3, a genotype relative risk of 1.5, and a default significance level of 7 × 10^-6, we achieved an expected power of approximately 75%. With a balanced sample size of 950 cases and 950 controls, power would exceed 90%, but it would drop significantly with a smaller balanced cohort of 590 cases and 590 controls. Given these results, we proceeded with our analysis to maximize statistical power despite the case-control imbalance.” 

Author response image 2.

Minor comments 

(1) Referencing around key points of TB epidemiology and disease states seems out of date, given recent epidemiology reviews and seminal nature or lancet review articles. Please update.  

Thank you for your suggestion. We have included the following recent publications in the introductory paragraph: 

Zaidi, S. M. A., Coussens, A. K., Seddon, J. A., Kredo, T., Warner, D., Houben, R. M. G. J., & Esmail, H. (2023). Beyond latent and active tuberculosis: a scoping review of conceptual frameworks. EClinicalMedicine, 66, 102332. https://doi.org/10.1016/j.eclinm.2023.102332

Menzies, N. A., Swartwood, N., Testa, C., Malyuta, Y., Hill, A. N., Marks, S. M., Cohen, T., & Salomon, J. A. (2021). Time Since Infection and Risks of Future Disease for Individuals with Mycobacterium tuberculosis Infection in the United States. Epidemiology, 32(1), 70–78. https://doi.org/10.1097/EDE.0000000000001271  

Cudahy, P. G. T., Wilson, D., & Cohen, T. (2020). Risk factors for recurrent tuberculosis after successful treatment in a high burden setting: a cohort study. BMC Infectious Diseases, 20(1), 789. https://doi.org/10.1186/s12879-020-05515-4  

Escombe, A. R., Ticona, E., Chávez-Pérez, V., Espinoza, M., & Moore, D. A. J. (2019). Improving natural ventilation in hospital waiting and consulting rooms to reduce nosocomial tuberculosis transmission risk in a low resource setting. BMC Infectious Diseases, 19(1), 88. https://doi.org/10.1186/s12879-019-3717-9  

Laghari, M., Sulaiman, S. A. S., Khan, A. H., Talpur, B. A., Bhatti, Z., & Memon, N. (2019). Contact screening and risk factors for TB among the household contact of children with active TB: a way to find source case and new TB cases. BMC Public Health, 19(1), 1274. https://doi.org/10.1186/s12889-0197597-0  

Matose, M., Poluta, M., & Douglas, T. S. (2019). Natural ventilation as a means of airborne tuberculosis infection control in minibus taxis. South African Journal of Science, 115(9/10). https://doi.org/10.17159/sajs.2019/5737

Smith, M. H., Myrick, J. W., Oyageshio, O., Uren, C., Saayman, J., Boolay, S., van der Westhuizen, L., Werely, C., Möller, M., Henn, B. M., & Reynolds, A. W. (2023). Epidemiological correlates of overweight and obesity in the Northern Cape Province, South Africa. PeerJ, 11, e14723. https://doi.org/10.7717/peerj.14723  

(2) Lines 46 to 48 appear to have two contradictory statements next to each other. The first says there are numerous GWAS investigating TB susceptibility; the second says there are sparse. Please clarify.

Thank you for bringing this to our attention. We have amended the lines as follows: 

“Numerous genome-wide association studies (GWASs) investigating TB susceptibility have been conducted across different population groups. However, findings from these studies often do not replicate across population groups (Möller & Kinnear, 2020; Möller et al., 2018; Uren et al., 2017).”

(3) Add ref in line 69 for two SAC populations.

Thank you for your recommendation. We have included the citation for the ITHGC meta-analysis paper here: 

“The authors described possible reasons for the lack of associations, including the smaller sample size compared to the other ancestry-specific meta-analyses, increased genetic diversity within African individuals and population stratification produced by two admixed cohorts from the South African Coloured (SAC) population (Schurz et al. 2024).”

(4) Write out abbreviations the first time they appear (Line 121).

Thank you for your recommendation. We have corrected the sentence as follows: 

“Monomorphic sites were removed. Individuals were screened for deviations in Hardy-Weinberg Equilibrium (HWE) for each SNP and sites deviating from the HWE threshold of 10-5 were removed.”

(5) It would be good in the supplement to see if there is a SNP peak in chromosome 20 with a hit that reached significance in the Bantu-speaking African ancestry.

Thank you for your recommendation. We have included a regional plot for the lead variant identified on chromosome 20 originating from Bantu-speaking African ancestry in the supplementary material (Supplementary Figure 3).

(6) It would be good to mention the p-values of rs28383206 from the ITHGC paper in this cohort for KhoeSan and Bantu-speaking African ancestries. 

Thank you for your suggestion. We have included the following paragraph from line 352:

“The lead variant identified in the ITHGC meta-analysis, rs28383206, was not present in our genotype or imputed datasets. The ITHGC imputed genotypes using the 1000 Genomes (1000G) reference panel (4). Variant rs28383206 has an alternate allele frequency of 11.26% in the African population subgroup within the 1000G dataset (https://www.ncbi.nlm.nih.gov/snp/rs28383206). However, rs28383206 is absent from our in-house whole-genome sequencing (WGS) datasets, which include Bantu-speaking African and KhoeSan individuals. This absence suggests that rs28383206 might not have been imputed in our datasets using the AGR reference panel, potentially due to its low alternate allele frequency in southern African populations. Our merged dataset contained two variants located within 800 base pairs of r_s28383206: rs482205_ (6:32576009) and rs482162 (6:32576019). However, these variants were not significantly associated with TB status in our cohort (Supplementary Table 1).” Supplementary Table 1 can be found in the supplementary material:

(7) It would improve the readability of the ancestry proportions listed on lines 236 and 237 if these population groups were linked with the corresponding specific population used in Figure 1, as has been done in Table 2.

Thank you for your suggestion. We have amended Figure 1 to include the corresponding population labels mentioned in Table 2.  

(8) In line 209, it is not clear why the number of alleles of a specific ancestry at a locus is referred to as a covariate in admixture mapping when the corresponding marginal effect is the parameter of interest. 

Thank you for bringing this to our attention. We have amended the description as follows: 

“(2) Local ancestry (LA) model:

This model is used in admixture mapping to identify ancestry-specific variants associated with a specific phenotype. The LA model evaluates the number of alleles of a specific ancestry at a locus and includes the corresponding marginal effect as a covariate in association analyses.”

(9) Table 3 would benefit from a column on whether the SNP was genotyped or imputed. 

Thank you for your suggestion. We have included a column indicating whether the SNP was genotyped or imputed, as well as an additional column with the INFO score for imputed genotypes. 

(10) The authors should remove the print and download icons in Figure 1 on lines 240 and 241.

Thank you for your suggestion. We have amended the figure as requested.  

(11) In the quality control, the authors use a more relaxed threshold for missingness in individuals (90%) and genotypes (5%) and have strayed away from the conventional 97%-98%. An explanation of the choice of these thresholds will be helpful to the reader.

Thank you for your suggestion. We aimed to use similar genotype and individual missingness thresholds outline by the ITHGC meta-analysis (which utilised a threshold of 10% for both genotype and individual missingness) and the previous LAAA analysis paper performed by Swart et al. in 2021. We have amended line 116 for more clarity: 

“Individuals with genotype call rates less than 90% and SNPs with more than 5% missingness were removed as described previously (5).”

References  

(1) Swart Y, van Eeden G, Uren C, van der Spuy G, Tromp G, Moller M. GWAS in the southern African context. Cold Spring Harbor Laboratory. 2022;

(2) Byeon YJJ, Islamaj R, Yeganova L, Wilbur WJ, Lu Z, Brody LC, et al. Evolving use of ancestry, ethnicity, and race in genetics research-A survey spanning seven decades. Am J Hum Genet. 2021 Dec 2;108(12):2215–23.

(3) Majara L, Kalungi A, Koen N, Tsuo K, Wang Y, Gupta R, et al. Low and differential polygenic score generalizability among African populations due largely to genetic diversity. HGG Adv. 2023 Apr 13;4(2):100184.

(4) Schurz H, Naranbhai V, Yates TA, Gilchrist JJ, Parks T, Dodd PJ, et al. Multi-ancestry metaanalysis of host genetic susceptibility to tuberculosis identifies shared genetic architecture. eLife. 2024 Jan 15;13.

(5) Swart Y, Uren C, van Helden PD, Hoal EG, Möller M. Local ancestry adjusted allelic association analysis robustly captures tuberculosis susceptibility loci. Front Genet. 2021 Oct 15;12:716558.

eLife Assessment

Studying several allergens in different mouse strains, the authors assessed the role of IgM in airway inflammatory responses and show that IgM deficient mice have reduced airway hyperresponsiveness. Although the findings are useful and interesting and among others show the expression of a protein that regulates actin in smooth cells, the study remains incomplete as the data and analyses only partly support their primary claim.

Reviewer #1 (Public review):

Summary:

The authors of this study sought to define a role for IgM in responses to house dust mites in the lung.

Strengths:

Unexpected observation about IgM biology.<br /> Combination of experiments to elucidate function.

Weaknesses:

Would love more connection to human disease

Reviewer #2 (Public review):

Summary:

The manuscript by Hadebe and colleagues describes a striking reduction in airway hyperresponsiveness in Igm-deficient mice in response to HDM, OVA and papain across the B6 and BALB-c backgrounds. The authors suggest that the deficit is not due to improper type 2 immune responses, nor an aberrant B cell response, despite a lack of class switching in these mice. Through RNA-Seq approaches, the authors identify few differences between the lungs of WT and Igm-deficient mice, but see that two genes involved in actin regulation are greatly reduced in IgM-deficient mice. The authors target these genes by CRISPR-Cas9 in in vitro assays of smooth muscle cells to show that these may regulate cell contraction. While the study is conceptually interesting, there are a number of limitations, which stop us from drawing meaningful conclusions.

Strengths:

Fig. 1. The authors clearly show that IgMKO mice have striking reduced AHR in the HDM model, despite the presence of a good cellular B cell response.

Weaknesses:

Due to several technical and experimental limitations, it is unclear what leads to the reduction in airway hyperresponsiveness in IGM-KO mice. The limitations as outlined previously remain.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary: The authors of this study sought to define a role for IgM in responses to house dust mites in the lung.

Strengths:

Unexpected observation about IgM biology

Combination of experiments to elucidate function

Weaknesses:

Would love more connection to human disease

We thank the reviewer for these comments. At the time of this publication, we have not made a concrete link with human disease. While there is some anecdotal evidence of diseases such as Autoimmune glomerulonephritis, Hashimoto’s thyroiditis, Bronchial polyp, SLE, Celiac disease and other diseases in people with low IgM. Allergic disorders are also common in people with IgM deficiency, other studies have reported as high as 33-47%. The mechanisms for the high incidence of allergic diseases are unclear as generally, these patients have normal IgG and IgE levels. IgM deficiency may represent a heterogeneous spectrum of genetic defects, which might explain the heterogeneous nature of disease presentations. 

Reviewer #2 (Public Review):

Summary:

The manuscript by Hadebe and colleagues describes a striking reduction in airway hyperresponsiveness in Igm-deficient mice in response to HDM, OVA and papain across the B6 and BALB-c backgrounds. The authors suggest that the deficit is not due to improper type 2 immune responses, nor an aberrant B cell response, despite a lack of class switching in these mice. Through RNA-Seq approaches, the authors identify few differences between the lungs of WT and Igm-deficient mice, but see that two genes involved in actin regulation are greatly reduced in IgM-deficient mice. The authors target these genes by CRISPR-Cas9 in in vitro assays of smooth muscle cells to show that these may regulate cell contraction. While the study is conceptually interesting, there are a number of limitations, which stop us from drawing meaningful conclusions.

Strengths:

Fig. 1. The authors clearly show that IgMKO mice have striking reduced AHR in the HDM model, despite the presence of a good cellular B cell response.

Weaknesses:

Fig. 2. The authors characterize the cd4 t cell response to HDM in IGMKO mice.<br /> They have restimulated medLN cells with antiCD3 for 5 days to look for IL-4 and IL-13, and find no discernible difference between WT and KO mice. The absence of PBS-treated WT and KO mice in this analysis means it is unclear if HDM-challenged mice are showing IL-4 or IL-13 levels above that seen at baseline in this assay.

We thank the Reviewer for this comment. We would like to mention that a very minimal level of IL-4 and IL-13 in PBS mice was detected. We have indicated with a dotted line on the Figure to show levels in unstimulated or naïve cytokines. Please see Author response image 1 below from anti-CD3 stimulated cytokine ELISA data. The levels of these cytokines are very low and are not changed between WT and IgM^-/- mice, this is also true for PMA/ionomycin-stimulated cells.

Author response image 1.

The choice of 5 days is strange, given that the response the authors want to see is in already primed cells. A 1-2 day assay would have been better.

We agree with the reviewer that a shorter stimulation period would work. Over the years we have settled for 5-day re-stimulation for both anti-CD3 and HDM. We have tried other time points, but we consistently get better secretion of cytokines after 5 days.

It is concerning that the authors state that HDM restimulation did not induce cytokine production from medLN cells, since countless studies have shown that restimulation of medLN would induce IL-13, IL-5 and IL-10 production from medLN. This indicates that the sensitization and challenge model used by the authors is not working as it should.

We thank the reviewer for this observation. In our recent paper showing how antigen load affects B cell function, we used very low levels of HDM to sensitise and challenge mice (1 ug and 3 ug respectively). See below article, Hadebe et al., 2021 JACI. This is because Labs that have used these low HDM levels also suggested that antigen load impacts B cell function, especially in their role in germinal centres. We believe the reason we see low or undetectable levels of cytokines is because of this low antigen load sensitisation and challenge. In other manuscripts we have published or about to publish, we have shown that normal HDM sensitisation load (1 ug or 100 ug) and challenge (10 ug) do induce cytokine release upon restimulation with HDM. See the below article by Khumalo et al, 2020 JCI Insight (Figure 4A).

Sabelo Hadebe, Jermaine Khumalo, Sandisiwe Mangali, Nontobeko Mthembu, Hlumani Ndlovu, Amkele Ngomti, Martyna Scibiorek, Frank Kirstein, Frank Brombacher. Deletion of IL-4Ra signalling on B cells limits hyperresponsiveness depending on antigen load. doi.org/10.1016/j.jaci.2020.12.635).

Jermaine Khumalo, Frank Kirstein, Sabelo Hadebe, Frank Brombacher. IL-4Rα signalling in regulatory T cells is required for dampening allergic airway inflammation through inhibition of IL-33 by type 2 innate lymphoid cells. JCI Insight. 2020 Oct 15;5(20):e136206. doi: 10.1172/jci.insight.136206

The IL-13 staining shown in panel c is also not definitive. One should be able to optimize their assays to achieve a better level of staining, to my mind.

We agree with the reviewer that much higher IL-13-producing CD4 T cells should be observed. We don’t think this is a technical glitch or non-optimal set-up as we see much higher levels of IL-13-producing CD4 T cells when using higher doses of HDM to sensitise and challenge, say between 7 -20% in WT mice (see Author response image 2, lung stimulated with PMA/ionomycin+Monensin, please note this is for illustration purposes only and it not linked to the current manuscript, its merely to demonstrate a point from other experiments we have conducted in the lab).

Author response image 2.

In d-f, the authors perform a serum transfer, but they only do this once. The half life of IgM is quite short. The authors should perform multiple naïve serum transfers to see if this is enough to induce FULL AHR.

We thank the reviewer for this comment. We apologise if this was not clear enough on the Figure legend and method, we did transfer serum 3x, a day before sensitisation, on the day of sensitisation and a day before the challenge to circumvent the short life of IgM. In our subsequent experiments, we have now used busulfan to deplete all bone marrow in IgM-deficient mice and replace it with WT bone marrow and this method restores AHR (Figure 3).

This now appears in line 165 to 169 and reads

“Adoptive transfer of naïve serum

Naïve wild-type mice were euthanised and blood was collected via cardiac puncture before being spun down (5500rpm, 10min, RT) to collect serum. Serum (200mL) was injected intraperitoneally into IgM-deficient mice. Serum was injected intraperitoneally at day -1, 0, and a day before the challenge with HDM (day 10).”

The presence of negative values of total IgE in panel F would indicate some errors in calculation of serum IgE concentrations.

We thank the reviewer for this observation. For better clarity, we have now indicated these values as undetected in Figure , as they were below our detection limit.

Overall, it is hard to be convinced that IgM-deficiency does not lead to a reduction in Th2 inflammation, since the assays appear suboptimal.

We disagree with the reviewer in this instance, because we have shown in 3 different models and in 2 different strains and 2 doses of HDM (high and low) that no matter what you do, Th2 remains intact. Our reason for choosing low dose HDM was based on our previous work and that of others, which showed that depending on antigen load, B cells can either be redundant or have functional roles. Since our interest was to tease out the role of B cells and specifically IgM, it was important that we look at a scenario where B cells are known to have a function (low antigen load). We did find similar findings at high dose of HDM load, but effects on AHR were not as strong, but Th2 was not changed, in fact in some instances Th2 was higher in IgM-deficient mice.

Fig. 3. Gene expression differences between WT and KO mice in PBS and HDM challenged settings are shown. PCA analysis does not show clear differences between all four groups, but genes are certainly up and downregulated, in particular when comparing PBS to HDM challenged mice. In both PBS and HDM challenged settings, three genes stand out as being upregulated in WT v KO mice. these are Baiap2l1, erdr1 and Chil1.

Noted

Fig. 4. The authors attempt to quantify BAIAP2L1 in mouse lungs. It is difficult to know if the antibody used really detects the correct protein. A BAIAP2L1-KO is not used as a control for staining, and I am not sure if competitive assays for BAIAP2L1 can be set up. The flow data is not convincing. The immunohistochemistry shows BAIAP2L1 (in red) in many, many cells, essentially throughout the section. There is also no discernible difference between WT and KO mice, which one might have expected based on the RNA-Seq data. So, from my perspective, it is hard to say if/where this protein is located, and whether there truly exists a difference in expression between wt and ko mice.

We thank the reviewer for this comment. We are certain that the antibody does detect BAIAP2L1, we have used it in 3 assays, which we admit may show varying specificities since it’s a Polyclonal antibody. However, in our western blot, the antibody detects 1 band at 56.7kDa and no other bands, apart from what we think are isoforms. We agree that BAIAP2L1 is expressed by many cell types, including CD45+ cells and alpha smooth muscle negative cells and we show this in our supplementary Figure 9. Where we think there is a difference in expression between WT and IgM-deficient mice is in alpha-smooth muscle-positive cells. We have tested antibodies from different companies, and we find similar findings. We do not have access to BAIAP2L1 KO mice and to test specificity, we have also used single stain controls with or without secondary antibody and isotype control which show no binding in western blot and Immunofluorescence assays and Fluorescence minus one antibody in Flow cytometry, so that way we are convinced that the signal we are seeing is specific to BAIAP2L1.

Fig. 5 and 6. The authors use a single cell contractility assay to measure whether BAIAP2L1 and ERDR1 impact on bronchial smooth muscle cell contractility. I am not familiar with the assay, but it looks like an interesting way of analysing contractility at the single cell level.

The authors state that targeting these two genes with Cas9gRNA reduces smooth muscle cell contractility, and the data presented for contractility supports this observation. However, the efficiency of Cas9-mediated deletion is very unclear. The authors present a PCR in supp fig 9c as evidence of gene deletion, but it is entirely unclear with what efficiency the gene has been deleted. One should use sequencing to confirm deletion. Moreover, if the antibody was truly working, one should be able to use the antibody used in Fig 4 to detect BAIAP2L1 levels in these cells. The authors do not appear to have tried this.

We thank the reviewer for these observations. We are in a process to optimise this using new polyclonal BAIAP2L1 antibodies from other companies, since the one we have tried doesn’t seem to work well on human cells via western blot. So hopefully in our new version, we will be able to demonstrate this by immunofluorescence or western blot.

Other impressions:

The paper is lacking a link between the deficiency of IgM and the effects on smooth muscle cell contraction.

The levels of IL-13 and TNF in lavage of WT and IGMKO mice could be analysed.

We have measured Th2 cytokine IL-13 in BAL fluid and found no differences between IgM-deficient mice and WT mice challenged with HDM (Author response image 1). We could not detected TNF-alpha in the BAL fluid, it was below detection limit.

Author response image 3.

IL-13 levels are not changed in IgM-deficient mice in the lung. Bronchoalveolar lavage fluid in WT or IgM-deficient mice sensitised and challenged with HDM. TNF-a levels were below the detection limit.

Moreover, what is the impact of IgM itself on smooth muscle cells? In the Fig. 7 schematic, are the authors proposing a direct role for IgM on smooth muscle cells? Does IgM in cell culture media induce contraction of SMC? This could be tested and would be interesting, to my mind.

We thank the Reviewer for these comments. We are still trying to test this, unfortunately, we have experienced delays in getting reagents such as human IgM to South Africa. We hope that we will be able to add this in our subsequent versions of the article. We agree it is an interesting experiment to do even if not for this manuscript but for our general understanding of this interaction at least in an in vitro system.

Reviewer #3 (Public Review):

Summary:

This paper by Sabelo et al. describes a new pathway by which lack of IgM in the mouse lowers bronchial hyperresponsiveness (BHR) in response to metacholine in several mouse models of allergic airway inflammation in Balb/c mice and C57/Bl6 mice. Strikingly, loss of IgM does not lead to less eosinophilic airway inflammation, Th2 cytokine production or mucus metaplasia, but to a selective loss of BHR. This occurs irrespective of the dose of allergen used. This was important to address since several prior models of HDM allergy have shown that the contribution of B cells to airway inflammation and BHR is dose dependent.

After a description of the phenotype, the authors try to elucidate the mechanisms. There is no loss of B cells in these mice. However, there is a lack of class switching to IgE and IgG1, with a concomitant increase in IgD. Restoring immunoglobulins with transfer of naïve serum in IgM deficient mice leads to restoration of allergen-specific IgE and IgG1 responses, which is not really explained in the paper how this might work. There is also no restoration of IgM responses, and concomitantly, the phenotype of reduced BHR still holds when serum is given, leading authors to conclude that the mechanism is IgE and IgG1 independent. Wild type B cell transfer also does not restore IgM responses, due to lack of engraftment of the B cells. Next authors do whole lung RNA sequencing and pinpoint reduced BAIAP2L1 mRNA as the culprit of the phenotype of IgM^-/- mice. However, this cannot be validated fully on protein levels and immunohistology since differences between WT and IgM KO are not statistically significant, and B cell and IgM restoration are impossible. The histology and flow cytometry seems to suggest that expression is mainly found in alpha smooth muscle positive cells, which could still be smooth muscle cells or myofibroblasts. Next therefore, the authors move to CRISPR knock down of BAIAP2L1 in a human smooth muscle cell line, and show that loss leads to less contraction of these cells in vitro in a microscopic FLECS assay, in which smooth muscle cells bind to elastomeric contractible surfaces.

Strengths:

(1) There is a strong reduction in BHR in IgM-deficient mice, without alterations in B cell number, disconnected from effects on eosinophilia or Th2 cytokine production

(2) BAIAP2L1 has never been linked to asthma in mice or humans

Weaknesses:

(1) While the observations of reduced BHR in IgM deficient mice are strong, there is insufficient mechanistic underpinning on how loss of IgM could lead to reduced expression of BAIAP2L1. Since it is impossible to restore IgM levels by either serum or B cell transfer and since protein levels of BAIAP2L1 are not significantly reduced, there is a lack of a causal relationship that this is the explanation for the lack of BHR in IgM-deficient mice. The reader is unclear if there is a fundamental (maybe developmental) difference in non-hematopoietic cells in these IgM-deficient mice (which might have accumulated another genetic mutation over the years). In this regard, it would be important to know if littermates were newly generated, or historically bred along with the KO line.

We thank the reviewer for asking this question and getting us to think of this in a different way. This prompted us to use a different method to try and restore IgM function and since our animal facility no longer allows irradiation, we opted for busulfan. We present this data as new data in Figure 3. We had to go back and breed this strain and then generated bone marrow chimeras. What we have shown now with chimeras is that if we can deplete bone marrow from IgM-deficient mice and replace it with congenic WT bone marrow when we allow these mice to rest for 2 months before challenge with HDM (new Supplementary Figure 6 a-c) We also show that AHR (resistance and elastance) is partially restored in this way (Figure 3 a and b) as mice that receive congenic WT bone marrow after chemical irradiation can mount AHR and those that receive IgM-deficient bone marrow, can’t mount AHR upon challenge with HDM. If the mice had accumulated an unknown genetic mutation in non-hematopoietic cells, the transfer of WT bone marrow would not make a difference. So, we don’t believe the colony could have gained a mutation that we are unaware of. We have also shipped these mice to other groups and in their hands, this strains still only behaves as an IgM only knockout mice. See their publication below.

Mark Noviski, James L Mueller, Anne Satterthwaite, Lee Ann Garrett-Sinha, Frank Brombacher, Julie Zikherman 2018. IgM and IgD B cell receptors differentially respond to endogenous antigens and control B cell fate. eLife 2018;7:e35074. DOI: https://doi.org/10.7554/eLife.35074 we have also added methods for bone marrow chimaeras and added results sections and new Figures related to this methods.

Methods (line 171-182).

“Busulfan Bone marrow chimeras

WT (CD45.2) and IgM^-/- (CD45.2) congenic mice were treated with 25 mg/kg busulfan (Sigma-Aldrich, Aston Manor, South Africa) per day for 3 consecutive days (75 mg/kg in total) dissolved in 10% DMSO and Phosphate buffered saline (0.2mL, intraperitoneally) to ablate bone marrow cells. Twenty-four hours after last administration of busulfan, mice were injected intravenously with fresh bone marrow (10x10⁶ cells, 100mL) isolated from hind leg femurs of either WT (CD45.1) or IgM^-/- mice(33). Animals were then allowed to complement their haematopoietic cells for 8 weeks. In some experiments the level of bone marrow ablation was assessed 4 days post-busulfan treatment in mice that did not receive donor cells. At the end of experiment level of complemented cells were also assessed in WT and IgM^-/- mice that received WT (CD45.1) bone marrow.”

Results (line 491-521)

“Replacement of IgM-deficient mice with functional hematopoietic cells in busulfan mice chimeric mice restores airway hyperresponsiveness.

We then generated bone marrow chimeras by chemical radiation using busulfan(33). We treated mice three times with busulfan for 3 consecutive days and after 24 hrs transferred naïve bone marrow from congenic CD45.1 WT mice or CD45.2 IgM^-/- mice (Fig. 3a and Supplementary Fig. 5a). We showed that recipient mice that did not receive donor bone marrow after 4 days post-treatment have significantly reduced lineage markers (CD45+Sca-1+) or lineage negative (Lin-) cells in the bone marrow when compared to untreated or vehicle (10% DMSO) treated mice (Supplementary Figure 5b-c). We allowed mice to reconstitute bone marrow for 8 weeks before sensitisation and challenge with low dose HDM (Figure 3a). We showed that WT (CD45.2) recipient mice that received WT (CD45.1) donor bone marrow had higher airway resistance and elastance and this was comparable to IgM^-/- (CD45.2) recipient mice that received donor WT (CD45.1) bone marrow (Figure 3b). As expected, IgM^-/- (CD45.2) recipient mice that received donor IgM^-/- (CD45.2) bone marrow had significantly lower AHR compared to WT (CD45.2) or IgM^-/- (CD45.2) recipient mice that received WT (CD45.1) bone marrow (Figure 3b). We confirmed that the differences observed were not due to differences in bone marrow reconstitution as we saw similar frequencies of CD45.1 cells within the lymphocyte populations in the lungs and other tissues (Supplementary Fig. 5d). We observed no significant changes in the lung neutrophils, eosinophils, inflammatory macrophages, CD4 T cells or B cells in WT or IgM^-/- (CD45.2) recipient mice that received donor WT (CD45.1/CD45.2) or IgM^-/- (CD45.2) bone marrow when sensitised and challenged with low dose HDM (Fig. 3c)

Restoring IgM function through adoptive reconstitution with congenic CD45.1 bone marrow in non-chemically irradiated recipient mice or sorted B cells into IgM^-/- mice (Supplementary Fig.  6a) did not replenish IgM B cells to levels observed in WT mice and as a result did not restore AHR, total IgE and IgM in these mice (Supplementary Fig.  6b-c).”

The 2 new figures are

Figure 3 which moved the rest of the Figures down and Supplementary Figure 5, which also moved the rest of the supplementary figures down.

Discussion appears in line 757-766 of the untracked version of the article.

To resolve other endogenous factors that could have potentially influenced reduced AHR in IgM-deficient mice, we resorted to busulfan chemical irradiation to deplete bone marrow cells in IgM-deficient mice and replace bone marrow with WT bone marrow. While it is well accepted that busulfan chemical irradiation partially depletes bone marrow cells, in our case it was not possible to pursue other irradiation methods due to changes in ethical regulations and that fact that mice are slow to recover after gamma rays irradiation. Busulfan chemical irradiation allowed us to show that we could mostly restore AHR in IgM-deficient recipient mice that received donor WT bone marrow when challenged with low dose HDM.

(2) There is no mention of the potential role of complement in activation of AHR, which might be altered in IgM-deficient mice 

We thank the reviewer for this comment. We have not directly looked at complement in this instance, however, from our previous work on C3-/- mice, there have been comparable AHR to WT mice under the HDM challenge.

(3) What is the contribution of elevated IgD in the phenotype of the IgM-deficient mice. It has been described by this group that IgD levels are clearly elevated

We thank the reviewer for this question. We believe that IgD is essentially what drives partial class switching to IgG, we certainly have shown that in the case of VSV virus and Trypanosoma congolense and Trypanosoma brucei brucei that elevated IgD drive delayed but effective IgG in the absence of IgM (Lutz et al, 2001, Nature). This is also confirmed by Noviski studies where they show that both IgM and IgD do share some endogenous antigens, so its likely that external antigens can activate IgD in a similar manner to prompt class switching.

(4) How can transfer of naïve serum in class switching deficient IgM KO mice lead to restoration of allergen specific IgE and IgG1?

We thank the Reviewer for these comments, we believe that naïve sera transferred to IgM deficient mice is able to bind to the surface of B cells via IgM receptors (FcμR / Fcα/μR), which are still present on B cells and this is sufficient to facilitate class switching. Our IgM^-/- mouse lacks both membrane-bound and secreted IgM, and transferred serum contains at least secreted IgM which can bind to surfaces via its Fc portion. We measured HDM-specific IgE and we found very low levels, but these were not different between WT and IgM^-/- adoptively transferred with WT serum. We also detected HDM-specific IgG1 in IgM^-/- transferred with WT sera to the same level as WT, confirming a possible class switching, of course, we can’t rule out that transferred sera also contains some IgG1. We also can’t rule out that elevated IgD levels can partially be responsible for class switched IgG1 as discussed above.

In the discussion line 804-812, we also added the following

“We speculate that IgM can directly activate smooth muscle cells by binding a number of its surface receptors including FcμR, Fcα/μR and pIgR(52-54). IgM binds to FcμR strictly, but shares Fcα/μR and pIgR with IgA(5,52,54). Both Fcα/μR and pIgR can be expressed by non-structural cells at mucosal sites(54,55). We would not rule out that the mechanisms of muscle contraction might be through one of these IgM receptors, especially the ones expressed on smooth muscle cells(54,55). Certainly, our future studies will be directed towards characterizing the mechanism by which IgM potentially activates the smooth muscle.”

We have discussed this section under Discussion section, line 731 to 757. In addition, since we have now performed bone marrow chimaeras we have further added the following in our discussion in line 757-766.

To resolve other endogenous factors that could have potentially influenced reduced AHR in IgM-deficient mice, we resorted to busulfan chemical irradiation to deplete bone marrow cells in IgM-deficient mice and replace bone marrow with WT bone marrow. While it is well accepted that busulfan chemical irradiation partially depletes bone marrow cells, in our case it was not possible to pursue other irradiation methods due to changes in ethical regulations and that fact that mice are slow to recover after gamma rays irradiation. Busulfan chemical irradiation allowed us to show that we could mostly restore AHR in IgM-deficient recipient mice that received donor WT bone marrow when challenged with low dose HDM.

We removed the following lines, after performing bone marrow chimaeras since this changed some aspects.

Our efforts to adoptively transfer wild-type bone marrow or sorted B cells into IgM-deficient mice were also largely unsuccessful partly due to poor engraftment of wild-type B cells into secondary lymphoid tissues. Natural secreted IgM is mainly produced by B1 cells in the peritoneal cavity, and it is likely that any transfer of B cells via bone marrow transfer would not be sufficient to restore soluble levels of IgM(3,10).

(5) Alpha smooth muscle antigen is also expressed by myofibroblasts. This is insufficiently worked out. The histology mentions "expression in cells in close contact with smooth muscle". This needs more detail since it is a very vague term. Is it in smooth muscle or in myofibroblasts.

Response: We appreciate that alpha-smooth muscle actin-positive cells are a small fraction in the lung and even within CD45 negative cells, but their contribution to airway hyperresponsiveness is major. We also concede that by immunofluorescence BAIAP2L1 seems to be expressed by cells adjacent to alpha-smooth muscle actin (Fig. 5b), however, we know that cells close to smooth muscle (such as extracellular matrix and myofibroblasts) contribute to its hypertrophy in allergic asthma.

James AL, Elliot JG, Jones RL, Carroll ML, Mauad T, Bai TR, et al. Airway Smooth Muscle Hypertrophy and Hyperplasia in Asthma. Am J Respir Crit Care Med [Internet]. 2012;185:1058–64. Available from: https://doi.org/10.1164/rccm.201110-1849OC

(6) Have polymorphisms in BAIAP2L1 ever been linked to human asthma?

No, we have looked in asthma GWAS studies, at least summary statics and we have not seen any SNPs can could be associated with human asthma.

(7) IgM deficient patients are at increased risk for asthma. This paper suggests the opposite. So the translational potential is unclear

We thank the reviewer for these comments. At the time of this publication, we have not made a concrete link with human disease. While there is some anecdotal evidence of diseases such as Autoimmune glomerulonephritis, Hashimoto’s thyroiditis, Bronchial polyp, SLE, Celiac disease and other diseases in people with low IgM. Allergic disorders are also common in people with IgM deficiency as the reviewer correctly points out, other studies have reported as high as 33-47%. The mechanisms for the high incidence of allergic diseases are unclear as generally, these patients have normal or higher IgG and IgE levels. IgM deficiency may represent a heterogeneous spectrum of genetic defects, which might explain the heterogeneous nature of disease presentations.

maybe his original goal- Britain a stopping point, have his power validated by the big imperial powers of the time

shown what they want him to see

Why bread and whipped cream??? like at Tous Les Jours…

Cute

eLife Assessment

This important study describes how a single effector of the Type Six Secretion System (T6SS) has two distinct functions, which may contribute to bacterial survival and the development of novel antibacterials. The authors utilized various methods in biochemistry, microbiology, and microscopy to produce convincing data supporting their claims about the protein's function; however, they could clarify the implications for non-experts to enhance the accessibility of this work. This manuscript is of interest to those studying T6SS, particularly those interested in effectors and bacterial enzymes.

Reviewer #1 (Public review):

Summary:

The manuscript performs a comprehensive biochemical, structural, and bioinformatic analysis of TseP, a type 6 secretion system effector from Aeromonas dhakensis that includes identification of a domain required for secretion and residues conferring target organism specificity. Through targeted mutations, they have expanded the target range of a T6SS effector to include a gram-positive species, which are not typically susceptible to T6SS attack. Although this is not the first dual domain effector to be described, this is the first time anyone has been able to modify a T6SS effector to have an expanded target species range.

Strengths:

The thorough dissection of TseP activity and modulation of target specificity represent a novel contribution to the field of antibacterial research.

Weaknesses:

Although the mechanistic activity of TseP is fully dissected here, there are some unaddressed questions regarding the importance/evolution of the dual activity domain organization. For example, does the modified Gram-positive targeting TseP effector still kill Gram-negative bacteria in bacterial mixtures? And if so, what is the evolutionary benefit of having a TseP that cannot target Gram-positives? And can something be inferred about the biology of Aeromonas from this?

Comments on revisions:

The comments and critiques from the initial submission have been addressed. However, some of them have only been addressed in the author's rebuttal. Some of the discussion particularly regarding the validity of using E. coli PG, the ability for TseP_C4+ to still kill E. coli, and the advantages of having dual domain function effectors probably should be present in the actual manuscript.

Reviewer #2 (Public review):

Summary:

Wang et al. investigate the role of TseP, a Type VI secretion system (T6SS) effector molecule, revealing its dual enzymatic activities as both an amidase and a lysozyme. This discovery significantly enhances the understanding of T6SS effectors, which are known for their roles in interbacterial competition and survival in polymicrobial environments. TseP's dual function is proposed to play a crucial role in bacterial survival strategies, particularly in hostile environments where competition between bacterial species is prevalent.

Strengths:

(1) The dual enzymatic function of TseP is a significant contribution, expanding the understanding of T6SS effectors.<br /> (2) The study provides important insights into bacterial survival strategies, particularly in interbacterial competition.<br /> (3) The findings have implications for antimicrobial research and understanding bacterial interactions in complex environments.

Weaknesses:

(1) The manuscript assumes familiarity with previous work, making it difficult to follow. Mutants and strains need clearer definition and references.<br /> (2) Figures lack proper controls, quantification, and clarity in some areas, notably in Figures 1A and 1C.<br /> (3) The Materials and Methods section is poorly organized, hindering reproducibility. Biophysical validation of Zn²⁺ interaction and structural integrity of proteins need to be addressed.<br /> (4) Discrepancies in protein degradation patterns and activities across different figures raise concerns about data reliability.

Comments on revisions:

The authors have addressed most of the comments, significantly improving the manuscript. They provided clear details of mutant constructs and strains, including additional references and a revised strain. Individual data points and statistical analyses were added to key figures, ensuring transparency and reproducibility. Supplemental data, such as protein purification details and loading controls, were included to address concerns about experimental reliability. However, the authors did not perform new experiments, such as isothermal titration calorimetry (ITC) to demonstrate the interaction between Zn²⁺ and TsePN or stop-flow spectroscopy to examine enzymatic kinetics, which could have further strengthened the manuscript. I trust these aspects will be addressed in future studies.

The revised Materials and Methods section was significantly improved, providing detailed protocols for bioinformatics analyses, microscopic imaging, and enzymatic assays.

These revisions provide a clearer and more robust presentation of TseP's dual enzymatic functions and their implications in bacterial competition. The manuscript now represents a significant contribution to understanding T6SS effectors, and I recommend it for publication in its current form.

Reviewer #3 (Public review):

Summary:

Type VI secretion systems (T6SS) are employed by bacteria to inject competitor cells with numerous effector proteins. These effectors can kill injected cells via an array of enzymatic activities. A common class of T6SS effector are peptidoglycan (PG) lysing enzymes. In this manuscript, the authors characterize a PG-lysing effector-TseP-from the pathogen Aeromonas dhakensis. While the C-terminal domain of TseP was known to have lysozyme activity, the N-terminal domain was uncharacterized. Here, the authors functionally characterize TsePN as a zinc-dependent amidase. This discovery is somewhat novel because it is rare for PG-lysing effectors to have amidase and lysozyme activity. In the second half of the manuscript, the authors utilize a crystal structure of the lysozyme TsePC domain to inform the engineering of this domain to lyse gram-positive peptidoglycan.

Strengths:

The two halves of the manuscript considered together provide a nice characterization of a unique T6SS effector and reveal potentially general principles for lysozyme engineering.

Weaknesses:

The advantage of fusing amidase and lysozyme domains in a single effector is not discussed but would appear to be a pertinent question.

Comments on revisions:

The authors have adequately addressed my previous comments. The authors did not conduct any additional experiments to address the comments made by other reviewers. However, in most cases it seems that paring down the strength of claims made in the text or adding data to the supplement is sufficient to address these concerns.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The manuscript performs a comprehensive biochemical, structural, and bioinformatic analysis of TseP, a type 6 secretion system effector from Aeromonas dhakensis that includes the identification of a domain required for secretion and residues conferring target organism specificity. Through targeted mutations, they have expanded the target range of a T6SS effector to include a gram-positive species, which is not typically susceptible to T6SS attack.

Strengths:

All of the experiments presented in the study are well-motivated and the conclusions are generally sound.

Thank you.

Weaknesses:

There are some issues with the clarity of figures. For example, the microscopy figures could have been more clearly presented as cell counts/quantification rather than representative images. Similarly, loading controls for the secreted proteins for the westerns probably should be shown.

Also, some of the minor/secondary conclusions reached regarding the "independence" of the N and C term domains of the TseP are a bit overreaching.

We thank the reviewer for pointing out the issues and have carefully revised the manuscript accordingly. We acknowledge the reviewer’s concern regarding the independence of the N- and C-terminal domains, and have toned down the relevant claims.

Reviewer #2 (Public review):

Summary:

Wang et al. investigate the role of TseP, a Type VI secretion system (T6SS) effector molecule, revealing its dual enzymatic activities as both an amidase and a lysozyme. This discovery significantly enhances the understanding of T6SS effectors, which are known for their roles in interbacterial competition and survival in polymicrobial environments. TseP's dual function is proposed to play a crucial role in bacterial survival strategies, particularly in hostile environments where competition between bacterial species is prevalent.

Strengths:

(1) The dual enzymatic function of TseP is a significant contribution, expanding the understanding of T6SS effectors.

(2) The study provides important insights into bacterial survival strategies, particularly in interbacterial competition.

(3) The findings have implications for antimicrobial research and understanding bacterial interactions in complex environments.

Thank you.

Weaknesses:

(1) The manuscript assumes familiarity with previous work, making it difficult to follow. Mutants and strains need clearer definitions and references.

Thank you for raising the issue. We have revised the manuscript accordingly to improve the clarity by including more detailed descriptions of the mutants and strains, along with references to prior work where relevant, to improve clarity.

(2) Figures lack proper controls, quantification, and clarity in some areas, notably in Figures 1A and 1C.

We have now added the controls as requested by reviewers.

(3) The Materials and Methods section is poorly organized, hindering reproducibility. Biophysical validation of Zn²⁺ interaction and structural integrity of proteins need to be addressed.

We have now included more details in the Materials and Methods section. While we recognize the importance of biophysical validation of the Zn²⁺ interaction, this analysis lies beyond the primary scope of the current study. We plan to investigate the role of Zn²⁺ interaction and the EF-hand domain in greater depth as part of our follow-up studies. Thank you for this suggestion.

(4) Discrepancies in protein degradation patterns and activities across different figures raise concerns about data reliability.

We acknowledge the concern about discrepancies in protein degradation patterns. TseP exhibits inherent instability, which might explain the observed variations. We have added an explanation in the detailed response letter and the manuscript.

Reviewer #3 (Public review):

Summary:

Type VI secretion systems (T6SS) are employed by bacteria to inject competitor cells with numerous effector proteins. These effectors can kill injected cells via an array of enzymatic activities. A common class of T6SS effector are peptidoglycan (PG) lysing enzymes. In this manuscript, the authors characterize a PG-lysing effector-TseP-from the pathogen Aeromonas dhakensis. While the C-terminal domain of TseP was known to have lysozyme activity, the N-terminal domain was uncharacterized. Here, the authors functionally characterize TsePN as a zinc-dependent amidase. This discovery is somewhat novel because it is rare for PG-lysing effectors to have amidase and lysozyme activity.

In the second half of the manuscript, the authors utilize a crystal structure of the lysozyme TsePC domain to inform the engineering of this domain to lyse gram-positive peptidoglycan.

Strengths:

The two halves of the manuscript considered together provide a nice characterization of a unique T6SS effector and reveal potentially general principles for lysozyme engineering.

Thank you.

Weaknesses:

The advantage of fusing amidase and lysozyme domains in a single effector is not discussed but would appear to be a pertinent question. Labeling of the figures could be improved to help readers understand the data.

Thank you for the suggestions. We have revised the manuscript and figures to improve clarity.

The advantage of having dual-domain functions relative to having just one of the two functions is likely for increasing competitive fitness. Although such dual functional cell-wall targeting effectors have not been characterized prior to this study, there are some examples that dual functions are encoded by the same secretion module, for example the VgrG1-TseL pair in Vibrio cholerae. The C-terminal of VgrG1 not only catalyzes actin crosslinking but also recognizes and delivers the downstream encoded lipase effector TseL through direct interaction. In this context, the VgrG1-TseL pair also represent a dual-functional module. Therefore, it is likely that fusing effector domains and coupling effector functions are parallel strategies for the evolution of T6SS effectors.

eLife Assessment

This manuscript reports an important new statistical method for calculating the significance of correlations between two time-series, which provides more accuracy than other methods when the data has few replicates. The proposed method solves a real-life problem that is frequently encountered and is broadly applicable to many realistic datasets in many experimental contexts. The technique is supported with compelling mathematical derivations as well as analysis of both computer-generated and previously published experimental data.

Both women were too preoccupied and engulfed by fear that they didn't even consider this as a possibility. Despite being previously described as "stiff" and thought of as soulless, we see the elder as quite human in this instance.

Especially when thinking about 1. oscillators and 2. various field like the electromagnetic field.

A famous type of example. Einstein wrote his first papers on relativity with explanations of street cars moving at various speeds relative to an observer on the sidewalk. 🙂

I came across an interesting discussion on Reddit titled “Reddit is valued at more than ten billion dollars, yet it is extremely dependent on mods who work for absolutely nothing. Should they be paid, and does this lead to power-tripping mods?” This was posted in November 2021 in the r/TheoryOfReddit subreddit and raised a thought-provoking point about the power dynamics in online communities.