i love quincey’s brain. like his thought process is so great it’s just:

i once saw a bat drink so much blood from a horse that the horse nearly died -> the woman i love is losing massive amounts of blood everytime there’s a bat outside her window -> i now shoot bats on sight

like forget “doing the math wrong but reaching the right conclusion” motherfucker barely did math At All but he got the answer Perfectly

original post

Mina: Oh, hello Mr. Swales.

Old Man: Listen, Mina, I'm sorry I upset you by talking about death before. It's just that death comes for us all, we can't escape it, we are all doomed forever, and I'm probably going to die right in front of you right now

Mina: -now openly weeping-

Old Man: no no no listen see it's fine, because everyone on earth could die at any second, really, even young lawyers who have fiancees waiting at home who really love them, so don't be upset

Old Man: because Death is always coming towards us on the horizon, like that ship over there in the storm

Dracula, presumably steering the Demeter: I DON'T KNOW HOW BOATS WORK

original post

Notation of a random process that has outcomes

The "universal set" aka "sample space" of all possible outcomes is sometimes denoted by \(U\), \(S\), or \(\Omega\): https://en.wikipedia.org/wiki/Sample_space

Probability theory & measure theory

From what I recall, the notation, \(\Omega\), was mainly used in higher-level grad courses on probability theory. ie, when trying to frame things in probability theory as a special case of measure theory things/ideas/processes. eg, a probability space, \((\cal{F}, \Omega, P)\) where \(\cal{F}\) is a \(\sigma\text{-field}\) aka \(\sigma\text{-algebra}\) and \(P\) is a probability density function on any element of \(\cal{F}\) and \(P(\Omega)=1.\)

Somehow, the definition of a sigma-field captures the notion of what we want out of something that's measurable, but it's unclear to me why so let's see where writing through this takes me.

Working through why a sigma-algebra yields a coherent notion of measureable

A sigma-algebra \(\cal{F}\) on a set \(\Omega\) is defined somewhat close to the definition of a topology \(\tau\) on some space \(X\). They're both collections of sub-collections of the set/space of reference (ie, \(\tau \sub 2^X\) and \(\cal{F} \sub 2^\Omega\)). Also, they're both defined to contain their underlying set/space (ie, \(X \in \tau\) and \(\Omega \in \cal{F}\)).

Additionally, they both contain the empty set but for (maybe) different reasons, definitionally. For a topology, it's simply defined to contain both the whole space and the empty set (ie, \(X \in \tau\) and \(\empty \in \tau\)). In a sigma-algebra's case, it's defined to be closed under complements, so since \(\Omega \in \cal{F}\) the complement must also be in \(\cal{F}\)... but the complement of the universal set \(\Omega\) is the empty set, so \(\empty \in \cal{F}\).

I think this might be where the similarity ends, since a topology need not be closed under complements (but probably has a special property when it is, although I'm not sure what; oh wait, the complement of open is closed in topology, so it'd be clopen! Not sure what this would really entail though 🤷‍♀️). Moreover, a topology is closed under arbitrary unions (which includes uncountable), but a sigma-algebra is closed under countable unions. Hmm... Maybe this restriction to countable unions is what gives a coherent notion of being measurable? I suspect it also has to do with Banach-Tarski paradox. ie, cutting a sphere into 5 pieces and rearranging in a clever way so that you get 2 sphere's that each have the volume of the original sphere; I mean, WTF, if 1 sphere's volume equals the volume of 2 sphere's, then we're definitely not able to measure stuff any more.

And now I'm starting to vaguely recall that this what sigma-fields essentially outlaw/ban from being possible. It's also related to something important in measure theory called a Lebeque measure, although I'm not really sure what that is (something about doing a Riemann integral but picking the partition on the y-axis/codomain instead of on the x-axis/domain, maybe?)

And with that, I think I've got some intuition about how fundamental sigma-algebras are to letting us handle probability and uncertainty.

Back to probability theory

So then events like \(E_1\) and \(E_2\) that are elements of the set of sub-collections, \(\cal{F}\), of the possibility space \(\Omega\). Like, maybe \(\Omega\) is the set of all possible outcomes of rolling 2 dice, but \(E_1\) could be a simple event (ie, just one outcome like rolling a 2) while \(E_2\) could be a compound(?) event (ie, more than one, like rolling an even number). Notably, \(E_1\) & \(E_2\) are NOT elements of the sample space \(\Omega\); they're elements of the powerset of our possibility space (ie, the set of all possible subsets of \(\Omega\) denoted by \(2^\Omega\)). So maybe this explains why the "closed under complements" is needed; if you roll a 2, you should also be able to NOT roll a 2. And the property that a sigma-algebra must "contain the whole space" might be what's needed to give rise to a notion of a complete measure (conjecture about complete measures: everything in the measurable space can be assigned a value where that part of the measurable space does, in fact, represent some constitutive part of the whole).

But what about these "random events"?

Ah, so that's where random variables come into play (and probably why in probability theory they prefer to use \(\Omega\) for the sample space instead of \(X\) like a base space in topology). There's a function, that is, a mapping from outcomes of this "random event" (eg, a role of 2 dice) to a space in which we can associate (ie, assign) a sense of distance (ie, our sigma-algebra). What confuses me is that we see things like "\(P(X=x)\)" which we interpret as "probability that our random variable, \(X\), ends up being some particular outcome \(x\)." But it's also said that \(X\) is a real-valued function, ie, takes some arbitrary elements (eg, events like rolling an even number) and assigns them a real number (ie, some \(x \in \mathbb{R}\)).

Aha! I think I recall the missing link: the notation "\(X=x\)" is really a shorthand for "\(X(\omega)=x\)" where \(\omega \in \cal{F}\). But something that still feels unreconciled is that our probability metric, \(P\), is just taking some real value to another real value... So which one is our sigma-algebra, the inputs of \(P\) or the inputs of \(X\)? 🤔 Hmm... Well, I guess it has the be the set of elements that \(X\) is mapping into \(\mathbb{R}\) since \(X\text{'s}\) input is a small omega \(\omega\) (which is probably an element of big omega \(\Omega\) based on the conventions of small notation being elements of big notation), so \(X\text{'s}\) domain much be the sigma-algrebra?

Let's try to generate a plausible example of this in action... Maybe something with an inequality like "\(X\ge 1\)". Okay, yeah, how about \(X\) is a random variable for the random process of how long it takes a customer to get through a grocery line. So \(X\) is mapping the elements of our sigma-algebra (ie, what customers actually end up experiencing in the real world) into a subset of the reals, namely \([0,\infty)\) because their time in line could be 0 minutes or infinite minutes (geesh, 😬 what a life that would be, huh?). Okay, so then I can ask a question like "What's the probability that \(X\) takes on a value greater than or equal to 1 minute?" which I think translates to "\(P\left(X(\omega)\ge 1\right)\)" which is really attempting to model this whole "random event" of "What's gonna happen to a particular person on average?"

So this makes me wonder... Is this fact that \(X\) can model this "random event" (at all) what people mean when they say something is a stochastic model? That there's a probability distribution it generates which affords us some way of dealing with navigating the uncertainty of the "random event"? If so, then sigma-algebras seem to serve as a kind of gateway and/or foundation into specific cognitive practices (ie, learning to think & reason probabilistically) that affords us a way out of being overwhelmed by our anxiety or fear and can help us reclaim some agency and autonomy in situations with uncertainty.

https://www.youtube.com/watch?v=FWiyKgeGWx0 Overall, "The Burial of the Dead" makes me think of "O Fortuna" by Carl Orff, they're both dreary with undertones of change, cycles, and the endless march of time, though, "O Fortuna" is much more cynical where "The Burial of the Dead" seems to hinge on the hope that things will get better. When it comes the highlighted area, the way Eliot described winter made me think of a wasteland covered in snow, but then shows how things start to change so suddenly so that it's summer again; bringing up the cycle that life goes through. In "O Fortuna", one of the lines (translated) is: Hateful life First oppresses And then soothes As fancy takes it; Poverty and power It melts them like ice. This line is why I chose "O Fortuna", because it shares the idea of life getting hard and oppressive (oppressing what? Dreams, memories, feelings?) and harsh (like winter) but then soothes the pain and oppression with warmth. But never does the song suggest that things will say prosperous, in fact it emphasizes that life gets easier and harder as fate dictates.

In "O Fortuna", fortune seems to be some sort of sentient/god like being who is blamed for all of the hardships mentioned in the poem, unlike in "The Waste Land", where a lot of the blame seems to be put on the cycle that life goes through. The original poet suggests there's a certain amount of helplessness for humans where they are held at the "fancy" of fate and the changes it might go through and the song itself mentions all of the cycles life (and it's inhabitants) go through with an emphasis on the harsher parts of life (poverty, health issues, death). With "The Waste Land", Eliot seems to cycle through the positive and negative parts of certain speakers lives and the harsher periods of nature, encapsulating the cycles that the world goes through that "O Fortuna" merely hinted at. When it comes to which one is more optimistic, I'd go with "The Waste Land" because there's always a lingering hope that something good is just around the corner in the poem, that something good could grow. In the end of the poem, a speaker asks another if something is growing from the corpse that was planted, "'Has it begun to sprout? Will it bloom this year?'". Meanwhile, "O Fortuna" ends with: So at this hour Without delay Pluck the vibrating strings; Since Fate Strikes down the strong man Everyone weep with me! The poem suggests that there's nothing anybody can do to stop the death that is approaching everyone. There’s no chance of saving nor does it provide any hope that things will turn out alright like Eliot did in “The Waste Land” and his suggestions of growth coming out of the frost.

https://www.youtube.com/watch?v=9CAz_vvsK9M

The lines about fearing mortality and death staring you in the face like your own shadow reminded me of the song "At The Door" by The Strokes, which is about the inevitability of death.

Some lines from the song:

"I can't escape it/ I'm never gonna make it out of this in time/ I guess that's just fine/ I'm not there quite yet/ My thoughts, such a mess"

These lines relate back to the beginning of the poem, where the speaker found comfort in the "forgetful snow, feeding/ A little life with dried tubers". The snow kept the speaker in a warm, sleepy state where they could forget about their anxiety over mortality. In contrast, the spring and summer "surprise" them by reminding them that life and death keep moving. Similarly, the lyrics to "At The Door" show a narrator who is afraid to face/accept death and is overwhelmed by its inevitability. Just as the speaker in "The Wasteland" is "surprised" by the summer, the narrator in "At The Door" says mortality "Struck [them] like a chord".

The narrator in the song also says:

"Sinkin' like a stone/ Use me like an oar/ And get yourself to shore"

Which is similar to the part in the poem where a corpse is planted and someone asks the speaker if it's begun to sprout yet. In both cases, a dead human body is being used in a utilitarian way, either to add compost to a garden or to help someone row themselves to shore, showing an interesting tension between nature and humans. Death is a part of nature, but we still feel disturbed and scared of it. Using a corpse as an oar is a morbid thought for us, and yet, a dog or animal wouldn't hesitate to "dig [a corpse] up again" as the poem says.

comments for tag: undefined vs. null: Technically this is undefined (unset, !diff) vs. true (diff), but it's similar enough that don't need a separate tag just for that.

annotation meta: may need new tag: undefined/unset vs. null/set

why does the account have to be in the merkle tree? Is it because only certain accounts are allowed to guess? So it's just specific to this app's goals and not necessary?

The news value being represented here is conflict. This is an example of conflict because of how spokeswoman Natalie Edelstein spoke about how a piece of news could be interpreted one way that is false and has a hidden message that is meant to support only Republican propaganda.

A metaphor to show that people were thankful that the tour dates did not affect their wedding plans. They did not breather a sigh of relief literally, it’s just a figure of speech.

I think this can go outside of nav because it's not technically part of navigation. That way you don't need a div for the logo, it can just be in header

This passage stuck out to me because it highlights the insidiousness of structural bias in institutions of power. A phrase I've often heard is that "if it's not in writing, then it's not legally binding". I would that this is evident here because if there were to be no explicit mention of women's rights as human rights then in the context of war crimes, gendered violence would just be seen as another footnote or supplemental fact in the history books because what's not illegal could legitimately be argued not enforceable by law. Otherwise, due to lack of gender affirming language, the patriarchy would be maintained.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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Reply to the reviewers

We really appreciate the reviewers’ insightful comments, which help improve the quality of this work. We have responded to the reviewers’ questions/comments point by point in the following text and made the corresponding changes in the revised manuscript. Lastly, we added one more figure (Fig. 7) with lineage tracing experiments demonstrating the conversion of id2a+ liver ductal cells to hepatocytes in extreme hepatocyte loss condition.

Reviewer #1 (Evidence, reproducibility and clarity):

Mi and Andersson describe a method for creating efficient 3' knock-ins in zebrafish using a combination of end-modified dsDNA and Cas9/gRNA RNPs. They tested their method on four genetic loci where they introduced Cre recombinase endogenously, and obtained high F0 mosaicism and germline transmission. The authors included fluorescent proteins with self-cleaving peptides to determine that endogenous expression patterns are observed. By crossing their knock-in Cre lines with lineage tracing reporter lines, the authors temporally traced lineage divergences in zebrafish liver and pancreas.

The authors should clarify the following points before I can recommend publication:

Overall, I suggest that the authors consider paring down their figures. Throughout the paper, multiple figure panels convey the same point but for different genes. Furthermore, many construct configurations are shown that are not used in the subsequent panels. For example, the mNeonGreen only (no Cre) constructs and the EGFP constructs are largely not used in downstream experiments. The authors could pick the important constructs and show the relevant data, and summarize all their other constructs in one supplementary figure. The authors also jump around in different parts of the paper with regards to using iCre or CreERT2 and ubi:Switch or ubi:CSHm. It's not clear to me why they're doing that? It makes the paper hard to follow. For example, why use iCre - it's not temporal if I understand correctly (and I'm not sure what improved Cre is - could they reference a paper and include a small explanation) so CreERT2 seems suitable especially for their temporal lineage tracing experiments. Why not limit the description to CreERT2 in the main text/figures? Also, isn't ubi:Switch and ubi:CSHm pretty similar except the latter is nuclear mCherry due to H2B? Why not only focus on ubi:CSHm experiments? I found the paper to be unnecessarily long and think it would benefit from editing to describe the most important concepts and experiments.

Response: Thank you for your constructive and helpful comments. We do agree that sometimes the schematic constructs seem redundant. This is because the krt4, nkx6.1, and id2a genes have similar gRNA targeting sites (all spanning over the stop codon). However, we prefer to keep these schematic constructs as we have all the statistical results showing the knock-in efficiency in the subsequent figure panels. Such layout can allow readers to make comparisons and better understand the efficacy of this method. However, combined with the comments from the second reviewer, we indeed need to add more detailed information, including the sequence and the length of the short left and right homologous arms in the schematics, to enable the readers to follow this strategy more easily. Meanwhile, we added a new supplementary figure with the sequences of the long left and right homologous arms, as well as the genetic cassettes/point mutations for krt92 knock-in (Figure EV1).

As for the color switch lines we used, we appreciate your comments and replaced Fig. 5E-G with new fluorescent images using zebrafish larvae carrying the ubb:CSHm transgene. For most of the lineage tracing experiments in this study, we used Tg(ubb:CSHm) as the H2BmCherry is more stable, located in the nucleus, and the fluorescence intensity is stronger than in Tg(ubb:Switch). However, for the lineage tracing experiments in the liver injury model, we believe that Tg(ubb:switch) is a better option than Tg(ubb:CSHm). In the absence of a hepatocyte specific far-red reporter line, we can distinguish the hepatocytes derived from the id2a+ origin using the Tg(ubb:Switch) line, as the cells with Cre recombination express mCherry in the cytoplasm; i.e. we can tell the cell types based on the cell morphology in combination with the ductal anti-vasnb staining. This strategy was previously used by Dr. Donghun Shin’s group in their 2014 Gastroenterology paper (Figure 4B, DOI: 10.1053/j.gastro.2013.10.019). Therefore, we still kept the ubb:switch in the Fig. 1F schematic, and we have elaborated on why we chose Tg(ubb:switch) line for the id2a+ cell conversion experiments in Fig. 7 and Figure EV14.

The iCre we used is a codon-improved Cre (iCre). The original cDNA sequence was from pDIRE (Addgene plasmid #26745; provided by Dr. Rolf Zeller, University of Basel) (Osterwalder et al., 2010).

At the beginning of this project, we actually didn’t know whether there were any differences between iCre and CreERT2 in labelling of the cells of interest. Here, using both the iCre and CreERT2 lines, we for the first time, formally show the developmental lineage path of nkx6.1-expressing cells in the zebrafish pancreas. Our data suggested that the early nkx6.1-expressing cells are multipotent pancreatic progenitors giving rise to all three major cell types in the pancreas (endocrine, ductal and acinar cells, shown by nkx6.1 knock-in iCre) and gradually the nkx6.1-expressing cells become restricted in the ductal/endocrine lineages (shown by the nkx6.1 knock-in CreERT2 treated with 4-OHT at different timepoints). In addition, we also aim to use these knock-in lines for multiple studies in which we need to perform many quantitative experiments. As expected, we are unable to reach 100% labeling using the knock-in CreERT2 lines, even if we treated the larvae with very high concentration of 4-OHT over a long period of time. This means that the CreERT2 induced recombination will introduce more variation for quantitative experiments (for instance, the number of regenerated beta-cells from the ductal origin). As we were quite confident with the efficiency of this knock-in strategy, we decided to make both iCre and CreERT2 lines in krt4, nkx6.1, and id2a locus and just observe how they performed. We often use iCre knock-in lines for lineage tracing experiments, because the iCre lines reach near 100% labeling efficiency. Such iCre lines are particularly useful if they only label terminally differentiated cell types. Thus, the near 100% labeling efficiency in iCre lines can be of great help for initial experiments, which later can be confirmed by temporal labeling using CreERT2 lines.

1. Could the authors describe the purpose of the 5'AmC6 modification earlier in the paper? I didn't see much text about it until the discussion. It seems that the speculation is that it provides end protection and prevents degradation (based on in vitro studies in human). This should be inserted into the introduction as a reader might be wondering about this and won't find an answer until near the end. Also, is this the first in vivo use of this modification for knock-ins? If so, that should be highlighted in the text.

Response: This is a helpful comment. In the revised manuscript, we elaborate more on why we chose 5'AmC6 modification in our donors. To our knowledge, this is the first time this 5’ modification is used in vivo, however, bulky 5’modification (5'Biotin - 5x phosphorothioate bonds) has been used in medaka (DOI: https://doi.org/10.7554/eLife.39468.001, 2018 Elife, as we previously referenced). The cell division rate is much faster in zebrafish embryos compared with medaka embryos during early development, so we speculate that such modification might be of more importance in zebrafish to achieve early integration. Another advantage is that the 5'AmC6 modification is commercially available, allowing researchers to prepare the donor dsDNA in a handy fashion. We have now expanded on these details and advantages in the introduction.

1. The authors do not show any sequencing data confirming that their insert was knocked-in as designed with no disruption to the immediate upstream and downstream endogenous sequences. Can they sequence the loci to confirm?

Response: This is indeed a question we frequently get – thank you for making us relay this information more clearly! We have put the raw Sanger sequencing data in a public repository (mentioned in the Data Availability section), and included the sequencing primers in the method paragraph. Now we also refer to this data in the discussion section in conjunction to highlighting that the integrations were correctly placed in the loci. If you think there are better ways to show the sequencing results, please let us know.

1. I found the descriptions of the long and short HA to be confusing when describing the results, especially since the first tested gene krt92 only has long and all subsequent ones are short. The discussion made it more clear that short HA is more efficient and applicable when gRNAs span the stop codon. Perhaps that wasn't possible with krt92, but the authors could prevent the confusion by clearly stating the design requirements of long and short HA and that they wanted to test which is more efficient before starting to describe the data. I also didn't see a description of what the length difference between long and short HA is? How short is short HA?

Response: This is a great question that is well worth discussing. In the revised manuscript, we changed the order in which the parts are described, with nkx6.1 knock-in in front of krt4 knock-in. Here we explain why we would like to do that:

At the beginning of this project, we did not know if the 5’ modified dsDNA could be an effective donor. To test our hypothesis, we chose the krt92 gene as our first target, as this is a keratin protein and expressed in the epithelial cells. We can easily detect the fluorescence in the epithelial cells (most notably in the skin), which allow us to sort the F0 mosaic embryos with high percentage of integration. Notably, from our experience, the most difficult part of the knock-in method is the sorting step (usually performed during 1-3 dpf). This is because the fluorescence signal is highly dependent on the endogenous gene expression level and is usually dimmer with an overall integration efficiency that is lower compared to canonical transgenesis. Therefore, we thought that targeting an epithelial cell marker would be informative and help us to evaluate the validity and reproducibility of the method. If it worked, then we could move on targeting genes expressing in more restricted tissues or cell types. For krt92 gene, the gRNA targets the region upstream of the stop codon. To prevent the cleavage of the donor template, we had to introduce several point mutations and at the same time keep the amino acid sequence intact. However, such mutations can restrict the knock-in and lower the integration efficiency when using shorter arms (due to the sequence mismatch).

After we managed to make the krt92 knock-in, our next question was, what about using a gRNA spanning over the stop codon region? In this way, we don’t need to introduce point mutations on neither the left nor the right homologous arm. Also, for the purpose of our biological study, the nkx6.1 were on top of our gene list for lineage tracing experiments and we luckily identified that there is very good gRNA targeting this locus. After we successfully made the nkx6.1 knock-in, we were thinking that we could simplify the protocol even further, i.e. switching to short homologous arms so that we can prepare the donor by a one-step PCR instead of making complicated constructs. We tested that hypothesis in nkx6.1, krt4, and id2a sites and obtained very promising efficiency. Also, we did some further testing with dsDNA without the 5’ modifications and showed that the 5’ modifications indeed greatly increased integration efficiency. Therefore, although the short homologous arm method is a highlight here, we also point out that it was not planned from the beginning. In the revised manuscripts, we want to convey our method in a logical way and show how we modify the method in a step-by-step fashion.

Moreover, with regards to the comments from the second reviewer, we now added the length of the homologous arms as well as the mutation site on the schematics. We chose short homologous arm because in previous literature it was suggested that short homologous arms (36-48 bp, which we now write out in both the results and the methods) can promote microhomology-mediated end joining (doi: 10.1096/fj.201800077RR). We also noticed that the recent Geneweld method (DOI: 10.7554/eLife.53968) also adheres to a similar length for homology mediated integration. In this study, HAs even shorter than 36 bp also perform well.

1. The authors state that they could not use in situs to confirm krt92 endogenous and knock-in expression overlap, but rather say that they match based on data from an intestine scRNA-seq dataset. Can they elaborate on this? Which clusters/cell types show overlap? Furthermore, is there any krt92:GFP transgenic line that can be used as a reference for expression as well? This point is also applicable for krt4 described in Fig.2

Response: We appreciated this point. In the beginning, we contacted Molecular Instruments to synthesize krt92 HCR3.0 in situ hybridization probes. However, the technical staff there told us that they are unable to make specific probes due to high sequence similarity to other keratin protein families. We can see that the sequence similarity mostly occurs in the middle of krt92 genes, and the HCR3.0 probes rely on a probe set (preferably 20-30 probes with different sequences) to target the mRNA.

The scRNA-seq data that we referenced are from 10X platform, which is based on a 3’enrichment methodology. The reads mapping to krt92 genes are mostly located on the 3’ end. This is good as there is much less similarity to other cytoskeleton genes in the 3’ end of the gene. Unfortunately, there is no krt92 transgenic lines available, so we relied on the single-cell data to correlate expression patterns in this case.

There are two zebrafish intestine single-cell data sets available, with the following links:

(1): https://singlecell.broadinstitute.org/single_cell/study/SCP1675/zebrafish-intestinal-epithelial-cells-wt-and-fxr?genes=krt92#study-visualize

(2): https://singlecell.broadinstitute.org/single_cell/study/SCP1623/zebrafish-intestine-conventional-and-germ-free-conditions?genes=krt92#study-visualize

We can see that krt92 is widely expressed in different types of intestinal epithelial cells (absorptive enterocytes, secretory enteroendocrine/goblet cells and ionocyte).

For the krt4 gene, we now added the HCR3.0 in situ hybridization and immunofluorescence for both krt4 knock-in EGFP-t2a-CreERT2 lines and the Tg(krt4:EGFP-rpl10a) transgenic line (a construct from Anna Huttenlocher, https://www.addgene.org/128839/, which has been widely used to label skin cells). The results are shown in Figure EV9. We show that krt4 has very high expression in the intestinal bulb and hindgut based on the HCR3.0 in situ. The Immunofluorescence of the krt4 knock-in fully recapitulate the krt4 expression pattern in the intestine, while there is almost no fluorescence signal in Tg(krt4:EGFP-Mmu.Rpl10a). We believe this is another advantage of using the knock-in method, over transgenics, for cellular labeling and lineage tracing. Classical transgenics often rely on short promoters of the proximal/enhancer region upstream of ATG with various length (arbitrarily or based on clues from motif analysis/DNA methylation sites). However, different tissues/cell types tend to use different cis-_regulatory elements and the chromatin structure/enhancer-promoter loops might differ dramatically among different cell types. It is hard to predict the exact region of the regulatory sequences that is sufficient for driving the gene expression in a certain cell type. Thus, such reasoning consolidates with that our knock-in lines recapitulate the endogenous _krt4 gene expression. Therefore, we believe that the knock-in based genetic lineage tracing will become the standard in the zebrafish field, as theoretically it avoids both the lack of relevant expression and leakage problems of transgenics.

1. I think Figure 2A needs the dotted lines on the last construct to be fixed (points to p2A)

Response: Thank you for noticing! This was due to a bug in the IBS software, and we changed it manually using Adobe Illustrator in the revised manuscript.

1. There are a few instances where the authors describe performing 4-OHT treatment for long period (e.g. over a 20 hour or 24 hour period). Is fresh 4-OHT added after a certain amount of time or is it a one-time addition? Is such long periods of 4-OHT required or has maximal recombination already occurred within a few hours after addition of 4-OHT?

Response: For 4-OHT treatment, we referred to the method described by Dr. Christian Mosimann (DOI: 10.1371/journal.pone.0152989). We actually tried different conditions (dosage, duration, refresh or not). This is particularly important for the knock-in CreERT lines because the level of CreERT2 is highly dependent upon the endogenous gene expression level. In our case, the nkx6.1 and id2a are transcriptional regulators and relatively lowly expressed compared with structural proteins. We maximized the labeling efficiency by using the highest concentration and longest duration suggested for 4-OHT treatment. The 4-OHT was stored in -20 ℃ and it would become less effective after 30 days of storage. Therefore, we first incubated the 4-OHT in 65 ℃ for 10 min (as recommended by Dr. Christian Mosimann) in order to convert it to a bioactive form. Next, we treated the zebrafish embryos with 4-OHT using a final concentration of 20 μM for 24 hours. We didn’t refresh the 4-OHT since there was no significant difference compared with a one-time addition. Moreover, using higher dosage or longer treatment time can lead to less survival and increased deformity rate. 20 μM 4-OHT treatment for shorter time periods (6 or 12 hours) can cause high labeling variability (some larvae have good labeling while others not). In the end, after several rounds of experiments, we settled on 20 μM 4-OHT treatment for 24 hours as it can reach the highest labeling efficiency, lower variability, and good survival.

1. For Figures 4-6 where confocal images of lineage tracing experiments are shown, there is no indication of how many times the experiments were repeated, how many sections were images, how many animals used, how many cells counted. All of this information should be included in the figure legends and plots should be added showing quantification and statistical analysis (where appropriate).

Response: The reviewer makes a good point and we have now added the number of larvae used and statistical results for the quantitative experiments. The quantification of experiments in Figure 3E-H (originally Figure 4E-H) are shown in Figure EV6D using box/dotplot. We randomly selected 3 secondary islets of different sizes (large, middle, and small) from each juvenile fish (n=5) and pooled the number of mCherry/ins double positive cells and ins positive cells together. The quantification of the lineage-tracing efficiency in the experiments in Figure 6 are shown in Figure EV13.

1. Figure 4 C, C' - I'm not sure what to look for. Is the message that there is no Cherry positive cells that are vasnb negative when labelling is done at 8 somite? But the vasnb positive cells that are also Cherry positive remain? The vasnb staining seems much weaker/harder to see in C C' compared to B, B'. As mentioned above, these data should be quantified and statistical significance indicated.

Response: Thank you for pointing this out; the second reviewer made a similar point. We redid the experiments using zebrafish larvae carrying the ptf1α:EGFP transgene to indicate the acinar cells (Figure 3B-D, Figure EV4G). We also quantified the results and performed statistical testing.

1. I recommend the authors include a short section in the discussion comparing the efficiency of their method to other knock-in strategies used in zebrafish. This is an important claim of the paper yet it is not clear how much better it is (if at all) in terms of frequency of F0 mosaicism and identification of founders relative to other methods. I do appreciate the relative simplicity of the molecular steps of construct design/generation.

Response: This is indeed important. It is also tricky since we are unable to make head-to-head comparisons between different methods as we are targeting different genetic loci and do not have the other methods up and running in our lab. However, the general comparison is based on the statistics shown in the hallmark papers describing these other methods, regardless of which genes were selected for targeting. In the discussion, we added a list of points that are novel/improved with our method versus previous ones, including that: 1) we simplify the knock-in methodology circumventing complicated molecular cloning; 2) we have very high germline transmission rate, which means that one morning of injection is often enough to get a founder; and the expression of fluorescence proteins avoids tedious work in identifying founders, which also saves a lot of space in the fish facility; 3) our lines can be applied for multiple utilities; 4) the method does not disrupt the endogenous gene product. We believe this is critical for the field of developmental biology, regenerative medicine, and disease modeling in zebrafish – and perhaps a similar 3’ knock-in based lineage-tracing method can become commonly used to delineate the cell differentiation and plasticity during homeostatic and diseased conditions in additional organisms.

Reviewer #1 (Significance):

Overall, the study contributes a new knock-in strategy in zebrafish that appears to be more user-friendly and results in high germline transmission. The authors also identify nkx6.1+ ductal cells as progenitors of endocrine cells in the pancreas highlighting the biological applications of their method. I think this study represents an important advancement in zebrafish genetics and will have future impact in lineage tracing during development, regeneration, and disease.

Reviewer #2 (Evidence, reproducibility and clarity):

Summary:

Here, the authors present a strategy where they performed knock-in at the level of the STOP codon, taking care of not perturbing the coding region. They integrate cassettes coding for fluorescence protein and Cre recombinase, which are separated from the endogenous gene and each other by two self-cleavable peptides.

The cassettes are done by PCR with primers with 5' AmC6 modifications and they test short (36 to 46 bp) or long homologous arms (~950bp). For nkx6.1 gene, they observed a dramatic increase of recombination efficiency when injecting the donors with short Homology arms compared to long arms suggesting that short arms could be used. Indeed, short arms used with krt4 and id2a allow them to obtain K.I lines.

The techniques described here look promising. Indeed, even if the proportion of F0 showing adequate reporter expression is low (usually about 2%), the percentages of founders among these mosaic F0 were quite high (between 50% and 100%). And this is the most important aspect as it is usually the most time-consuming aspect of the work.

Major comment:

The authors claim that the knock-in lines can precisely reflect the endogenous gene expression, as visualized by optional fluorescent proteins. But are the authors sure that the integration of the cassettes coding for fluorescence protein and Cre recombinase, which are separated from the endogenous gene and each other by two self-cleavable peptides, will not affect the level of expression of the targeted genes . Indeed, it has been shown that sometimes self-cleavable peptides could affect the expression of the genes of the cassette like for example in this reference ([https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034980]. Therefore it is important that the authors check whether the cassette affect the level of expression of the targeted gene if they want to claim that the knock-in lines precisely reflect the endogenous gene expression.

Response: Thank you for your insightful comments. With regards to the endogenous gene expression, we now use qPCR for further validation. We added the qPCR results to the supplement material (Figure EV15) in the revised manuscript. In brief, we pooled 4 larvae in one tube per biological replicate and have 4 biological replicates for each knock-in line. We didn’t see a significant change in the endogenous expression for any gene. In addition, we have grown up homozygous knock-in lines to adulthood and they are fertile without any overt phenotype.

The highlighted reference is dealing with a cardiomyocyte specific transgenic line, and we assume figure 3-Supplementary figure 1 is what the reviewer is referring to. The altered level of erbb2 expression might be due to the experimental conditions (no treatment or 3 days post treatment). Also, it is possible multiple transgenic insertions occur, as well as gene silencing at some insertion sites. However, such issues would not present, or very limited, with knock-in methods.

Minor comments:

General points:

I believed that the authors should improve the presentation of their data. Indeed, based on what they present, it would be impossible for me to reproduce their technique. Indeed, it is not clear at all how they design the short and long arm, where they are exactly located, which mutations they have done (for fig1), where is located the guide RNA compared to the STOP codon and the HA arms. Graphics that exactly place all these sequences are absolutely required to understand the strategy used and should be placed in figure 1, 2, 3 and 4.

Response: Thank you for these comments. In the revised version, we added the sequence information of the short homologous arms in each of the schematics. As for the krt92 gene, we added the sequence information in the first supplement results (Figure EV1) with the genetic cassettes and point mutation information. We list all the primer information in the methods. Also, we have uploaded our vector templates in the public repository (as listed in the Data availability section). Lastly, we added a key resource table in the supplement file with all the detailed information of reagents for the ease of reproducibility (including all the primers sequences used). We are also willing to share our constructs with the scientific community upon request.

Specific points:

Introduction:

"In zebrafish, the NHEJ-mediated methods have been intensively investigated in 5'knock-in upstream of ATG using donor plasmid containing in vivo linearization site flanking the insertion sequences (11,12,17-20). The 3' knock-in method has also been examined using circular plasmid as the donor with either long or short homologous arms (HAs) flanked by in vivo linearization sites (14, 21-23). Recently, intron-based and exon-based knock-in approaches have remarkably expanded the knock-in toolbox by targeting genetic loci beyond the 5' or 3' end (8-10,13,24-26)."<br /> This part should be explained better in order that the readers could really understand the differences between these old studies and this new one. And really insist on what is the novelty of their technique.

Response: Good points. In the revised version, we elaborated more on the previous discoveries, the major challenges, the knowledge gap in zebrafish knock-in methodology, and what is novel and improved with our new technique. Please, see clarifications and the expanded text in both the introduction and discussion.

Results:

Page 4: To my opinion, the first paragraph should be removed and the technique directly explained based on krt92 strategy as this paragraph does not allow to understand the technique. As indicated above, figure 1 should indicate more clearly the location of the long arms and which mutations they have done and where is located the guide RNA.

Figure 1G: The expression in the skin is far from obvious and the image should be improved (for example with some inset).

Response: Thank you for the comments. We added a new supplementary figure (Figure EV1) and show the sequences of left and right homologous arms, the genetic cassettes, as well as the point mutations with different background color highlight. We added the insets to show the magnified regions of interest. Also, we added the images from the fluorescent microscope used for sorting, to show the EGFP signals in live zebrafish embryos (Figure EV2D and Figure EV8D).

Figure 3E: The authors say that "cells expressing nkx6.1 (displayed by the green fluorescence) were located on the ventral side of the spinal cord whereas H2BmCherry positive cells, which include all the progenies of nkx6.1+ cells after the iCre recombination, resided in both the ventral and dorsal parts of spinal cord". This differential expression in the spinal cord is not obvious and a more closer view should be provided.

Response: Thank you for the comment. First, we changed the order and now describe all nkx6.1 content in Figure 2 and 3 and the krt4 content in Figure 4. We added insets to show the magnified regions and better display the expression pattern of the two fluorescence proteins in Figure 2E-G. One can now clearly see from the magnified insets that the green signals driven by the endogenous nkx6.1 gene are present in the ventral part of the spinal cord, while the red signals are present in both the ventral and the dorsal side of the spinal cord.

Fig S4H: The authors say that" using lineage tracing, we could trace back all three major cell types in the pancreas (acinar, ductal and endocrine cells) to nkx6.1 lineage (Figure 3H-H',Supplementary Figure S4G, H)". While this is obvious for endocrine, the colocalisation with ela3l:GFP is not obvious and the figure should be improved.

Response: This is a very good point, and the first reviewer gave similar suggestions. In the revised version (shown in Figure EV4H and I), we added the insets to show the magnified regions to better display the expression pattern of two fluorescence proteins. The ela3l reporter line is using a short promoter to drive the expression of H2B-EGFP (doi: 10.1242/dmm.026633). However, this short promoter cannot reach 100% labeling of acinar cells, so we also use the ptf1α:EGFP transgene for further validation (new Figure EV4G). Both transgenic reporter lines showed many EGFP and mCherry double-positive cells, indicating that these acinar cells are derived from a nkx6.1-expressing origin. Here we did not use the anti-GFP antibody, as our color switch lines contains CFP and anti-GFP antibody can also recognize CFP. However, the GFP signal is strong enough to show the expression. We hope the additional experiments and insets clarifies this point.

Page 8: the authors say that "The immunostaining at 6 dpf showed that both intrapancreatic ductal cells and a portion of acinar cells can be lineage traced when the 4-OHT treatment started at the 6 somite stage (Figure 4B and B'). The identification of the acinar cells has been done based on the absence of the ductal marker vasnb. To trace efficiently the acinar cells, this should be done with an acinar marker.

Response: Another good point also mentioned by reviewer one. We redid the analyses using zebrafish larvae containing the ptf1α:EGFP transgene to indicate the acinar cells and the co-expression pattern with the lineage-tracing (the data is shown in new Figure 3B-D).

Reviewer #2 (Significance):

I do not have enough expertise in the KI field to evaluate whether this strategy is really novel and as mentioned above, the authors should better explain what is really the novelty of their strategy.

Response: In our answers to the comments of the first reviewer, we elaborated more on the points that are novel/improved with our method vs previous methods, as reiterated here:

“…including that: 1) we simplify the knock-in methodology circumventing complicated molecular cloning; 2) we have very high germline transmission rate, which means that one morning of injection is often enough to get a founder; and the expression of fluorescence proteins avoids tedious work in identifying founders, which also saves a lot of space in the fish facility; 3) our lines can be applied for multiple utilities; 4) the method does not disrupt the endogenous gene product.”

Moreover, the first reviewer asked about the difference between the krt4 knock-in and krt4 transgenics, and based on the in situ data, we showed that our krt4 knock-in can fully recapitulate the endogenous gene expression, while the krt4 transgenics can hardly label the intestinal bulb and hindgut. This might be due to that different tissues/cell types may depend on different _cis-_regulatory elements to drive the gene expression. The chromatin structure and the enhancer/promoter loop might also differ dramatically among different tissues. Therefore, the transgenics might be useful for one type of cells, while they might be not useful at all for other cell types. In the future, we believe that, similar to the mouse field, the 3’ knock-in based lineage tracing methods might become the standard method in the zebrafish field, to delineate cellular differentiation and plasticity during homeostatic and diseased conditions.

= call for = in call means of Indy Threads for Conversations - bunch of people share a conversation - kernel-esque - in this conversations - lot of threads arise - people add useful things - consider how to utilize recording?

= answer : - curate extracts and annotions - both on the margins with docdrop - curate collate salient highlights and annotations in in Indy Page to share for interpersonal collaboration that are continuouis without being synchronous - next time in a call - each participant can have their on Indy Self Space - for note taking - a shared Messaging board to innitiate and partake in threaded conversations from the comforst of their own space link and share

" Re-establish our spiritual interdependence to the sacredness of our Mother Earth." I think that it's very interesting and important to note the use of "Spiritual Independence" because I think that is a thing that is rarely talked about when it comes to fights for independence and autonomy. Being spiritually free is just as important as being physically free, and you can't really have one without the other.

It's cool to know that just two different words can drastically change your search results.

This article explains the economic market. Creating 528,000 jobs is an outstanding aspect for the American people. But It also needs to explain the bad parts of creating jobs in this situation. Because challenging the rules of economics should not make a better situation, There are also high risks.

I don't think that this is good - I love that programming is expressive and indicative of a style of a programmer! It's absolutely not the case that different programming styles are necessarily better or worse - obviously there are good and bad design decisions, but some tradeoffs are just different.

PMs/POs are supposed to mediate externally, but at [last company] they just seemed to mediate conversations and expectations internally. It's important for people to talk to each other - and it's important for the person building the product to be able to speak directly to their end user ad-hoc!

Why are people so seduced by this dumb argument—to the point that they almost seem proud of it?

First, it's presumptuous. Who says we're even using glibc instead of some other libc—which I just might choose to include in the projects I work on? Who says we're even using Linux, for that matter?

Secondly, even if we were, let's assume that we're not, and then see if that teaches us anything about the overall line of reasoning. The original comment was about NPM. NPM is used a fair bit for not just backend stuff but for managing packages used in the browser, too. Let's assume, for simplicity, that our program is entirely a browser-based JS+HTML+CSS app with no backend to speak of. Would the same people argue that, among other things, the Web browser sources would need to be included? Does it even make sense to argue that? Asking the system software question betrays a failure to accurately grapple with the classes of software artifacts we're dealing with, their role in the overall project, and our responsibility for them.

Computing is natural bottom-up. Top-down programming, by contrast, is an entirely different skill; poking around existing abstractions and developments to extract some relevant glue.

But it's clear that no matter what language you're using, you are programming with an abstraction - the computer is running your program, after all - so the framing of this writing isn't really the problem. The abstraction that the user interacts with just has to be better - it has to feel natural, like a part of the language, and not accomplish too much or too little so as to distract from the point. The building blocks should provide you help without getting in your way.

I love this. It's such an eye-opener, because till I read it, I hadn't really realized the power the Arabic language gives to events of importance, occurrences, and people, in comparison to English. just like sh-h-d, shahed, mashhad, and shaheed, there is zahr, to revolt, mozaher, the revolter, and mozahara, the revolution, another strong act of seeking justice and voicing concerns, opinions, and demanding rights.

This reminds me of a game called Pathologic about curing a plague, it is also a kind of depressing game in that lots of odds are stacked against you and sometimes you can do everything right and still not get a good result (I haven't actually played just heard a lot about it). I sometimes wonder what the point of these games are because usually people wants stories for the escapism right? I think as this creator talks about, there is a sort of power being able to face horrible things head on and video games can help you do that emotionally while still remaining physically safe.

content-addressability is Magic

https://t.co/oEzMb6sxa3

https://twitter.com/MindDriveCo/status/1586034166855966721

This is a great observation - platforms are typically described today as "walled / closed gardens" - Plunkett observes that they are in fact an "open substrate" - it's just that it's open to but a few platform owners / controllers.

For example, if you see someone's face, you can "simply see" they are angry. You don't know how you recognized it, even if anger itself is a hypothesis that you somehow have inferred from just seeing light reflecting off their face.

In fact, it took a long time for cognitive psychology to even get started, because it's so obvious that you can "simply see". It took a long time for humans to see that "simply seeing" is not so simple after all.

Why? Because it costs too much for the brain to model how it models the outside world. Since the brain doesn't model how it models the outside world, it treats its model of the outside world as the outside world -- naive realism as a way to save computing power.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

2RE: Review Commons Refereed Preprint #RC-2022-01651

Overall author response to reviewers:

We appreciate the enthusiasm of reviewer 3, who remarks on how our study’s toolset allows us to tackle a longstanding question in the field. We also appreciate the fair criticisms of reviewers 1 and 2.

We have taken into account all the comments of the reviewers by adding new data and new figures, but also by better underlining the limitations of our study design. Here, we underline the main points (see below for points to points answer).

We have addressed the reviewer’s concerns by including sex-specific data in the supplemental for each figure. We also hope the reviewers can see the value in not making this manuscript about sex-specific lifespan effects given our arguments below. We believe that additional experiments using a co-housing design, with far more replication per sex per genotype, would be needed to make strong claims about sex-specific effects. In designing our study, we included both sexes to avoid biasing our overall results to only one sex. In our study, we did not design our experiments in a way that allows sex*genotype interactions to be distinguished from vial effects. We believe our current evidence is sufficient to make claims strictly at the genotype level. But at the sex-specific level, not only is our study not designed for a fair comparison of the sexes by virtue of keeping them in independent vials (with independent microbiomes developing over long timeframes), but such comparisons would also be less robust, effectively based on only half the data per treatment compared to comparisons at the genotype level.

Of note, we do report Cox mixed model statistics in the figures for the interested reader. We just focused on median lifespans for data presentation clarity, and to ensure we focused only on strong trends we could be confident were not statistical Type I errors (falsely rejecting a true null hypothesis). While relying on median lifespan for insights is non-standard, focusing on and showing median lifespans also allows us to better display inter-experiment variation by showing individual data points reflecting each experiment. Sum survival curves with error bars/shading would force us to make an arbitrary decision about what error range to display (SE? SD? 95%CI?), and would not permit the reader to see inter-experiment variation. Median lifespan graphs also let us show just how repeatable our experiments were. We also chose to analyse median lifespan to make it easier to consider the effect of multiple hypothesis testing, so we could better ensure that trends in our data are really striking enough to be worth comment, particularly given the genetics caveats we draw attention to (despite our isogenization efforts, e.g. DefSK3).

We hope with the revisions we provide, reviewers 1 and 2 can, if not agree, at least acknowledge our concern with claiming many sex*genotype interactions. Such effects are lost if we look at other genotypes containing those mutations (e.g. GrA vs. GrAC, or GrB vs. GrBC). It is a challenge to use the fly lines we have generated, which systematically combines 8+ mutant loci, requiring constant reflection. Taking into consideration the complexities of life span studies, we prefer to focus our attention only on the key and robust results of our study.

Nevertheless, in the revised version, we also now provide additional data, and we soften our language regarding the impact of nora virus on aging. We have included new data with additional genotypes that were nora-infected to reinforce our claim that nora virus impacts lifespan. These data greatly strengthen the correlation of nora virus and lifespan reduction. We now also make it explicit that our statement that nora virus infection shortens lifespan is strictly correlation-based, and does not reflect intentional infection experiments. Of note, our study confirms a previous observation done by Ayebed et al. (2009) in the lab of Dan Hultmark, though we find a more striking effect in the DrosDel iso w1118 background.

Overall, we believe we have reinforced the claims of our manuscript after the revisions and addressed the constructive comments of the reviewers. Changes from the original manuscript are highlighted in yellow.

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Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary:

Whether and how age-associated immune hyperactivation affects the ageing process is a key question in the ageing and immunology fields. Using a CRISPR-based knockout approach, Hanson and Lemaitre address the effect of antimicrobial peptides (AMPs), essential immune effectors, on the ageing process. They compared the lifespan and climbing ability of flies deficient for groups of, or individual, AMPs to their isogenic control and the null mutants of the major immune pathways IMD and Toll. Although deletion of individual AMPs had limited effects, the authors detected an association between deletion of a group of AMPs on bacterial proliferation, and examined possible causality using antibiotic treatment. Interestingly, this causal link was missing in the IMD deficient strain, suggesting a potential AMP-independent mechanism of innate immune signalling in the ageing process. The topic is ambitious and exciting; however, the work has some quite serious technical limitations that would need to be addressed with further data analysis and experimentation.

We appreciate Reviewer 1 and Reviewer 2’s concerns regarding the sex-specific effects, which is an important consideration in aging studies. We de-emphasized sex specific effects for reasons outlined in the supplemental text, which we will reiterate here: Per experiment, per genotype, we used ~20 males in one vial and ~20 females in another vial. As a result, the interaction of sex*experiment within a genotype is effectively the same as vial effects. We have to be especially careful of vial effects in our study because we are studying immune-deficient flies susceptible to stochastic microbiome dysbiosis, causing vial stickiness and mass mortality events. Moreover, these mass mortality events are not randomly distributed over aging: they were most likely to occur on Mondays. This is because microbes accrue in the vial over 3 days during the weekend, but only over 2 day time periods for flies flipped between Monday-Friday. As a result, in genotypes that suffer dysbiosis (many AMP group mutants), dysbiosis vial effects can change that experiment’s male/female relative lifespan by ~7 days just by stochastic chance. This issue likely affects other aging studies less, as microbiome dysbiosis is a uniquely important consideration in our immune-deficient flies, and we emphasized this point by noting mass mortality events in Figure 3 as “{“ annotations in the survival curves. We have added these considerations in the revised version (new lines 209-214).

Microbe density in vials is also affected by remaining fly density. As an example of how this can greatly impact the final data at the sex*genotype level: let’s say 4 males died or were censored over the first 50 days randomly in a given experiment. That male vial (16 flies) is less dense than its paired female vial (20 flies), and would have persistent lower microbe load every flip as fewer flies are transferring microbes to the next vial through feces. Then on say… day 60 (a Monday) of that experiment there’s a mass mortality event. Specifically, at day 60 the female vial, which is more densely populated and has had days-to-weeks of higher microbe transfer rate during flipping, has 11/20 females die at once. On the other hand, the males pull through without mass mortality owing to their lower vial density. Suddenly, the female median lifespan in that experiment is 60. The male mortality is most likely to pass the median the next Monday, when another microbiota-induced mass mortality event occurs. Exactly when these critical weekends occurred was stochastic by experiment, but the fact that they occurred was consistent in all experiments in AMP mutant stocks. In this example, female median lifespan would be 60, while males would likely be 67 or so… Importantly, this isn’t a genuine sex-specific effect, it’s variance from how early stochastic mortality affects late-experiment vial density due to microbiome development. It is a vial effect that can look like a sex*genotype effect given our study design, which is exacerbated here because we work on immune deficient flies given that can exhibit microbiome dysbiosis (Marra et al., 2021; mBio).

Given the importance of AMPs in combatting infection, we chose to keep sexes separate to avoid random mating, which can introduce stochastic sexually-transmitted infections. We would also then have had to worry about differences in mating rate amongst genotypes impacting the likelihood of transmitting infections, and differently imposing stress on females by having constant suitors even when they were not receptive. A future study using a co-housing setup would be better equipped to tackle microbiome vial effects. We have now performed the study with sexes kept separately, that can provide a reference for that future study. A future study would also benefit from focusing on fewer genotypes, with far more replication per sex per genotype, to ensure AMP*sex*lifespan effects were robust.

We have now provided supplementary analyses of the genotype-specific ratios of male/female lifespans for each figure (new Table S1-S4). By using the ratio of male lifespan to female lifespan, we focus on how within-genotype comparisons work. We want to emphasize the stochasticity of this ratio depending on experiment batch, even within genotype. For instance, the iso w1118 wild-type male/female lifespan ratio was 0.87 for the six experiment in Figure 2, or 0.93 for the four experiments in Figure 3. It’s a reminder that any collection of experiments is just a sampling of the true population mean. Still, we do see some striking departures in the paper from these ratios. For instance, if we focused on striking departures from this sexual dimorphism, we could comment on a couple genotypes in Figure 3 (AMP groups figure): Group B had a ratio of 1.04 (Only females had reduced lifespan, while males remained comparable to iso w1118). Yet in Group AB the ratio is 0.91, with both sexes having proportionally reduced lifespan. And then Group BC, the ratio is 0.86, with both sexes living to wild-type lifespans. ∆AMP10 and ∆AMP14, which contain all Group B mutations, also have ratios of 0.87 and 0.93 respectively. We also did not see a striking effect in individual mutations from Group B, which all have lifespan and male/female ratios comparable to wild-type. What are we to make of Group B alone then? Is it a genuine effect that combining the Group B mutations uniquely reduces female lifespan? Or is this simply an outlier, perhaps caused by vial effects, that is not supported by all other individual or combinatory genotypes that include Group B mutations?

With all that said, the supplemental tables now included for each figure note these data trends, and includes our cautious view of how to best interpret them. We prefer to merge the sexes in the main article to simplify data display, which is not unique to our study (e.g. see Kounatidis et al. 2017; Cell Rep). We feel our study is not robust at the sex*genotype level to make assertions on sex-specific effects.

Major comments:

1. Figure 1A shows a large increase in fly lifespan in response to deletion of 8 AMPs on Chr II. However, the labelling on the figure shows that data from different experimental runs and from the two sexes have been combined to produce the result, and this problem runs through subsequent results. This is not an acceptable approach. Males and females have different average lifespans and may respond differently to the deletion. Repeats of the same lifespan measurement at different times often give significantly different absolute lifespans between runs, even if the differences between experimental treatment are consistent. The procedure used to construct this Figure therefore lumps heterogeneous data, which in this case is both biologically and, more generally, statistically invalid. We need to see an analysis where the results for the sexes and for different runs of the experiment are disaggregated. To assess the effect of the deletion, comparison should be made only within a single replicate of the experiment, and within each sex. It needs to be made clear how many experimental runs were done and with how many flies in each, since replicability is important with these labile traits. Data should be appropriately analysed, for instance with Log Rank test, or, if the assumptions of the test are fulfilled, with Cox Proportional Hazards. The authors mention analysis of median lifespans, but it is not clear what experimental data the medians were derived from, and this is not a gold standard approach to analysis of lifespan data. This problem also applies to the comparison made between lifespans of iso w1118 flies in Figure 1A and 1B - these were evidently measured in different experiments, so direct comparison cannot be made.

We hope the above response reassures the reviewer about our statistical approach, which we agree is non-standard. We feel it is less likely to result in false-positive claims (Type I error) given the many genotypes we screen and the high stochasticity of individual vials affecting some AMP mutant genotypes more than others (i.e. statistically: unequal variance, requiring an alternate data treatment). Still, we did perform Cox mixed models, and we report those genotype-specific p-values in the figures. But we prefer to make inferences only based on trends robust enough to show in median lifespan comparisons.

1. Figure 2A reports data from mutants that were not backcrossed into a standard genetic background. The authors were aware that this can be a problem because they took care of it with their own mutants, but the data reported in this Figure 2A could be entirely attributable to difference in genetic background between stocks rather than the mutants themselves.

Agreed. The reviewer may be confused regarding our intent in showing these genotypes. We used lines as used in other papers to show how control genotypes behave in our hands. This makes our study easier to compare to the broader literature encompassing immunity*aging studies. The intent is not to test if those mutations’ effects are true in another background. They are there to provide context for our lab settings compared to other studies. They are inter-study calibrator controls.

We have added some text in the study to better highlight the utility of including these control genotypes for making our work more comparable for the field at large (e.g. Line 298, Line 515).

1. Sexual dimorphism is widely reported in many ageing and immunology studies (reviewed in Belmonte et al., Front. Immunol., 2020 and Garschall et al., F1000Res., 2018). This is a problem for the data discussed on line 317, where it is suggested that Group A mutations are shorter lived than the control group because Group A carries the DefSK3 mutation. However, if the results are split by sex, the lifespan difference between Group A vs control is solely contributed by the shorter-lived male flies in Group A. The female flies in Group A live as long as the female control flies. However, the lifespans of both genders of the DefSK3 mutant in Figure 2B are all shorter than control flies. There appears to be a complicated interaction, with diverse function of individual AMPs during ageing, which cannot be summarised by the statement that "deleting single AMP genes has no effect on lifespan" on line 295.

The reviewer is correct that Group A shows a male-specific lifespan reduction. But taking the example of Group B above, we could argue that Group B uniquely reduces female lifespan; except when Group B mutations are found alone, or small combinations (e.g. Dro-AttAB, DptSK1), or in the Group AB, Group BC, ∆AMP10, and ∆AMP14 backgrounds. The same contradictory results are true of the Group A result the reviewer highlights, as Group AC has the opposite effect on male/female lifespan ratios and wild-type lifespan, and ∆AMP10 and ∆AMP14 have normal male/female lifespan ratios.

1. The authors claimed nora virus regulates fly lifespan but they do not produce any direct evidence that this is the case. Bleaching can remove many bacteria and viruses, including many pathogens. To establish the causal role of nora virus on lifespan, reinfection studies with a range of microbes including nora virus would be needed.

We absolutely agree. These experiments were not intended to test for the effect of nora at the outset. Nora is only highlighted because its presence is strictly correlated, in our hands, with strongly reduced lifespan and also the bloating phenotype seen upon aging. We did not previously show other nora-infected data in the manuscript, but in retrospect, we can see that additional context is needed to reassure the reader of why we suspect nora so strongly. We have modified Figure 1B to include nora-positive data from four additional genotypes that were infected with nora at various times in the lab (previously did not show).

To explain the process of how we detected nora in some of our AMP stocks: we randomly screened them for a set of common viruses as part of a research workshop in 2019 hosted by Luis Teixeira. The results of that initial screen were not formally recorded, though we screened all the individual and group AMP mutants available at the time. The viruses we screened for were: Drosophila sigma virus, DCV, DAV, and Drosophila nora virus. Out of the stocks relevant to this study (~20 genotypes), we detected nora virus in iso w1118, AttC, Group C, and Bom. The new data we provide even includes an experiment where AttC with/without nora was included at the same timeframe (re: batch effect concern). We also had instances where OR-R became newly infected with nora virus just from random lab inoculation (not intentional infection).

Importantly, we had noted AttC, Group C and Bom as lines with exceptionally poor lifespan that also showed a bloating phenotype upon aging for ~2 years before we had nora virus on our radar. We were operating on a hypothesis of cryptic microsporidia infection at the time based on a set of chitin stainings of hemolymph that didn’t fully pan out. What convinces us this is nora virus and not a co-infecting virus/microbe is that we only saw the reduced lifespan and bloating upon aging in these confirmed nora-positive lines out of the ~20 we were screening at the time: a 5/5 correlation. We were even able to go back and screen RNA collected from those stocks from earlier experiments to confirm nora infection (informing which experiments we could objectively censor from our analyses). Upon bleaching, we rescued the lifespan of iso w1118, AttC, Bom, and Group C to the levels reported in the main figures. We hope this adds weight to the correlation between nora and lifespan, even if we haven’t done proper reinfection experiments. We also thank the reviewers for commenting, as including these data improves our study by providing more context on the variability of the nora lifespan effect in different genotypes: for instance, we did not collate the nora-infected OR-R data previously, but upon analyzing those data, the negative effect is clearly present but to a lesser extent than iso w1118 (and more in line with what was seen in Habayeb et al. (2009)).

To address the reviewer’s comment, we have also softened our language to be very clear we find only an association, and do not provide a demonstration (new lines 191-193). But the results were sufficiently striking, and poor lifespan associated with bloating was perfectly predictive of nora presence in stocks in our hands. We fully believe the result and feel it is important to include this in the main text to make the field more aware of this important aging study confounding variable.

1. The authors reported a potential AMPs-independent mechanism of the IMD/Relish pathway on ageing, which would be important. However, in Figure 4A and the associated raw data, the authors compared the lifespans of flies from different experiments, and this seems to be a problem. Using ΔAMP14 as an example of a more general problem: the authors assayed conventional feeding ΔAMP14 flies between 07.2021 and 11.2021; however, the antibiotic treated flies were assayed between 12.2021 and 02.2022. There is an obvious batch effect: the median lifespan of ΔAMP14 is 50d on 29.07.2021, 73d on 20.08.2021, 65d on 06.11.2021, and 61d on 10.11.2021. There is therefore a confounding of batch effects with the biological function of AMPs. Some groups seem to have extremely limited sample size, such as only two female flies and six male flies were recorded in "ATM8 23-07-2021 f" in Figure 2A-associated raw data.

We show this inter-experiment variation explicitly by presenting individual experiments as data points in median lifespan graphs. It is not hidden, it is emphasized by our median lifespan data presentation style. The experiments from 06.11.2021 and 10.11.2021 were from independently kept stocks of ∆AMP14, although naturally they come from similar timeframes and would have used food prepared at roughly the same time. If the reviewer feels we should merge the two experiments, we can do that. Merging the experiments and having only 3 entered replicates for conventionally-reared ∆AMP14 changes the one-way ANOVA result in Fig. 4B from “p = .006” to “p = .004,” so we don’t feel having those two experiments entered independently is biasing the analysis. We do feel the parents, and the flies measured, were independent at the rearing level.

ATM8 specifically had poor homozygous viability. This is the only genotype with such a significant departure in sample size per experiment. ATM8 flies are also not relevant to the core message of the study, and their reduced lifespan was extremely clear and in line with previous studies. We prefer to keep the genotype in the study to allow comparison of our lab conditions to studies using ATM8. We have added this note in the Fig. 2 caption (Line 1029).

Regarding how much can be attributed to batch effects: there are many elements of laboratory studies that can contribute to batch effects. However antibiotic food would undergo independent food preparation from standard food regardless of what we do, and parents and flies would be reared in independent vials from independent food preps regardless of what we do. This really just leaves the seasons as the batch effect, which ought to be controlled for by our incubators controlling temperature and humidity. We do appreciate the valid concern, and that incubators aren’t perfect. But we want the importance of the concern to be viewed in the light that even had we done the experiments at the same time, the conventionally-reared and antibiotic-reared flies would still have been given totally different preparations and conditions. Given this consideration, we will further note:

1) ∆AMP14 flies were already known to suffer dysbiosis with aging (Marra et al., 2021; mBio). Our present study only quantifies the effect of this dysbiosis on lifespan.

2) Dysbiosis is associated with flies gut barrier dysfunction and gut content leakage into the hemolymph (Rera et al., 2012; PNAS)(Clark et al., 2015; Cell Rep).

3) Imd-mediated barrier dysfunction leads to microbiome invasion into the hemolymph (Buchon et al., 2009; Genes Dev).

4) Systemic infection by microbiota-derived Acetobacter kills AMP mutants (Marra et al., 2021; mBio)(Hanson et al., 2022; Proc R Soc B), which suggests microbiome invasion into the hemolymph will kill AMP mutants more readily than wild-type flies.

The result that AMP mutants have improved lifespan in antibiotic conditions is therefore not especially surprising – it is important to actually test this, but it is by no means unexpected. One of the curious findings of our study is that we could not rescue Relish to the same extent. If batch effects were affecting the antibiotic rescue experiments by having different intrinsic lifespans during those months, we would expect this to also be visible in iso w1118 and Relish. Antibiotics (or time period batch effects) did not affect lifespan of our wild-type flies, which was very repeatable across all experiments across many years, agreeing with antibiotic-reared fly data from Ren et al. (2007).

Minor comments:

1. The authors reported that the mutant flies lacking 14 AMPs are short-lived due to dysbiosis. Interestingly antibiotic treatment rescued the shortened lifespan of ΔAMP14 but not RelE20. Considering that some of these AMPs, such as lDef, Dro and Drs, are controlled by both the IMD and Toll pathways. It would be worth exploring if the axenic environment could improve the lifespan of Toll mutant flies, which might point to a distinct function of the Toll pathway on the microbiome and ageing process.
2. Line 169: The authors stated that they screened the existence of common viruses but did not provide the results.
3. Line 179: The authors need to include the quantitative results of nora virus in their 44 stocks.
4. Lines 311-314 should be combined with the next paragraph as they are all about "screening".
5. Line 319: Please indicate the associated panel "Fig. 2B" instead of using "Fig. 2".
6. Lines 353 and 358 and in Figure 3C: The authors should provide the quantification of the sticky food in AMPs mutant and Relish mutant flies.
7. Line 389: Please indicate the associated panel "Fig. 2A-B" instead of using "Fig. 2".
8. Line 435: As discussed above, the authors cannot rule out an effect of individual AMPs on lifespan based on their current data and interpretation.
9. Line 507: "During our study, we experienced a number of challenges to lifespan data interpretation." As discussed in the major comments, unless the authors re-perform and re-analyse the lifespan assays this problem will persist.
10. Line 514: As discussed above, the authors cannot rule out the effect of other pathogens on lifespan.

We appreciate the reviewer’s request for spz axenic lifespans. The effect was more striking in Relish, and so we focused on Relish, which regulates antibacterial peptides in the gut. Repeating the experiment with spz will cause non-essential delays and would not affect our main conclusions that focus on AMPs genes.

Line 179: we have, in Fig. 1C. Negatives are not shown, but positives are included in the “Stocks” column. Is the reviewer requesting the exact stock names? We can provide most of these… although the screen was organized by having lab members submit stocks that were in use, and so the spreadsheet of those results is organized with labels provided by lab members (e.g. FM1, rather than the genotype exactly). So we cannot provide exactly which stocks were positive in our hands, but we also don’t feel this is important for the reader. We can share the spreadsheet of this screen (conducted in Dec 2020) with the reviewer if desired.

We were happy to revise the manuscript according to all other requests/comments, highlighted in yellow, and hope our explanations above are sufficient to give the full weight and meaning of the statement in Line 507.

Reviewer #1 (Significance (Required)):

Significance: The crosstalk between AMPs and ageing is a long-standing contradictory topic in the immunology and ageing field (Loch et al., PLoS One, 2017; Badinloo et al., Arch. Insect Biochem. Physiol., 2018; Garschall et al., F1000Res., 2018). This work is a potential step in determining whether and how AMPs regulate ageing. This research may open a door toward yet uncharacterized AMP-independent mechanisms of innate immune signalling in the ageing process.

I am familiar with Drosophila ageing and its relationship with innate immunity, although I am not an insect immunologist.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary: In this study, Hanson and Lemaitre generated AMP single or compound mutants, and subjected them to lifespan analyses. They showed that deletion of all AMPs but not individually (except for Defensin) reduced Drosophila lifespan. The observations that ΔAMP14 suffered microbial dysbiosis and antibiotic treatment rescued the decreased lifespan in ΔAMP14 flies implied a link between AMP-controlled lifespan and host microbiome. Thus, the authors would like to conclude that AMPs contribute to fly lifespan via modulating microbiome.

Comments: This reviewer deeply appreciates that the authors generated AMP mutants (individually or with various combinations). However, these mutants have been reported in their previous publications (eLife, 2019; Genetics, 2021). It seems that the authors carefully analyzed the lifespan of indicated flies, but many pieces of data are inconsistent with previous findings (for instance the lifespan under axenic condition). The explanation about the fly medium (Line 476-477) is not convincing (if yes, who not analyze the lifespan using the same diet as described in previous studies?). The lifespan analyses are of course the most pivotal part in this study, but it is a pity that the results are not shown in an appropriate way, making them rather difficult to interpret. For example, the reviewer is surprised to note that the lifespan of males and females are not analyzed and shown separately (e.g. Figures 1A, 1B, 2A, 2B, 3A, 4A), even the authors declared that they did this before lifespan examination. It is also unclear how many males and females were utilized individually in most assays.

We do not believe the axenic lifespans are inconsistent with previous findings. There have been papers both finding an effect of antibiotics, or finding no effect of antibiotics, on wild-type flies (ex: refs 56 and 57: Ren et al., 2007 and Brummel et al., 2004). Our study would support no major effect. Given our note on nora virus, one might wonder if papers where antibiotics affected lifespan might in part be explained by pathogens (like viruses) that were confounding results in the initial stock, and cleared by the initial bleaching common to those experiments. This would imply that commensal bacteria/fungi community do not have a major role in wild-type lifespans, but rather some stocks are stochastically infected with opportunistic viral pathogens, giving the impression that antibiotic treatment was the key, when in fact it was the bleaching pre-treatment that removed an opportunistic viral pathogen. This is only speculation, but it emphasizes the importance of openly discussing the effect nora virus can have on lifespan for future studies.

Regarding the diet comment: which standard diet should we have chosen? There are many standard diets. We have provided our recipe and we have used our diet for decades. Of note, we did try a set of experiments on a molasses-based food, which showed similar lifespan for wild-type flies in our hands (Response supplemental figure below).

We hope our answers to the first reviewer regarding our care to not include sex*genotype interactions as major claims in our study will satisfy reviewer 2’s concerns.

SEE ATTACHED RESPONSE TO REVIEWERS FOR FIGURE

Response supplemental figure: rearing on molasses food (recipe per lab of Brian McCabe) does not drastically alter wild-type lifespan. In this pilot experiment (n = 1) iso w1118 and OR-R lifespan remain comparable to our standard diet, including the relative lifespans of those wild-types to each other.

Another weakness is the study regarding AMP and microbiome. The authors observed that both ΔAMP14 and RelE20 flies became sticky during aging and their foods in the vials were also sticky and discolored, implying the proliferation of microbiome in the gut and/or the external environment of these flies. To test the microbial load, the authors performed an assay of the bacterial abundance on the fly medium. They further performed antibiotic treatment in the food to remove microbiome as they declared in the study and examined the effect on the lifespan of ΔAMP14 flies. According to the knowledge of the reviewer, antibiotic treatment in food can restrict the microbiome in the gut. The authors have also mentioned in the manuscript the important role of gut microbiota in impacting Drosophila aging and lifespan. Thus, a more direct and widely-utilized way is to dissect the guts for microbial analysis (qPCR, 16S-seq, etc.), which is lacking in this study without reasonable explanations.

We published a paper in mBio on the relationship of AMPs and the microbiome with aging in much greater detail previously (Marra et al., 2021; mBio, ref25). That study did not perform lifespan experiments, but rather compared microbiome communities at 10 and 29 days. The novel aspect of this study is properly following survival. However, the reviewer is correct in their critique that our study is not especially innovative nor are our findings strikingly novel. We do have, we feel, an important set of experimental results to contribute to the field at large.

Reviewer #2 (Significance (Required)):

This study utilized various AMP mutants, but these mutants have been reported in the previous publications, making this study somehow lacking the novelty in this context. The IMD pathway has been shown to be involved in regulating fly lifespan, so the findings in this study are not that surprising. Additionally, this study doesn't show any creative improvements in terms of methodology and model system.

Admittedly, in some ways, much of our study is a “negative results” study. Given the contradictory nature of the literature claiming positive or negative effects of immunity and AMPs on lifespan, we feel this is a particularly valuable contribution to avoid publication bias focusing only on significant results in this controversial field. Still, we believe that the AMP/microbiome/lifespan interactions we uncover in our article will have an impact in the immune-aging field. Thus, while not being fully creative, our article is an important step for better characterizing of immune-aging relationships, which is of broad interest.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Gene knockouts provide definitive loss of function for individual and collective AMPs. This eliminates ambiguity caused by the partial efficiency of RNAi, and it disambiguates the pleiotropy caused by mutants of relish. Survival assays are conducted with and without the resident microbiome. This combined design leads to a clear conclusion: loss if individual (mostly so) AMPs does not affect adult survival either way, demonstrating these peptides likely function redundantly. Knock out of 14 AMPs (but still not all of those encoded in the genome) reduces survival when adults must cope with a microbiome. Depleting this microbiome sends survival back to normal. Perhaps these are not surprising results in that they show immune function is essential when challenged with pathogens, but the results are important because they unambiguously show that AMPs themselves are not a cause of intrinsic aging. This will finally put away that lingering hypothesis.

Overall, I like the scholarship of the work, of how it uses the literature, and its quality experimental execution. Cohort size, replication and survival analyses meet current, high standards. Demographic aging is supplemented with data on climbing rate as a function of age. It is a strength of the study that it is simple but comprehensive.

Reviewer #3 (Significance (Required)):

Aging across animal systems is strongly associated with changes in immunity and inflammation, both innate and adaptive. Overall, we want to understand if such changes are underlying causes of morbidity and mortality with age, or are consequences and compensation to underlying aging and cumulative pathogen exposure. These are difficult questions to address in mammalian systems but amenable using Drosophila which possesses a robust innate immune system. Researchers have used the fly for this end but still have mixed and ambiguous results. Now Hanson and Lemaitre provide a substantial design that fully controls the two essential ingredients: expression of antimicrobial peptides and the microbiome.

We thank the reviewer for their positive assessment. In particular, we appreciate the nod to the importance of the question on intrinsic contributions of AMPs to lifespan. We believe this is the key strength of our study’s mutant approach, which has been challenging to assess using previously-existing tools.

Additional Author changes (not requested by reviewers):

We realised there was a copy/paste error in data used in Figure 2. Specifically, extra OR-R experiments were included in these data for this figure that were not intended to be part of this figure. We have removed these OR-R experiments, which are experiments common to Figure S4 and remain visible in Figure S4. This does not impact any of the conclusions in the manuscript.

Absolutely. We see this a lot within children but that is a great thing. Having this at schools protects them from anything happening so being able to have that permission is great. Not just through pictures but through lots of activities within school.

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Reply to the reviewers

Reviewer #1

SUMMARY

The manuscript by Smoak et al., provides an analysis of the Hyr/Iff-like (Hil) genes in Candida species with a strong focus on C. auris. The authors demonstrate a repeated expansion of these genes in unique lineages of fungal species, many of which are associated with stronger clinical disease. There is evidence of selection operating on the gene family in the primary domain used for identification. These genes include a repeat just downstream of that core domain that changes frequently in copy number and composition. The location of these genes tends to cluster at chromosome ends, which may explain some aspects of their expansion. The study is entirely in silico in nature and does not include experimental data.

MAJOR POINTS

Altogether, many of the general findings could be convincing but there are some aspects of the analysis that need further explanation to ensure they were performed correctly. To start, a single Hil protein from C. auris was used as bait in the query to find all Hil proteins in yeast pathogens. Would you get the same outcome if you started with a different Hil protein? What is the basis for using Hil1 as the starting point? It also doesn't make sense to me to remove species just because there are already related species in the list. This may exclude certain evolutionary trends. Furthermore, it would be helpful to know how using domain presence and the conservation of position changes the abundance of the gene family across species? (beginning of results).

We appreciate the reviewer’s criticisms on our strategy for identifying Hil proteins. In response, we have significantly revised our pipeline. In particular, we now combine the search results from three queries: in addition to C. auris Hil1’s Hyphal_reg_CWP domain (XP_028889033), we added the Hyphal_reg_CWP sequences from C. albicans Hyr1 and C. glabrata Hyr1. They were chosen as representatives in the two phylogenetic groups distinct from the one containing C. auris in order to avoid the bias due to the query’s phylogenetic position. Using the same criteria as we did for the original search, we identified three additional hits compared with the original 104 homologs list. In response to the criticism of the arbitrary exclusion of some species, we now include any species from the BLASTP search results as long as it is part of the 332 yeast species studied by Shen et al. 2018 (PMID: 30415838). The reason for this criterion is so that we can use the high-quality species phylogeny generated by Shen et al. 2018 to properly study the gene family evolution by reconciling the gene tree with the species tree. We additionally include the species in the MDR clade closely related to C. auris and used Muñoz et al. 2018 (PMID: 30559369) as the basis for the species phylogeny in the clade. Lastly, we no longer require the particular domain organization in classifying Hil family members. All BLASTP hits satisfying the E-value cutoff of 1x10-5 and query coverage > 50% are included.

A major challenge in the analysis like this one is in dealing with repetitive sequences present in amplified gene families. For example, testing modes of selection on non-conserved sites is fraught. It's not clear if all sites used for these tests are positionally conserved and this should be clarified. Alignments at repeat edges will need to maintain this conservation and relatively good alignments as stated in lines 241-242 are concerning that this includes sequence that does not retain this structure necessary for making predictions of selection.

We appreciate the reviewer’s comment. In the original manuscript, we performed two different types of analyses, one on the conserved and well-aligned Hyphal_reg_CWP domain and another on the rapidly evolving repeat region. For the former, we performed phylogenetic dN/dS analyses using maximum-likelihood, for which a reliable alignment is crucial and is the case here. The Hyphal_reg_CWP domain alignment for C. auris Hil1-Hil8 is shown below and also included as Fig. S7 in the revised manuscript: (figure in the response file)

In the text, we added this sentence to emphasize this point: “We chose to focus on the Hyphal_reg_CWP domain because of its potential importance in mediating adhesion and also because the high-quality alignment in this domain allowed us to confidently infer the evolutionary rates (Fig. S7).”

For the repeat domain, what we did in the original version was to calculate the pairwise dN/dS between individual repeat units found in Hil1 and Hil2. This didn’t require aligning the entire repeat regions in the two proteins, but instead relied on the alignment of the individual ~44 aa repeat units, which were highly conserved (see below). In the revised manuscript, however, we decided to focus our analyses on the Hyphal_reg_CWP domain because of a different concern, namely gene conversions between paralogs could distort the evolutionary history of the repeats (the same concern was addressed for the effector domain using an additional step of detecting recombination breakpoints, but the same analysis would be challenging for the repeat region due to alignment issues).

(figure in the response file)

It's also unclear to me why Figure S12 is here. The parameters for this analysis should be tested ahead of building models so only one set of parameters should be necessary to run the test. The evolutionary tests within single genes and across strains is really nice!

We appreciate the reviewer’s suggestion. Based on the reviewer’s suggestion, we removed Fig. S12 and describe the model set up in the Materials and Methods section. We were not sure if the last point was a comment or a suggestion. We didn’t perform a population level selective sweep scan in C. auris. Such an analysis has in fact been attempted by Muñoz et al. 2021, who identified several members of the Hil family as the top candidates for positive selection (PMID: 33769478). We cited this in our Discussion:

“Lastly, scans for selective sweep in C. auris identified Hil and Als family members as being among the top 5% of all genes, suggesting that adhesins are targets of natural selection in the recent evolutionary history of this newly emerged pathogen (Muñoz et al. 2021).”

A major challenge for expanded gene families is rooting based on the inability to identify a strong similarity match for the full length sequence. The full alignment mentioned would certainly include significant gaps. If those gaps are removed and conserved sites only are used, does it produce the same tree? Inclusion of unalignable sequences would be expected to significantly alter the outcomes of those analysis and may produce some spurious relationships in reconciling with the species trees. Whether or not there are similar issues in the alignment of PF11765 need to be addressed as well. There's nothing in the methods that clarifies site selection.

We appreciate reviewer’s comment and agree with the concern about alignment quality affecting phylogenetic reconstruction. To clarify, all phylogenetic analyses in this work are based on the alignment of the Hyphal_reg_CWP domain, which is well aligned (shown above for the subset of eight homologs in C. auris). Alignment of all 215 homologs is provided for readers to review (shorturl.at/kDEJ3). To clarify this choice, we now include the following in Results:

“To further characterize the evolutionary history of the Hil family, including among closely related Candida lineages, we reconstructed a species tree-aware maximum likelihood phylogeny for the Hil family based on the Hyphal_reg_CWP domain alignment (Fig. 1C, Fig. S2).”

We also included detailed steps for reconstructing the gene tree in Materials and Methods.

To test the effect of gaps in the alignment on phylogenetic tree inference, we used two trimming programs, ClipKit (PMID: 33264284) and BMGE (PMID: 20626897), with author-recommended modes. They resulted in consistent gene tree results. We present the tree based on the ClipKit trimmed alignment in the main results. The root of the gene tree was inferred by jointly maximizing the likelihood scores for the gene tree based on the alignment and the evolution of the gene family within the species tree, using GeneRax (Morel et al. 2020, PMID: 32502238).

Figure 1A: the placement of evolved pathogenesis is a little arbitrary. It's just as feasible that a single event increased pathogenesis in the LCA of C. albicans and C. parapsilosis that was subsequently lost in L. elongisporus. These should be justified or I'd suggest removing. The assignment of Candida species here also seems incomplete. The Butler paper notes both D. hansineii and C. lusitaniae as Candida species whereas they are excluded here.

We removed Figure 1 entirely based on this and another reviewer’s comment. We note that there is broad consensus that opportunistic yeast pathogens have independently arisen multiple times, such as C. auris, C. albicans and C. glabrata. Whether Candida pathogens that are more closely related evolved separately or not are subjects of ongoing research (PMID: 24034898).

It is tricky to include scaffolds in analysis of chromosomal location of the HIL genes. The break in the scaffold may be due to the assc repeats of these proteins alone or other, nearby repeats. Any statistics would be best done to include only known chromosomes or those that are strongly inferred by Munoz, 2021. This will change the display of Figure 7, but is unlikely to change the take home message.

We agree with the reviewer’s concern. In the revised manuscript and with more species included, we now only analyze genomes assembled to a chromosomal level, with the exception of C. auris B8441, which is supported by Muñoz et al. 2021 as having chromosome-length sequences. The revised Figure 7 now only includes these results. We also removed the accompanying supplementary figure that showed results based on scaffold-level assemblies.

MINOR POINTS

Line 18: "spp." Should be "spps."

Addressed throughout the revised manuscript.

Line 41: I might rephrase this as "how pathogenesis arose in yeast..."

Accepted (line 43 in revised manuscript).

I might use a yeast-centric example around line 40 for duplication and divergence. This could include genes for metabolism of different carbon sources in S. cerevisiae.

Accepted (lines 47-48)

The Butler paper referenced on line 51 compared seven Candida species and 9 Saccharomyces species

Changed (line 48)

The autors state no other evolutionary analysis of adhesins has been performed but do not acknowledge this study: https://academic.oup.com/mbe/article/28/11/3127/1047032

We appreciate the reviewer pointing this important reference to us. We now cite it in the introduction (line 64) and discussion (line 340)

The first paragraph of the Results could be condensed

Addressed.

How was the species tree in Figure 1A obtained?

The previous figure 1 is now removed. The species tree used throughout the manuscript is based on Shen et al. 2018 with MDR clade species added, based on Muñoz et al. 2018.

Figure 2: In panel A, "DH" and "SS" are not defined. I'd be careful with use of "non-albicans Candida" in Figure 2B. This usually includes C. tropicalis and C. dubliniensis and may confuse the reader.

We removed the DH and SS labels. Instead, we highlighted three clades, which were defined in previous studies. These are the Candida/Lodderomyces clade (based on NCBI taxonomy database), the MDR clade (e.g., Muñoz et al. 2018, PMID: 30559369) and the glabrata clade (e.g., Gabaldón et al. 2013, PMID: 24034898).

How was the binding domain defined to extract those sequences are produce a phylogeny? In building a ML model, how were parameters chosen?

We now provide the following details in the Materials and Methods section:

“To infer the evolutionary history of the Hil family, we reconstructed a maximum-likelihood tree based on the alignment of the conserved Hyphal_reg_CWP domain. First, we used hmmscan (HmmerWeb version 2.41.2) to identify the location of the Hyphal_reg_CWP domain in each Hil homolog. We used the “envelope boundaries” to define the domain in each sequence, and then aligned their amino acid sequences using Clustal Omega with the parameter {--iter=5}. We then trimmed the alignment using ClipKit with its default smart-gap trimming mode (Steenwyk et al. 2020). RAxML-NG v1.1.0 was compiled and run on the University of Iowa ARGON server with the following parameters on the alignment: raxml-ng-mpi --all --msa $align --model LG+G --seed 123 --bs-trees autoMRE.”

The parameters for the ML tree reconstruction is listed on the last line above. The main parameter was the evolutionary model (LG+G), which accounts for rate variations using a gamma distribution. Other protein evolution models, e.g., VT+I+G, were tested and resulted in nearly identical tree topologies.

Figure 3C/D could be just one panel.

The structure predictions are now reorganized and presented on their own in the new Figure 3.

Can you relate more the fungal hit to the Hil proteins conveyed in lines 152-154?

We appreciate the reviewer’s comment, which referred to CgAwp1 and CgAwp3, whose effector domain structures were reported in a recent study (Reithofer et al. 2021, PMID: 34962966). We now discuss them in relation to the predicted Hyphal_reg_CWP structure, by showing them in Figure 3 and describing them in the Results (lines 181) “crystal structures for the effector domains of two Adhesin-like Wall Proteins (Awp1 and Awp3b) in C. glabrata, which are distantly related to those in the Hil family were recently reported, and the predicted structure of one of C. glabrata’s Hil family members (Awp2) was found to be highly similar to the two solved structures (Reithofer et al. 2021)”

Line 168: Should read "Hence, ..."

The original sentence was removed, but this grammatical error was checked for and corrected.

Label proteins along the top of Figure 4 too.

Accepted (in new Figure 4).

Figure 5: for tests of selection, were sites conserved across the group? What does the black number at each node indicate? Dn and Ds are given as decimals. This is based on what attribute? For panel B, it is unclear what each tip denotes i.e., Hil1_tr6. Hil1 is the gene but what is "tr6"?

In the revised manuscript, we provide the multiple sequence alignment for the Hyphal_reg_CWP domain used for the selection analysis as Fig. S7 to illustrate the level of conservation. The black numbers at the internal nodes are numeric indices used to refer to those nodes. In the revised manuscript, we use some of them to refer to the internal branches, e.g., 12…14 in the legend. In the new Figure 5, we do not list the numeric values of Dn and Ds (aka Ka, Ks). Instead, we use a color gradient to represent the estimated dN/dS ratios. The raw estimates are available in the project github repository. Panel B in the original Figure 5 and other panels related to the evolution of the repeats are now removed.

It's unclear why comparison of the PF11765 domain includes the MRD proteins when those aren't included in the comparison to the repeats alone. Could that skew the comparison due to unequal sample numbers or changed variation frequencies in MDR relative to the other two groups?

These results pertaining to the evolution of the repeats are now removed.

Table 2 doesn't add much. This section could probably be reduced to a few sentences since it's highly speculative (intraspecies variation).

Table 2 is now Table S5. We also simplified the result section in the revised version. While the functional implications of the intraspecific variable number of tandem repeats (VNTR) is speculative, it is founded on two bases: 1) the identification of the VNTR is credible, as the copy number variation is consistent within clades but differ between clades, which is not expected if they are caused by assembly errors; 2) experimental studies in S. cerevisiae for the Flo family strongly supported a direct impact of adhesin length on the adhesive phenotype of the cells (PMID: 16086015).

Table 3 is not needed.

Table 3 is now removed.

Figure 6 - color coding in 6A needs to be explained. I'm assuming this is a taxonomical coding.

In the revised Figure 6A, the coloring scheme is consistent with what we used in Figure 1 based on the three clades, and a legend is provided.

Figure 1B is unnecessary. A Model of the protein indicating domains is sufficient here. Figure 1C needs labels for all termini, not just the pathogenic red branches. The figure doesn't provide clear association between adhesin families and the associated species. This could be omitted, especially since Flo is often associated with Saccharomyces species. Figure 1D is unnecessary.

We have removed the original Figure 1.

SIGNIFICANCE

The work here is sorely needed in expanded gene families and in fungi specifically. No analysis at this level has been performed, to the best of my knowledge, in any fungal associated gene family and certainly not in relationship to pathogenic potential. The authors do a good job in citing the foundational literature upon which their study builds in most cases (one exception is noted above). It would be of general interest to those interested in the evolution of virulence, but the analysis is tricky. This is the biggest drawback I currently have as some of the information to assess the results is missing.

We really appreciate the reviewer's positive comments. We agree and plan to explore the relationship between the adhesin family evolution and virulence phenotypes.

Expertise: gene families, evolution dynamics, human fungal pathogens

Reviewer #2

SUMMARY

Gene duplication and divergence of adhesin proteins are hypothesized to be linked with the emergence of pathogenic yeasts during evolution; however, evidence supporting this hypothesis is limited. Smoak et al. study the evolutionary history of Hil genes and show that expansion of this gene family is restricted to C. auris and other pathogenic yeasts. They identified eight paralogous Hil proteins in C. auris. All these proteins share characteristic domains of adhesin, and the structural prediction supports that their tertiary structures are adhesin-like. Evolutionary analysis of protein domains finds weak evidence of positive selection in the ligand-binding domain, and the central domain showed rapid changes in repeat copy number. However, performed tests cannot unambiguously distinguish between positive selection and relaxed selection of paralogs after gene duplication. Some alternative tests are suggested that may be able to provide more unambiguous evidence. Together with these additional tests, the detailed phylogenetic analyses of Hil genes in C. auris might be able to better support the hypothesis that the expansion and diversification of adhesin proteins could contribute to the evolution of pathogenicity in yeasts.

We appreciate the reviewer’s comments and will address specific points below.

MAJOR COMMENTS

The authors present extensive analyses on the evolution of Hil genes in C. auris. There is significant merit in these analyses. However, the analyses conducted so far are incomplete, lacking proper consideration of other confounding factors. Detailed explanations of our major comments are listed below.

1. First, the authors restricted genes in the Hil family to those only containing the Hyphal_reg_CWP domain. Yet, previous work included genes containing the ligand-binding domain or the repeat domain as Hil genes. More justification is needed whether the author's choice represents the natural evolutionary history of Hil genes appropriately. For instance, are the genes only containing the ligand-binding domain monophyletic or polyphyletic? We recommend including the phylogeny of all the Hil candidate genes, to discern whether evolutionary histories of the repeat domain and ligand-binding domain are congruent. Authors can use this phylogeny as justification to focus only on the ligand-binding domain containing genes.

Butler et al. 2009 (PMID: 19465905) defined the Als family and the Hyr/Iff family as having either the N-terminal effector domain or the intragenic tandem repeats (ITR). Their rationale for the latter was that the ITS sequences were often conserved across species. Upon close inspection (Fig. S19,20 in that paper), however, we found that the ITS tend to be conserved in closely related species, but diverged among more distantly related species. Moreover, proteins in those figures that only contain the ITS and not the ligand-binding domains are all missing either the signal peptide, the GPI-anchor or both. This raises questions as to whether these proteins sharing the ITS sequence alone act as adhesins.

More generally, defining the evolutionary history of proteins with multiple domains is complicated by recombination, which causes different parts (e.g., domains) of the protein to have distinct evolutionary histories. In fact, our study and others show that there exist “chimeras” that combine the effector domain from one adhesin family and the repeat sequence found in another (Zhao et al. 2011, PMID: 21208290, Oh et al. 2019, PMID: 31105652). In these cases, one phylogenetic tree is insufficient to describe the evolutionary history of the whole protein. We chose to define the Hil family by the Hyphal_reg_CWP domain and thus focus on the evolutionary history of this region because 1) while tandem repeat regions also contribute to adhesion in yeasts (Rauceo et al. 2006, PMID: 16936142), the effector domain likely plays a more important role in ligand binding and specificity. Therefore, we believe using the effector domain to define a protein family is more likely to group proteins with similar functional properties than if the repeat sequences were used. Also, while putative fungal adhesins lacking a recognizable ligand-binding domain exist, they are rare (Lipke 2018, PMID: 29772751); 2) The repeat region evolved much more rapidly than the effector domain, as we illustrate in Figures 2, 4 and 6 in our revised manuscript. While some repeat units are highly conserved, e.g., the ~44 aa unit found in Hil1-4 in C. auris and close relatives in the MDR clade, many others are short and degenerate, making it difficult to reliably identify homologs sharing the repeat. Besides, since each protein could contain many distinct repeats, it is not clear how one defines two sequences as belonging to the same family if they share one out of six types of repeats. We acknowledge that this definition leaves out the evolutionary history defined by the tandem repeats, which may reveal intriguing evolutionary dynamics, with functional implications. A recent review for the Als family discussed similar definition challenges and partly supported our choice (Hoyer and Cota, 2016, PMID: 27014205).

In the analysis of positive selection, the authors do not adequately control for the effect of recombination on the evolutionary histories of protein sequences, especially given that Hil genes are rich in repetitive sequences. To account for recombination, GARD, an algorithm detecting recombination, should be performed to detect any recombination breakpoints within a protein domain. If recombination did occur within a protein domain, the authors should treat the unrecombined part as a single unit and use the phylogenetic information of this part to proceed with PAML analysis, instead of using the phylogeny of the entire protein domain. The authors should consider doing GARD analysis for the ligand-binding and repeat domains. For the repeat domain, low BS values in Fig. 5C indicate recombination between repeat units. Thus, the authors should analyze each repeat unit with GARD and re-analyze dN/dS.

We deeply appreciate the reviewers’ criticism here. In the revised manuscript, we removed the analysis of the repeat units and followed the reviewers’ suggestion to carry out GARD analysis on the effector domain, which we now show reveals evidence of intra-domain recombination. Using the inferred breakpoints (Fig. S8), we identified two putatively non-recombining partitions and performed all downstream phylogenetic analyses on them separately. The results are presented in Fig. 5 and Table S6. Compared with the previous result based on the entire Hyphal_reg_CWP domain alignment, the new results reveal clearer patterns, including significantly elevated dN/dS on a subset of the branches. Newly added branch-site test results support a role of positive selection on the effector domain during the expansion of the Hil family in C. auris, suggesting functional diversification following gene duplications.

The authors concluded positive selection in the ligand-binding domain based on the branch-wise model of PAML. Yet, w values were not higher than one, and it's unclear whether the difference in selective pressures the authors claimed here is biologically significant. Overall, what the authors present so far seems to be weak evidence of positive selection but is much more consistent with variation in the degree of purifying selection or evolutionary constraint. Using the site-wise model (m7 vs. m8) in PAML would allow the authors to detect which residues of the ligand-binding domain underwent recurrent positive selection. Combining the evolutionary information of protein residues and the predicted 3D structure will provide molecular insights into the biological impact of rapidly evolving residues. This would be a significant addition and raise the significance of the study, besides providing potentially stronger evidence of positive selection.

We appreciate the reviewers’ criticism and suggestions. In the revised manuscript, we performed site tests comparing models M2a vs M1a, M8 vs M7 and M8a vs M8. For partition 1 (P1-414), all three tests were insignificant. For partition 2 (P697-987), the M2a vs M1a test was insignificant (P > 0.05) but M8 vs M7 and M8a vs M7a were both significant at a 0.01 level, and the omega estimate for the positively selected category was estimated to be ~15. The site tests require all branches to evolve under the same selection regime. To relax this constraint, we performed additional branch-site tests by designating the branches with an estimated dN/dS > 10 as the foreground (based on the free-ratio model estimates). This test was significant for both branches at a 0.01 level and the Bayes Empirical Bayes (BEB) procedure identified a total of 5 residues as having been under positive selection. Although three of the five residues, located in the C-terminus of the Hyphal_reg_CWP domain, are part of the α-crystallin domain, we refrain from drawing any functional conclusions because 1) the BEB procedure is known to be lacking power in identifying positively selected residues and 2) we still lack structure-function relationship for the α-crystallin domain. But we agree and believe that this line of analysis is promising in yielding functional insight into the evolution of the effector domain in the family.

MINOR COMMENTS

1. In Fig 1c, the figure legend should include more specific details: which adhesin proteins are shown here? Please specify species names on the species tree

Figure 1 is removed in the revised manuscript

In Fig 3E, secondary structures are labeled with the wrong colors. Sheet: purple, helix: yellow

In the revised manuscript, the structures of SRRP-BR (original 3E) is now colored in a single color.

What's the ligand-binding activity of the b-solenoid fold? How structurally similar are C. auris PF 11765 domains compared to C. glabrata Awp domains? This information will support the role of adhesin for the ligand-binding domain of Hil genes.

We discuss the ligand-binding activity of the β-solenoid as follows in Discussion:

“The elongated shape and rigid structure of the β-helix are consistent with the functional requirements of adhesins, including the need to protrude from the cell surface and the capacity for multiple binding sites along its length that facilitate adhesion. In some bacterial adhesins, such as the serine rich repeat protein (SRRP) from the Gram-positive bacterium, L. reuterii, a protruding, flexible loop in the β-helix was proposed to serve as a binding pocket for its ligand (Sequeira et al. 2018). Such a feature is not apparent in the predicted structure of the Hyphal_reg_CWP domain. Further studies are needed to elucidate the potential substrate for this domain and its mechanism of adhesion.”

We also compare the structures of the C. auris Hil1/Hil7 Hyphal_reg_CWP domain and the CgAwp1/3 in Figure 3, with this in the legend “(C) Crystal structure of the C. glabrata Awp1 effector domain, which is highly similar to C. auris Hil1 and Hil7, but with the disulfide bond in a different location.”

We added a section in the Discussion to comment on the structure-function relationship based on known β-helix (aka β-solenoid) structures. The main insight comes from similar structures identified through DALI searches, many of which are bacterial and viral surface proteins mediating adhesion. The ligand binding pocket and specificity would require additional structural studies to elucidate.

In lines 248-249, the authors should also consider the influence of evolutionary history. For instance, repeats within the same Hil protein appeared later in evolution, compared to Hil gene duplication, and therefore these repeats experience less time for sequence divergence.

In the revised manuscript, we removed the analyses pertaining to the evolution of the repeat regions due to multiple challenges including alignment, potential of gene conversion and recombination. This is an important and intriguing aspect of adhesin family evolution that we plan to follow up in future work.

Although the bioinformatic evidence of C. auris Hil genes acting as adhesins is strong, it is still worthwhile to discuss the experiments of confirming the function of adhesins.

We agree with the reviewer and acknowledge in the revised manuscript the limitation of our work:

“Future experimental tests of these hypotheses will be important biologically for improving our understanding of the fungal adhesin repertoire, important biotechnologically for inspiring additional nanomaterials, and important biomedically for advancing the development of C. auris-directed therapeutics.”

SIGNIFICANCE

Overall, this study is interesting to investigate the evolutionary history of a crucial virulent gene in C. auris. Such evolutionary understanding will help us identify critical molecular changes associated with the pathogenicity of an organism during evolution, providing insights into the emergence of pathogens and novel strategies to cure fungal infections. The research question is important; however, the current analyses on the positive selection are incomplete, so the conclusion is modest so far. We recommend that the authors re-do the PAML analysis with the above considerations. This work will bring more significance to the mycology field if the functional impact of rapid evolution in protein domains can be supported or inferred.

This manuscript is well-written, and the authors also did a great job specifying all the necessary details in the M&M.

We appreciate the reviewers’ positive comments.

Reviewer #3

Summary:

The manuscript by Smoak et al. provides considerable information gleaned from analysis of HYR/IFF genes in 19 fungal species. A specific focus is on Candida auris. The main conclusion is that this gene family repeatedly expanded in divergent pathogenic Candida lineages including C. auris. Analyses focus on the sequences encoding the protein's N-terminal domain and tracts of repeated sequences that follow. The authors conclude with the hypothesis that expansion and diversification of adhesin gene families underpin fungal pathogen evolution and that the variation among adhesin-encoding genes affects adhesion and virulence within and between species. The paper is easy to read, includes clear and attractive graphics, as well as a considerable number of supplementary data files that provide thorough documentation of the sources of information and their analysis.

We appreciate the positive comment.

MAJOR COMMENTS:

• Are the key conclusions convincing?

Overall, the authors' conclusions are supported by the information they present. However, the overall conclusion is stated as a hypothesis and that hypothesis is not particularly novel. The idea that expansion of gene families associated with pathogenesis occurs in the pathogenic species dates back at least to Butler et al. 2009, who first presented the genome sequences for many of the species considered here.

We appreciate the reviewer’s comment. Our main conclusions are 1) the Hil family is strongly enriched in distinct clades of pathogenic yeasts after accounting for phylogenetic relatedness. This enrichment results from independent duplications, which is ongoing between closely related species; 2) the protein sequence of the Hil family homologs diverged rapidly following gene duplication, driven largely by the evolution of the tandem repeat content, generating large variation in protein length and β-aggregation potentials; 3) there is strong evidence for varying levels of selective constraint and moderate evidence for positive selection acting on the N-terminal effector domain during the expansion of the family in C. auris as our focal species. Based on these observations, we propose that expansion of adhesin gene families is a key preliminary step towards the emergence of fungal pathogenesis.

Indeed, some version of this hypothesis has been proposed by several groups before us. We fully acknowledged this in our previous as well as the revised manuscript, by citing Butler et al. 2009 (PMID: 19465905), Gabaldón et al. 2013, 2016 (PMID: 24034898, 27493146). Our study built on these earlier efforts and extended them by addressing several limitations. First, we performed phylogenetic regression to test for the association between gene family size and the life history trait (pathogen or not) in order to properly account for the phylogenetic relatedness. This was not done in previous studies. Second, most earlier studies didn’t construct a family-wide gene tree to fully investigate the evolutionary history of the family. Gabaldón et al. 2013 did a phylogenetic analysis for the Epa family and a few others within the Nakaseomycetes, revealing highly dynamic expansions. In the present study, we expanded this effort by comprehensively identifying homologs within the Hil family in all yeasts and beyond. Third and perhaps the most important novelty in our study is our detailed analysis of sequence divergence and role of natural selection during the evolution of the family post duplication. This allowed us to present a complete picture of the family’s evolution, not just in its increase in copy number but also its diversification after the duplications, which is a key part of how gene duplications contribute to the evolution of novel traits. As such, we believe our study provides strong support for the above hypothesis.

One key issue with a manuscript of this type is whether genome sequence data are accurate. The authors are not the first research group to take draft genome sequence data at face value and attempt to draw major conclusions from it. The accuracy of public genome data continues to improve, especially with the emergence of PacBio sequencing. Because the IFF/HYR genes contain long tracts of repeated sequences, genome assemblies from short-read data are frequently inaccurate. For example, is it reasonable to have confidence that the number of copies of a tandemly repeated sequence in a specific ORF is exactly 21 (an example taken from Table 2) when each repeat is 40+ amino acids long and highly conserved? Table S6 would benefit from inclusion of the type of sequence data used to construct each draft genome sequence. It is also reasonable to question whether the genome of the type strain is used as a template to construct the draft genomes of the other strains. If that was standard practice, conservation of the repeat copy number among strains might be an artefact. Conservation of repeat sequences to the degree shown is not a feature of the ALS family, a point of contrast between gene families that could be explored in the Discussion.

We appreciate the reviewer’s comment and agree strongly that a key limitation in gene family evolution studies like ours is the quality of the genome assembly. In the original manuscript, we took several steps to ensure the completeness and accuracy of the Hil family homologs, primarily by basing our results on the high quality RefSeq collection of assemblies, and supplementing it with two fungi-specific databases. In the revised manuscript, we performed further quality analyses to assess and correct for inaccuracy in the BLASTP hits. Because RefSeq aims to provide a stable reference, it is often slow in replacing older assemblies with newer ones based on improved technologies. We thus compared the RefSeq hits for species in which a newer, long-read based assembly had become available. The results are documented in Text S1 and in summary, while we did find examples of missing homologs and inconsistent sequences, the problems were isolated to specific species and the inconsistency pertains only to the tandem repeat regions. Regarding the specific example of within-species variable number of tandem repeats (VNTR) in C. auris Hil1-Hil4, we are confident of both the copy number and the sequence variation for two reasons. First, all C. auris strain genomes analyzed in this study were assembled de novo rather than based on a reference genome, and all were long-read based (PacBio) (Table S4). Second, empirically, we found the VNTR identified in Hil1-Hil4 agree among strains within one of the four clades of C. auris while differing between clades (Table S5). Since assembly errors are not expected to produce clade-specific patterns, we believe this is strong evidence for the VNTR identified being real.

We also appreciate the reviewer’s suggestion on discussing the conservation of the repeats as an interesting trait for a group of Hil proteins in comparison to the Als family. We now added a section in Discussion focusing on the special properties of this group of Hil proteins.

• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

Due to the nature of my comments, this review will not be anonymous. I will include some of the data from my laboratory to further illustrate the point about the quality of draft genome sequences, especially for gene families that contain repeated sequences. My laboratory group has spent the past several years looking at the families of cell wall genes in these species and know that the C. tropicalis genome sequence used in the current analysis is highly flawed. There is even a manuscript from several years ago that documents problems in the assembly (doi: 10.1534/g3.115.017566). There is a new PacBio sequence available that has considerably improved data for this group of genes, but still is not perfect. We designed primers and amplified the various coding regions to verify whether the IFF/HYR were correct in the draft genome sequences. For C. tropicalis, we know that 7 of the genes listed in this paper are broken (i.e. prematurely terminated) giving a false impression of their construction. The current study did not verify any gene sequences, so broken/incomplete genes are a stumbling block for developing conclusions.

We deeply appreciate the reviewer pointing out the flaws in the C. tropicalis genome. Using the PacBio sequence-based new assembly, we were able to confirm the reviewer’s comment on the sequence and annotation error in the RefSeq assembly for C. tropicalis. We listed the comparisons between the two assemblies in Table S8. Because the differences reside outside of the Hyphal_reg_CWP domain, they don’t impact our phylogenetic analyses, which are based on the effector domain alignment. To determine if this is a widespread issue affecting all genome assemblies based on older technologies, and in response to the reviewer’s criticism, we systematically checked the sequences of BLASTP hits based on the RefSeq assemblies against newer, long-read based ones when available. As detailed in Text S1 in the revised manuscript, the problems seen in C. tropicalis were not observed in four other species. While the sample size is small, we believe the issues with C. tropicalis are likely due to a combination of specific issues with the original assembly and special properties of the genome.

Similarly, the recent work from Cormack's lab features a PacBio C. glabrata sequence (doi: 10.1111/mmi.14707). The paper details how the authors focused on accurate assembly of the types of genes studied here. Sequences from the current project should be compared to the PacBio assembly to determine if they provide the same results.

We compared the sequences of the three C. glabrata Hil homologs identified in the RefSeq assembly (GCF_000002545.3) to the best BLAST hits in one of the new Cormack lab assemblies for (BG2 strain, GCA_014217725.1). Two of the three proteins showed identical sequences between the assemblies. One of them is longer in the new assembly than in the RefSeq (1861 vs 1771 aa, XP_447567.2, QHS67215.1). The main difference, however, was the number of hits recovered. Performing BLASTP searches in the new assembly recovered 13 hits vs 3 from the RefSeq assembly, of which 12 were in the subtelomeric region. For this reason, we used the new assembly as the basis for the Hil homologs in our subsequent analyses. To determine if we missed homologs in other genomes due to incomplete subtelomeric regions in the RefSeq assemblies, we repeated the BLASTP search in four other genomes (Text S1). In one of the four species, C. nivariensis, we recovered one more homolog than in the RefSeq. In all other three, we identified the same number (S. cerevisiae: 0, K. lactis: 1, C. albicans: 12), suggesting that the issues seen in C. glabrata is likely specific to this species and its RefSeq assembly.

Another part of the study that deserves additional attention or perhaps altered presentation is the idea that the Iff/Hyr N-terminal domain binds ligands. The literature on the Iff/Hyr proteins is limited. In my opinion, though, the authors of this paper could more completely present the information that is known. The paper by Uppuluri et al. is cited (doi: 10.1371/journal.ppat.1007056), but I did not see any information about their data regarding interaction of C. albicans Hyr1 with bacterial proteins mentioned in the manuscript under review. It is formally possible that the N-terminal domain of Iff/Hyr proteins does not bind a ligand. The current manuscript includes a great deal of speculation on that point, suiting it better to a Hypothesis and Theory format rather than other types of publications.

We appreciate the reviewer’s criticism and suggestion. We made two revisions based on the comments. First, we no longer refer to the Hyphal_reg_CWP domain as ligand-binding. Instead, we refer to it as the effector domain, following existing practices in the field (Lipke 2018, PMID: 29772751, de Groot et al. 2013, PMID: 23397570). Second, during the description of the predicted structure for the domain, we mentioned that it lacks an apparent binding pocket as suggested/identified in other β-solenoid proteins with carbohydrate binding abilities. Therefore, we suggest that the potential substrate and mechanism of binding by this domain remain to be determined with further experiments. We do, however, believe that there is strong evidence for the domain being involved in adhesion. A recent study (Reithofer et al. 2021) presented structural and phenotypic characterization of three Adhesin wall-like proteins (Awp1,2,3) in C. glabrata. In particular, experimental studies of CgAwp2, a Hil family protein, showed that its deletion resulted in the reversion of the hyperadhesive phenotype in one of the C. glabrata strains. Plastic was one of the substrates being evaluated, although, as the reviewer’s work pointed out, adhesion to plastics doesn’t indicate ligand binding, as it can be mediated by non-specific hydrophobic interactions (Hoyer and Cota 2016, PMID: 27014205). Nonetheless, the results presented in Reithofer et al. 2021 and other lines of evidence presented in the current work strongly supported adhesin functions of the Hil family.

Table 1 attempts to offer evidence that the Iff/Hyr N-terminal domain has adhesive function but falls short of convincing the reader. One of the example structural templates is a sugar pyrophosphorylase that seems irrelevant to the current discussion. In the column called "Function", the word adhesin is found several times, but no detail is presented. The only entry that offers an example ligand indicates that the domain binds cellulose which is not likely relevant for mammalian pathogenesis, the main focus of the work. Other functions listed include self-association and cell aggregation--using the N-terminal domain. It is formally possible that Iff/Hyr proteins drive aggregation using the N-terminal domain and beta-aggregation sequences in the repeated region. The authors should develop these ideas further. Discussion of adhesive/aggregative function related to the ALS family can be found in Hoyer and Cota, 2016 (doi: 10.3389/fmicb.2016.00280).

We appreciate the reviewer’s comments. In the revised manuscript, we removed Table 1, which was based on I-TASSER identified templates. Instead, we identified similar structures in the PDB50 database to the AlphaFold2 prediction for the Hyphal_reg_CWP domain in C. auris Hil1 using DALI (Table S3). We described the functional implications based on this list as follows:

“We identified a number of bacterial adhesins with a highly similar β-helix fold but no α-crystallin domain (Table S3), e.g., Hmw1 from H. influenzae (PDB: 2ODL), Tāpirins from C. hydrothermalis (PDB: 6N2C), TibA from enterotoxigenic E. coli (PDB: 4Q1Q) and SRRP from L. reuteri (PDB: 5NY0). For comparison, the binding region of the Serine Rich Repeat Protein 100-23 (SRRP100-23) from L. reuteri was shown in Fig. 3F (Sequeira et al. 2018). Together, these results strongly suggest that the Hyphal_reg_CWP domain in the C. auris Hil family genes mediate adhesion.”

One line of evidence that suggest the Hyphal_reg_CWP domain may have ligand-binding activity is from the L. reuteri SRRP-BR, which is one of the bacterial adhesins identified as having a highly similar β-helical structure (but missing the α-crystallin domain). In Sequeira et al. 2018 (PMID: 29507249), the authors showed via both in-vitro and in-vivo experiments that this domain “bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH”. However, the predicted structure for the Hyphal_reg_CWP domain in C. auris Hil1 and Hil7 lack a protruding, flexible loop in the β-helix, which was proposed to serve as a binding pocket for the ligand in SRRP-BR. We therefore commented in the text “Such a feature is not apparent in the predicted structure of the Hyphal_reg_CWP domain. Further studies are needed to elucidate the potential substrate for this domain and its mechanism of adhesion.”

We also appreciate the reviewer’s suggestion to discuss the potential role of the Hil proteins in mediating adhesion vs cell aggregation. We now have a section in Discussion that focuses on the potential role of the β-aggregation sequences especially in the subset of Hil proteins led by C. auris Hil1-Hil4, which have an unusually large number of such sequences. We discuss the recent literature suggesting the potential of such features mediating cell-cell aggregation.

The incredibly large number of figures that focus on the repeated sequences in the genes does not appear to include mention of the idea that these regions are frequently highly glycosylated. Knowing how much carbohydrate is added to these sequences in the mature protein would also have bearing on whether the beta-aggregation potential is realized. The Iff/Hyr proteins could stick to other things based on ligand binding (adhesion), hydrophobicity, aggregative activity, etc. Not much is really known about protein function so the conclusions are only speculative. The authors are largely accurate in presenting their conclusions as speculative, but the conclusions are not developed fully and always land on the idea that the N-terminal domain has adhesive function when that aspect clearly is not known.

We appreciate the reviewer’s comment. We have performed N- and O-glycosylataion predictions for the Hil family proteins in C. auris as a focal example and presented the results in Figure 2 of the revised manuscript. Briefly, we found that all eight proteins are predicted to be heavily O-glycosylated (Fig. 2C). N-glycosylation is rare except in Hil5 and Hil6, in regions with a low Ser/Thr content (Fig. 2C). We also deemphasized the ligand-binding ability of the effector domain and its importance in assessing the adhesin function of the Hil family proteins. At the same time, we highlighted other mechanisms as the reviewer pointed out, such as aggregative activities, in our discussion on the potential importance of the large number of β-aggregation motifs.

Another aspect of the analysis that is not mentioned is that several of the species discussed are diploid. What effect does ploidy have on the conclusions? Most draft genomes for diploid species are presented in a haploid display, so are not completely representative of the species. Additionally, some species such as C. parapsilosis are known to vary between strains in their composition of gene families, with varying numbers of loci in different isolates.

We appreciate the reviewer raising this issue. The potential impact of having diploid genomes represented as haploids is twofold. First, if the genome sequencing was performed on a diploid cell sample with some highly polymorphic regions, that would present difficulties to the assembly and could result in poorly assembled sections. Second, either because of the first issue, or because the researchers used the haploid phase of the organism for sequencing, the representative haploid genome will not be “completely representative of the species” as the reviewer suggested. The second problem is not specific to diploids – even for haploids, any single or collection of genomes would represent just a slice of the genetic diversity in the species. We did two things to look into this. First, we analyzed multiple strains in C. auris to reveal both Hil family size variation and also sequence polymorphism, particularly in the tandem repeat region. We also, as part of the quality control, compared and searched assemblies for different strains of some species when available. We agree that characterizing multiple genomes in a species is important for fully revealing the gene pool diversity and could have important consequences on our understanding of the emergence of novel yeast pathogens.

Regarding the first issue, we checked the original publications for two large-scale yeast genome sequencing projects that included 10 of the 32 species in the present study (Dujon et al. 2004, PMID: 15229592 and Butler et al. 2009, PMID: 19465905). In Dujon et al. 2004, the authors stated that haploid cells were used in cases where the species has both haploid and diploid phases. In Butler et al. 2009, the authors said in the Methods that “for highly polymorphic regions of diploid genomes, initial sequence assemblies were iteratively re-assembled in regions of high polymorphism to minimize read disagreement from the two haplotypes while maximizing coverage.”. Therefore, the potential issue of heterozygosity is likely minimal. In addition, many diploid yeasts have large regions in their genomes being homozygous, both as a result of clonal expansion and also due to loss of heterozygosity (LOH), as documented in C. albicans and other Candida species (e.g., PMID: 28080987). Nonetheless, we acknowledge that this issue is yet another challenge to having high-quality, complete genome assemblies. In the discussion, we fully acknowledge the limitation of our study by genome assemblies, and believe that ongoing improvement thanks to the development of long-read technologies will allow more in-depth studies, particularly in the subtelomeric regions and for repeat-rich sequences.

The manuscript concludes that having more genes is better, that the gene family represents diversification that must be driven by its importance to pathogenesis, without recognizing that some species evolve toward lower pathogenesis. This concept could be explored in the Discussion. …My own experience makes me wonder if the authors found any examples of species that provide an exception to the idea that having more genes is better and positively associated with pathogenesis. The parallel between IFF/HYR and ALS genes is made many times in the manuscript. Spathaspora passalidarum, a species that is not pathogenic in humans, but clearly within the phylogenetic group examined here, has 29 loci with sequence similarity to ALS genes. How many IFF/HYR genes are in S. passalidarum?

We appreciate the reviewer’s comment. We will address the two comments above together as they are related. First of all, S. passalidarum is now included in our extended BLAST search list and we identified a total of 3 homologs in this species. When compared with the related Candida/Lodderomyces clade, which includes C. albicans, the Hil family in this species is relatively small (3 vs. >10). More generally, we observe a significant correlation between the Hil family size and the species’ pathogenic potential (Figure 1B and the phylogenetic regression result in the text).

Regarding the first comment, we did identify two species that had a large Hil family (>8 based on C. auris) and yet were not known to infect humans. One of them, M. bicuspidata, has 29 Hil homologs and is interestingly a parasite for freshwater animals, such as Daphnia. The other species, K. africana, has 10 homologs and its ecology is not well described in the literature. With respect to the relationship between adhesin family and pathogenicity, we would like to make two points. First, as mentioned above, we observed a strong correlation between the Hil family size and the pathogen status, after correcting for phylogenetic relatedness, suggesting that expansion of the Hil family is a shared trait among pathogenic species. This doesn’t rule out the possibility that some species may have an expanded adhesin family, such as the example the reviewer mentioned, for reasons other than infecting a human host. Second, a key point in our work is that expansion of the adhesin family is only the first step – the crucial contribution of gene duplications to adaptation is not just in the increase in copy number, but also in providing the raw materials for selection to generate novel phenotypes. On that front, we documented the rapid divergence of the central domains both between and within species, as well as signatures of relaxed selective constraint and positive selection acting on the effector domain following gene duplications in C. auris, both of which support the above theme.

There are several current taxonomies for the species in this region of the tree. The source of the names used in this paper could be specific more completely.

We appreciate the reviewer’s comment. We now gave the complete Latin names for all species in Figure 1 and only use abbreviated names, e.g., C. auris, after the first occurrence. For species with multiple names in the literature, we followed the species name and phylogenetic placement in Shen et al. 2018 (PMID: 30415838).

The Results and Discussion sections are largely redundant. The tone of the paper is conversational, making it easy to read, but there seems little left to say in the Discussion that has not already been mentioned as the background for the various types of analyses. The authors should revise the paper to eliminate discussions of published literature from the Results and expand the Discussion to include some of the themes that have not been mentioned yet.

We appreciate the reviewer’s comment. In the revised manuscript, we have moved discussion points from the Result to the Discussion section. We also overhauled the Discussion to focus on the implications based on, but not already covered, in the Result part, including the points the reviewer suggested, e.g., the implications of the structure on adhesion mechanism.

Another point that the authors do not mention is documented recombination between IFF and ALS genes (doi: 10.3389/fmicb.2019.00781) and the effect of that process on evolution among these gene families.

We appreciate the reviewer’s comment. We now mention this and related observations in Discussion as part of the discussion on the mutational mechanisms for the evolution of the family:

“Diversification of adhesin repertoire within a strain can arise from a variety of molecular mechanisms. For example, chimeric proteins generated through recombination between Als family members or between an Als protein’s N terminal effector domain and an Hyr/Iff protein’s repeat region have been shown (Butler et al. 2009; Zhao et al. 2011; Oh et al. 2019). Some of the adhesins with highly diverged central domains may have arisen in this manner (Fig. S10).”

My reading of the work by Xu et al. 2021 (doi: 10.1111/mmi.14707) does not match the direction of its presentation in the current paper. Oh et al., 2021 (doi: 10.3389/fcimb.2021.794529) discussed that point recently, providing another point for the Discussion in the current paper.

We appreciate the reviewer’s comment and agree that our original reading of Xu et al. 2021 was incorrect. Instead of suggesting a higher mutation rates in the subtelomeric region, the authors instead suggested the evolution of the Epa family in the subtelomere was driven by Break-Induced Replication. We now update our discussion in the following way, also citing Oh et al. 2021

“Finally, as reported by (Muñoz et al. 2021), we found that the Hil family genes are preferentially located near chromosomal ends in C. auris and also in other species examined (Fig 7), similar to previous findings for the Flo and Epa families (Teunissen and Steensma 1995; De Las Peñas et al. 2003; Xu et al. 2020; Xu et al. 2021) as well as the Als genes in certain species (Oh et al. 2021). This location bias of the Hil and other adhesin families is likely a key mechanism for their dynamic expansion and sequence evolution, either via ectopic recombination (Anderson et al. 2015) or by Break-Induced Replication (Bosco and Haber 1998; Sakofsky and Malkova 2017; Xu et al. 2021). Another potential consequence of the subtelomeric location of Hil family members is that the genes may be subject to epigenetic silencing, which can be derepressed in response to stress (Ai et al. 2002). Such epigenetic regulation of the adhesin genes was found to generate cell surface heterogeneity in S. cerevisiae (Halme et al. 2004) and leads to hyperadherent phenotypes in C. glabrata (Castaño et al. 2005).”

I might have missed it, but I could not find what constitutes a BLAST-excluded sequence (Table S7). Additional explanation (or making the explanation easier to find) would help the reader.

We apologize for the inadvertent mistake of leaving out Table S7. In the revised manuscript, we include all hits from species that are part of the 322 species phylogeny in Shen et al. 2018. Thus, we removed the original Table S7.

• Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

• Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

Ideally, validation of all sequences would provide a stronger foundation for the work. However, that request is not realistic in terms of time or resources.

We agree with the reviewer and appreciate the understanding. In the revised manuscript, we performed additional analyses to evaluate the accuracy and correct the sequences of the BLASTP hits from RefSeq database by comparing them to long-read based assemblies when possible. Please see previous replies to reviewers’ comments and Text S1 for details.

• Are the data and the methods presented in such a way that they can be reproduced?

Yes, the data and methods are documented clearly and perhaps too thoroughly in many places. A considerable amount of confidence is placed in sequences that might not be accurate and tracking details down to the amino acid residue may not be reasonable in this context. A disclaimer might help--everyone probably already knows that genome sequences are not perfect but stating that the analysis is only as good as the genome sequence acknowledges that fact.

We appreciate the reviewer’s comment. In the revised manuscript, we tried to strike a balance between providing enough methodological details for the readers to assess the conclusions and yet also keeping the flow of the paper. We also accepted the reviewer’s suggestion by adding a disclaimer in the Discussion:

“we acknowledge the possibility of missing homologs in some species and having inaccurate sequences in the tandem-repeat region. We believe the expected improvements in genome assemblies due to advances in long-read sequencing technologies will be crucial for future studies of the adhesin gene family in yeasts.”

• Are the experiments adequately replicated and statistical analysis adequate?

The idea of replicates does not really apply to this analysis. I think that the species sampled are reasonable to represent the region of the phylogenetic tree on which the analysis is focused. The authors clearly documented their computational methods in an admirable way.

We appreciate the reviewer’s comment.

MINOR COMMENTS:

Figure 1 has elements that would make a nice graphical summary, but most of it should not be part of the final manuscript. For example, Panel A is repeated in Figure 2. It is not clear what Panel C means until the reader gets to Figure 2. Panel D is unnecessary. The image in Panel B is a good graphic. Endothelial adhesion is not mentioned, though. It is also debatable whether the proteins bind directly to plastic or to the body fluids that coat the plastic.

Based on this and another reviewer’s comments, we removed Figure 1 from the revised manuscript.

Compared to Figure 1, the information in Figure 3 is inconsistent. The "central domain" in Panel A is not central to anything as drawn, located at the end of the protein. The figure should be revised to be consistent with the majority of the authors' results.

We appreciate the reviewer’s suggestion. The terminologies used to describe the different parts of a typical yeast adhesin vary in the literature. In the Als family literature, central domain refers to the region after the N-terminal effector domain and before the C-terminal Ser/Thr-rich stalk domain. In the Hil family proteins, there is not a clear distinction between a “central” and a “stalk” region. In Boisramé et al. 2011 (PMID: 21841123), the authors referred to the region between the Hyphal_reg_CWP domain and the GPI-anchor as the central domain. We adopted that use. We realize that this can lead to confusion especially for Als researchers. In some other literature, e.g., Reithofer et al. 2021, this part of the protein is referred to as the B-region. But we couldn’t find wide use of that term. We decided to stay with “central domain” in this work and hope that by defining the term in Figure 2A, we would avoid any confusion within the scope of this work.

Are the low-complexity repeats mentioned in the Figure 4 legend present anywhere else in the C. auris genome or elsewhere among the species used in this study? The answer to that question may also provide evolutionary clues.

We did find one other putative GPI-anchored cell wall protein containing this ~44aa repeat unit, but with a different effector domain (GLEYA, PF10528). This protein (PIS58185.1 in C. auris B8441), appears to be a hybrid between the repeat region of C. auris Hil1 and an N-terminal effector domain of a different family. This result fits the theme of the reviewer’s work in C. albicans and C. tropicalis on the chimeric adhesins formed between the Als and Hyr/Iff families. Due to the scope of the current work, we omitted this finding from the main result.

Figure S1 legend. How was the distance to C. glabrata measured to call it equal?

The original Figure S1 was removed in the revised manuscript. A consistent set of criteria was employed in deciding which BLASTP hits to include as Hil family members.

Figure S4 could be presented better. Both diagonals have the same information. One could be emptied or could alternatively present nucleotide identity.

The original Figure S4 was removed in the revised manuscript.

Italicize the species names in Panel C of Figure S8.

The original Figure S8C is now Figure S9 and we systematically checked to make sure that species Latin names are italicized. Thanks for pointing this out.

Lines 256-257: The paper selectively samples the Iff/Hyr family and does not examine the "entire" family. Please revise.

We appreciate the reviewer’s comment. In the revised manuscript, we no longer selectively sample species. Instead, we only exclude three species that are not part of the 322-yeast species phylogeny in Shen et al. 2018 and Muñoz et al. 2018, namely Diutina rugosa, Kazachstania barnettii and Artibeus jamaicensis. Our extensive BLASTP searches also indicated that the family as defined in this work is specific to the budding yeast subphylum. We therefore believe it is accurate to describe the work as examining the entire Hil family.

• Are prior studies referenced appropriately?

I was disappointed to see that the paper does not reference my laboratory's work at all. When ALS genes are featured so strongly in a report, it seems reasonable to include something we have done over 30+ years. Our most-recent ALS paper (Oh et al., 2021 doi: 10.3389/fcimb.2021.794529) would be a reasonable source for defending the gene numbers used in Figure 2A. Other examples of our work that directly relate to concepts in this paper were mentioned above.

We sincerely apologize for our negligence. We are new to the fungal adhesin field through an accidental finding, and despite our effort to digest the relevant literature, we did unfortunately overlook the extensive work done on the Als family, much of which came from the reviewer’s lab. We have carefully read the papers suggested by the reviewer as well as others, and now have better incorporated prior foundational and insightful work into our result and discussion sections (see previous replies to the reviewer’s comments).

• Are the text and figures clear and accurate?

Suggestions for improvement are incorporated into the comments above.

• Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

Please present Methods and Results in the past tense. I still make the same mistake when I try to get my ideas on the page but proofread one more time and ensure the verb tenses are accurate.

We appreciate the reviewer’s comments and have edited the Methods and Results sections accordingly.

SIGNIFICANCE

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

The paper reads as if it is presenting preliminary data for a grant proposal. Perhaps Prof. He's lab wants to seek functional evidence for the role of the Iff/Hyr proteins. The current paper provides an exhaustive background for such a pursuit. As presented, there is little functional data for these proteins, genome sequences are not 100% accurate, but the trends noted are defendable.

We appreciate the reviewer’s comments. We acknowledge that experimental studies will be needed to prove and further establish the functional importance of our findings. However, we believe our gene family evolutionary studies provided important novel insights and serve as an example for adhesin family evolution.

• Place the work in the context of the existing literature (provide references, where appropriate).

The ideas presented here are similar to those pioneered in the Butler et al. Nature paper in 2009 (doi: 10.1038/nature08064). We now have the benefit of more genome sequences so the analysis can encompass more species. C. auris adds a newer focus on part of the phylogenetic tree that was not previously emphasized. The idea of "more is better" is very simplistic, though. Parallel work for the ALS family shows complexity in gene expression levels, suggesting that some adhesins are poised to make a large contribution while others are likely to have a scant presence on the cell surface. Those concepts are not really explored in the current paper, either. See Hoyer and Cota 2016 (doi: 10.3389/fmicb.2016.00280); Oh et al. (doi: 10.3389/fmicb.2020.594531).

We appreciate the reviewer’s comments and have included a discussion about the potential diversity of the duplicated Hil family proteins, in terms of function and their regulation in the Discussion. Also see our response to the first comment of the reviewer regarding the novelty of our hypothesis and the significance of our findings.

• State what audience might be interested in and influenced by the reported findings.

Potential readers would come from the fields of fungal adhesion and pathogenesis, as well as evolutionary biology.

We appreciate the reviewer’s comments.

• Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

I discovered and named the ALS gene family in C. albicans and have spent 30+ years characterizing it. Most recently, my lab has focused on providing an accurate gene census and validated gene sequences for the cell wall "adhesinome" in the pathogenic Candida species. Some families are expanded and some are not. Some proteins appear only in a few species and demonstrate key roles in host-fungus interactions. There are many nuances to interpretation of what these fungi are doing from the standpoint of cell-surface adhesins and we look forward to exploring these ideas across many genomes, using validated gene sequences. We have a tremendous dataset that might make good fuel for a collaboration with Prof. He, given his enthusiasm for this area of study, as well as his outstanding expertise and perspectives on evolutionary analyses.

We sincerely thank the reviewer for the critical analysis of our manuscript and appreciate the many suggestions for improving the manuscript.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #1

Evidence, reproducibility and clarity

The manuscript by Smoak et al., provides an analysis of the Hyr/Iff-like (Hil) genes in Candida species with a strong focus on C. auris. The authors demonstrate a repeated expansion of these genes in unique lineages of fungal species, many of which are associated with stronger clinical disease. There is evidence of selection operating on the gene family in the primary domain used for identification. These genes include a repeat just downstream of that core domain that changes frequently in copy number and composition. The location of these genes tends to cluster at chromosome ends, which may explain some aspects of their expansion. The study is entirely in silico in nature and does not include experimental data.

Major points

Altogether, many of the general findings could be convincing but there are some aspects of the analysis that need further explanation to ensure they were performed correctly.

To start, a single Hil protein from C. auris was used as bait in the query to find all Hil proteins in yeast pathogens. Would you get the same outcome if you started with a different Hil protein? What is the basis for using Hil1 as the starting point? It also doesn't make sense to me to remove species just because there are already related species in the list. This may exclude certain evolutionary trends. Furthermore, it would be helpful to know how using domain presence and the conservation of position changes the abundance of the gene family across species? (beginning of results).

A major challenge in the analysis like this one is in dealing with repetitive sequences present in amplified gene families.

• For example, testing modes of selection on non-conserved sites is fraught. It's not clear if all sites used for these tests are positionally conserved and this should be clarified. Alignments at repeat edges will need to maintain this conservation and relatively good alignments as stated in lines 241-242 are concerning that this includes sequence that does not retain this structure necessary for making predictions of selection.
• It's also unclear to me why Figure S12 is here. The parameters for this analysis should be tested ahead of building models so only one set of parameters should be necessary to run the test. The evolutionary tests within single genes and across strains is really nice!
• A major challenge for expanded gene families is rooting based on the inability to identify a strong similarity match for the full length sequence. The full alignment mentioned would certainly include significant gaps. If those gaps are removed and conserved sites only are used, does it produce the same tree? Inclusion of unalignable sequences would be expected to significantly alter the outcomes of those analysis and may produce some spurious relationships in reconciling with the species trees.
• Whether or not there are similar issues in the alignment of PF11765 need to be addressed as well. There's nothing in the methods that clarifies site selection. Figure 1A: the placement of evolved pathogenesis is a little arbitrary. It's just as feasible that a single event increased pathogenesis in the LCA of C. albicans and C. parapsilosis that was subsequently lost in L. elongisporus. These should be justified or I'd suggest removing. The assignment of Candida species here also seems incomplete. The Butler paper notes both D. hansineii and C. lusitaniae as Candida species whereas they are excluded here. It is tricky to include scaffolds in analysis of chromosomal location of the HIL genes. The break in the scaffold may be due to the assc repeats of these proteins alone or other, nearby repeats. Any statistics would be best done to include only known chromosomes or those that are strongly inferred by Munoz, 2021. This will change the display of Figure 7, but is unlikely to change the take home message.

Minor points

Line 18: "spp." Should be "spps."

Line 41: I might rephrase this as "how pathogenesis arose in yeast..."

I might use a yeast-centric example around line 40 for duplication and divergence. This could include genes for metabolism of different carbon sources in S. cerevisiae.

The Butler paper referenced on line 51 compared seven Candida species and 9 Saccharomyces species The autors state no other evolutionary analysis of adhesins has been performed but do not acknowledge this study: https://academic.oup.com/mbe/article/28/11/3127/1047032

The first paragraph of the Results could be condensed

How was the species tree in Figure 1A obtained?

Figure 2: In panel A, "DH" and "SS" are not defined. I'd be careful with use of "non-albicans Candida" in Figure 2B. This usually includes C. tropicalis and C. dubliniensis and may confuse the reader. How was the binding domain defined to extract those sequences are produce a phylogeny? In building a ML model, how were parameters chosen?

Figure 3C/D could be just one panel.

Can you relate more the fungal hit to the Hil proteins conveyed in lines 152-154?

Line 168: Should read "Hence, ..." Label proteins along the top of Figure 4 too.

Figure 5: for tests of selection, were sites conserved across the group? What does the black number at each node indicate? Dn and Ds are given as decimals. This is based on what attribute? For panel B, it is unclear what each tip denotes i.e., Hil1_tr6. Hil1 is the gene but what is "tr6"?

It's unclear why comparison of the PF11765 domain includes the MRD proteins when those aren't included in the comparison to the repeats alone. Could that skew the comparison due to unequal sample numbers or changed variation frequencies in MDR relative to the other two groups?

Table 2 doesn't add much. This section could probably be reduced to a few sentences since it's highly speculative (intraspecies variation).

Table 3 is not needed.

Figure 6 - color coding in 6A needs to be explained. I'm assuming this is a taxonomical coding.

Figure 1B is unnecessary. A Model of the protein indicating domains is sufficient here. Figure 1C needs labels for all termini, not just the pathogenic red branches. The figure doesn't provide clear association between adhesin families and the associated species. This could be omitted, especially since Flo is often associated with Saccharomyces species. Figure 1D is unnecessary.

Significance

The work here is sorely needed in expanded gene families and in fungi specifically. No analysis at this level has been performed, to the best of my knowledge, in any fungal associated gene family and certainly not in relationship to pathogenic potential. The authors do a good job in citing the foundational literature upon which their study builds in most cases (one exception is noted above). It would be of general interest to those interested in the evolution of virulence, but the analysis is tricky. This is the biggest drawback I currently have as some of the information to assess the results is missing. Expertise: gene families, evolution dynamics, human fungal pathogens

this is usefully pithy

I wanted to share a content creator that looks at the psychology behind video games. I love his world and just shows you the depth behind video games. It's not just data and things for bland entertainment. https://www.youtube.com/c/DarylTalksGames

Reviewer #2 (Public Review):

Congratulations on producing a very nice study. Your study aims to examine the morphological diversity of different mammalian limb elements, with the ultimate goal seemingly to test expectations based on the different timing of development of the limb bones. There's a lot to like: the sample size is impressive, the methods seem appropriate and sound, the results are interesting, the figures are clear, and the paper is very well written. You find greater diversity and integration in distal limb segments compared to proximal elements, and this may be due to the developmental timing and/or functional specialization of the limb segments. These are interesting results and conclusions that will be of interest to a broad readership. And the large dataset will likely be valuable to future researchers who are interested in mammalian limb morphology and evolution. I have one major concern with how you frame your discussion and conclusions, which I explain below. But I think you can address this issue with some text edits.

Major concern - is developmental timing the best hypothesis?

You discuss two potential drivers for the relatively greater diversity in distal elements: 1) later development and 2) greater functional specialization. Your data doesn't allow you to fully test these two hypotheses (e.g. you don't have detailed evo-devo data to infer developmental constraints), and I think you realize this - you use phrases like "consistent with the hypothesis that ...". You seem to compromise and conclude that both factors (development + function) are likely driving greater autopod diversity (e.g. Lines 302-306). Being unable to fully test these hypotheses weakens the impact of your conclusions, making them a bit more speculative, but otherwise, it isn't a critical issue.

But my concern is that you seem to favor developmental factors over functional factors as the primary drivers of your results, and that seems backwards to me. For instance, early in the Abstract (Line 32) and early in the Discussion (Line 201) you mention that your results are consistent with the developmental timing hypothesis, but it's not until later in the Abstract or Discussion that you mention the role of functional diversity/specialization/selection. The problem with favoring the development hypothesis is that your integration results seem to contradict that hypothesis, at least based on your prediction in the Introduction (Line 126; although you spend some of the Discussion trying to make them compatible). Later in the paper, you acknowledge that functional specialization (rather than developmental factors) might be a better explanation for the integration results (Lines 282-284, 345-347), but, again, this is only after discussions about developmental factors.

When you first start discussing functional diversity, you say, "high integration in the phalanx and metacarpus, possibly favoured the evolution of functionally specialized autopod structures, contributing to the high variation observed in mammalian hand bones." (Line 282). This implies that integration led to functional diversity in the autopod. But I'd flip that: I think the functional specialization of the hand led to greater integration. Integration does not result solely from genetic/developmental factors. It can also result from traits evolving together because they are linked to the same function. From Zelditch & Goswami (2021, Evol. & Dev.): "Within individuals, integration is customarily ascribed to developmental and/or functional interdependencies among traits (Bissell & Diggle, 2010; Cheverud, 1982; Wagner, 1996) and modularity is thus due to their developmental and/or functional independence."

In sum, I think your results capture evidence of greater functional specialization in hands relative to other segments. You're seeing greater 1) disparity and 2) integration in hands, and both of those are expected outcomes of greater functional specialization. In contrast, I think it's harder to fit your results to the developmental timing hypothesis. Thus, I recommend that throughout the paper (Abstract, Intro, Discussion) you flip your discussion of the two hypotheses and start with a discussion on how functional specialization is likely driving your results, and then you can also note that some results are consistent with the development hypothesis. You could maintain most of your current text, but I'd simply rearrange it, and maybe add more discussion on functional diversity to the Intro.

Or, if you disagree and think that there's more support for the development hypothesis, then you need to make a better case for it in the paper. Right now, it feels like you're trying to force a conclusion about development without much evidence to back it up.

Limitations of the dataset

Using linear measurements is fine, but they mainly just capture simple aspects of the elements (lengths and widths). You should acknowledge in your paper the limitations of that type of data. For example, the deltoid tuberosity of the humerus can vary considerably in size and shape among mammals, but you don't measure that structure. The autopod elements don't have a comparable process, meaning that if you were to measure the deltoid tuberosity then you'd likely see a relative increase in humerus disparity (although my guess is that it'd still be well below that of the autopod). And you omit the ulna from your study, and its olecranon process varies considerably among taxa and its length is a very strong correlate of locomotor mode. In other words, your finding of the greatest disparity in the hand might be due in part to your choice of measurements and the omission of measurements of specific processes/elements. I recommend that you add to your paper a brief discussion of the limitations of using linear measurements and how you might expect the results to change if you were to include more detailed measurements and/or more elements.

In single parent cases, I am somewhat fed up with people blaming a child's problems on the fact that they come from a single parent, usually single mom, household. Too often working at schools do I hear "well they're always going to be struggling because they have just mom" and I think it's really disrespectful. It's good to hear some research that confirms it doesn't hinder bilingual development. I think people pigeonhole their thinking to find excuses or reasons why someone is struggling, when in all likelihood those students would be struggling if circumstances were different as well. There's many children with two parents who are struggling too.

Ghassane

In the Tongue Tied book, part 2 goes over the history of bilingualism relative to law. It's a timeline on the major events involved in it. Laws affect bilingualism super extensively.

Edit: When I originally wrote this annotation I hadn't read past the first few pages of part 2, and I didn't anticipate just how deeply rooted the history of the political factors go. I wanted to add that political factors affect many, many aspects of bilingual education, ranging from its availability to its effectiveness.

Ghassane

It's not just Appalachia, everyone gets hurt by stereotypes and misconceptions. Stereotypes and misconceptions act as a filter that can affect people's true judgments. For children who are growing up and have endless possibilities, stereotypes and misunderstandings can be devastating. If there are stereotypes and misconceptions in education, they are likely to undermine children's motivation and initiative.

the lost principle

Thoughts on Zettelkasten numbering systems

I've seen variations of the beginner Zettelkasten question:

"What happens when you want to add a new note between notes 1/1 and 1/1a?"

asked at least a dozen times in the Reddit fora related to note taking and zettelkasten, on zettelkasten.de, or in other places across the web.

Dense Sets

From a mathematical perspective, these numbering or alpha-numeric systems are, by both intent and design, underpinned by the mathematical idea of dense sets. In the areas of topology and real analysis, one considers a set dense when one can choose a point as close as one likes to any other point. For both library cataloging systems and numbering schemes for ideas in Zettelkasten this means that you can always juxtapose one topic or idea in between any other two.

Part of the beauty of Melvil Dewey's original Dewey Decimal System is that regardless of how many new topics and subtopics one wants to add to their system, one can always fit another new topic between existing ones ad infinitum.

Going back to the motivating question above, the equivalent question mathematically is "what number is between 0.11 and 0.111?" (Here we've converted the artificial "number" "a" to a 1 and removed the punctuation, which doesn't create any issues and may help clarify the orderings a bit.) The answer is that there is an infinite number of numbers between these!

This is much more explicit by writing these numbers as:<br /> 0.110<br /> 0.111

Naturally 0.1101 is between them (along with an infinity of others), so one could start here as a means of inserting ideas this way if they liked. One either needs to count up sequentially (0, 1, 2, 3, ...) or add additional place values.

Decimal numbering systems in practice

The problem most people face is that they're not thinking of these numbers as decimals, but as natural numbers or integers (or broadly numbers without any decimal portions). Though of course in the realm of real numbers, numbers above 0 are dense as well, but require the use of their decimal portions to remain so.

The tough question is: what sorts of semantic meanings one might attach to their adding of additional place values or their alphabetical characters? This meaning can vary from person to person and system to system, so I won't delve into it here.

One may find it useful to logically chunk these numbers into groups of three as is often done using commas, periods, slashes, dashes, spaces, or other punctuation. This doesn't need to mean anything in particular, but may help to make one's numbers more easily readable as well as usable for filing new ideas. Sometimes these indicators can be confusing in discussion, so if ever in doubt, simply remove them and the general principles mentioned here should still hold.

Depending on one's note taking system, however, when putting cards into some semblance of a logical sort-able order (perhaps within a folder for example), the system may choke on additional characters beyond the standard period to designate a decimal number. For example: within Obsidian, if you have a "zettelkasten" folder with lots of numbered and named files within it, you'll want to give each number the maximum number of decimal places so that when doing an alphabetic sort within the folder, all of the numbered ideas are properly sorted. As an example if you give one file the name "0.510 Mathematics", another "0.514 Topology" and a third "0.5141 Dense Sets" they may not sort properly unless you give the first two decimal expansions to the ten-thousands place at a minimum. If you changed them to "0.5100 Mathematics" and "0.5140 Topology, then you're in good shape and the folder will alphabetically sort as you'd expect. Similarly some systems may or may not do well with including alphabetic characters mixed in with numbers.

If using chunked groups of three numbers, one might consider using the number 0.110.001 as the next level of idea between them and then continuing from there. This may help to spread some of the ideas out as surely one may have yet another idea to wedge in between 0.110.000 and 0.110.001?

One can naturally choose almost any any (decimal) number, so long as it it somewhat "near" the original behind which one places it. By going out further in the decimal expansion, one can always place any idea between two others and know that there will be a number that it can be given that will "work".

Generally within numbers as we use them for mathematics, 0.100000001 is technically "closer" by distance measurement to 0.1 than 0.11, (and by quite a bit!) but somehow when using numbers for zettelkasten purposes, we tend to want to not consider them as decimals, as the Dewey Decimal System does. We also have the tendency to want to keep our numbers as short as possible when writing, so it seems more "natural" to follow 0.11 with 0.111, as it seems like we're "counting up" rather than "counting down".

Another subtlety that one sees in numbering systems is the proper or improper use of the whole numbers in front of the decimal portions. For example, in Niklas Luhmann's system, he has a section of cards that start with 3.XXXX which are close to a section numbered 35.YYYY. This may seem a bit confusing, but he's doing a bit of mental gymnastics to artificially keep his numbers smaller. What he really means is 3000.XXX and 3500.YYY respectively, he's just truncating the extra zeros. Alternately in a fully "decimal system" one would write these as 0.3000.XXXX and 0.3500.YYYY, where we've added additional periods to the numbers to make them easier to read. Using our original example in an analog system, the user may have been using foreshortened indicators for their system and by writing 1/1a, they may have really meant something of the form 001.001/00a, but were making the number shorter in a logical manner (at least to them).

The close observer may have seen Scott Scheper adopt the slightly longer numbers in the thousands (like 3500.YYYY) as a means of remedying some of the numbering confusion many have when looking at Luhmann's system.

Those who build their systems on top of existing ones like the Dewey Decimal Classification, or the Universal Decimal Classification may wish to keep those broad categories with three to four decimal places at the start and then add their own idea number underneath those levels.

As an example, we can use the numbering for Finsler geometry from the Dewey Decimal Classification wikipedia page shown as:

``` 500 Natural sciences and mathematics

510 Mathematics

    516 Geometry

        516.3 Analytic geometries

            516.37 Metric differential geometries

                516.375 Finsler geometry

```

So in our zettelkasten, we might add our first card on the topic of Finsler geometry as "516.375.001 Definition of Finsler geometry" and continue from there with some interesting theorems and proofs on those topics.

Of course, while this is something one can do doesn't mean that one should do it. Going too far down the rabbit holes of "official" forms of classification this way can be a massive time wasting exercise as in most private systems, you're never going to be comparing your individual ideas with the private zettelkasten of others and in practice the sort of standardizing work for classification this way is utterly useless. Beyond this, most personal zettelkasten are unique and idiosyncratic to the user, so for example, my math section labeled 510 may have a lot more overlap with history, anthropology, and sociology hiding within it compared with others who may have all of their mathematics hiding amidst their social sciences section starting with the number 300. One of the benefits of Luhmann's numbering scheme, at least for him, is that it allowed his system to be much more interdisciplinary than using a more complicated Dewey Decimal oriented system which may have dictated moving some of his systems theory work out of his politics area where it may have made more sense to him in addition to being more productive on a personal level.

Of course if you're using the older sort of commonplacing zettelkasten system that was widely in use before Luhmann's variation, then perhaps using a Dewey-based system may be helpful to you?

A Touch of History

As both a mathematician working in the early days of real analysis and a librarian, some of these loose ideas may have occurred tangentially to Gottfried Wilhelm Leibniz (1646 - 1716), though I'm currently unaware of any specific instances within his work. One must note, however, that some of the earliest work within library card catalogs as we know and use them today stemmed from 1770s Austria where governmental conscription needs overlapped with card cataloging systems (Krajewski, 2011). It's here that the beginnings of these sorts of numbering systems begin to come into use well before Melvil Dewey's later work which became much more broadly adopted.

The German "file number" (aktenzeichen) is a unique identification of a file, commonly used in their court system and predecessors as well as file numbers in public administration since at least 1934. We know Niklas Luhmann studied law at the University of Freiburg from 1946 to 1949, when he obtained a law degree, before beginning a career in Lüneburg's public administration where he stayed in civil service until 1962. Given this fact, it's very likely that Luhmann had in-depth experience with these sorts of file numbers as location identifiers for files and documents. As a result it's reasonably likely that a simplified version of these were at least part of the inspiration for his own numbering system. † ‡

Your own practice

At the end of the day, the numbering system you choose needs to work for you within the system you're using (analog, digital, other). I would generally recommend against using someone else's numbering system unless it completely makes sense to you and you're able to quickly and simply add cards to your system with out the extra work and cognitive dissonance about what number you should give it. The more you simplify these small things, the easier and happier you'll be with your set up in the end.

References

Krajewski, Markus. Paper Machines: About Cards & Catalogs, 1548-1929. Translated by Peter Krapp. History and Foundations of Information Science. MIT Press, 2011. https://mitpress.mit.edu/books/paper-machines.

Munkres, James R. Topology. 2nd ed. 1975. Reprint, Prentice-Hall, Inc., 1999.

This quote is art... :)

It's sorted. There's just a contingent pretending that there's something about it that isn't.

https://www.youtube.com/watch?v=R83vTGt0vg8 This scene made me recall a similar scene in Lord of the Rings: The Two Towers, where Frodo and his group walk through The Dead Marshes with the fallen soldiers from a long past war fought. The faces ghostly white and eyes clouded over with death as they hover just beneath the waterline. Eliot didn't describe the scene in so much detail, but it's still an interesting scene where the sailor is describes as having pearls for eyes (suggesting that the eyes reached the pallor mortis stage) similar to the eyes of the corpse floating in the marshes. By calling them pearls, it suggests that there's something that draws people towards them much like Frodo was before he fell into the marsh.

I’m wondering Is she a push over or is she just so afraid to loose her job that she doesn’t speak up. Because doesn’t she love Jean. And why would miss Julie purpose this was this to further distract John or was this to be flashy amongst her guests

Another powerful line! Vocabulary and diction are just one of many parts that enable the machine to work. Without purpose and depth within the words, we choose the words to mean nothing. Processes create results.

I agree fully with this list of flaws about the second season of Made in Abyss. The first season was terrific, but it flirted with excess in terms of violence and general unpleasantness (excess was achieved in the movie). The second season feels stagnant, sidelines the main characters, and the "aimless body horror" is a good way to describe much of the violence and grossness, which reached Elfen Lied levels of absurdity toward the end.

I've used Livescribe pens/paper for note taking (including with audio) before, and they've got OCR software to digital workflows. Or for the paper motivated, one could use their larger post it notes and just stick them to index cards as a substrate for your physical ZK with digitally searchable back ups? Now that I've thought about it and written this out, I may have to try it to see if it's better than my prior handwritten/digital experiments.

I think new developing technologies, especially within the gaming industry is really exciting. Having more people participate in the formation of different video games just means more video games, made by people like myself, usually just for the sake of enjoyment. (Which is my favorite type of art). Of course you might end up with bad video games here and there, but I think it's overall more of a positive move for humanity, and gives more people a chance at enjoying video games.

So long as there's people around to tell stories, stories are going to be told through whatever technology they had available to them. Using digital media just seems more efficient

It's interesting how this comes up in at least one of the readings each week. Before this class I never really thought about school in this sense. School to me was just something that had to be done, kind of like a chore. But now I really see how education can result in success.

Except that, seemingly, many historians have written and published about just this very topic!

People often talk about the "magic of note taking", but it's not frequent that they frame the methods as magician's secrets.

Gosling se dio cuenta que los chips estaban en todas partes pero se usaban muy mal (el mismo tenía hasta tres controles remotos)

Bank transfers require compliance processes

Know-your-client and anti-money-laundering compliance are based on laws that sanction individuals and criminal organizations. A blockchain version of bank transfers would require the same compliance workflows. As more money moves by blockchain, there will be more pressure on the intermediaries to comply with these laws. Unless you support the funding of criminal enterprises, I suppose.

Idk how good of an idea that is. Maybe it's just me but although salt water can be used to clean wounds doesn't it dry out your skin, especially when you've been exposed to the sun and salt water for a long time? Idk. Probably just me

Whenever I see paragraphs like this with someone fawnin over their master I can't help but see subtext here of a deeper relationship. Maybe sexually, or romantically, or maybe it's just a power dyanmic. Maybe it's just how to present these slaves as happily serving their masters that makes me think this way.Idk how else to put this...I can't find the right words

This is important to the field of psychology because without these works, psychology might have been just a branch of philosophy. It's interesting to me that the book neglects to mention these authors who tried to create a new science so early in human history.

Thank Cisneros for calling Manifest Destiny arrogant because it was and America is probably not better for it.

Despite that, I do love this painting from John Gast (1872) It's just pretty. https://billofrightsinstitute.org/activities/art-analysis-american-progress-by-john-gast-1872ast

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Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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Reply to the reviewers

Response to reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)): The authors develop a previously identified lead compound for the blocking of malaria transmission from humans to mosquitoes further and identified a protein target of the chemical. The protein target, Pfs16 is long known to be upregulated in gametocytes and has been speculated to be a target for small molecules. The work is well (if at time maybe too well/too detailed) described and potential shortfalls are highlighted.

My major comment is that without a deletion mutation of Pfs16, the paper will remain somewhat preliminary. I would strongly encourage the authors to generate such a mutant and compare it to the parasites treated with their drug candidate. I feel the text can be much shortened and a lot of information moved to the materials and methods. The conclusions should be toned down on several occasions (abstract, introduction, discussion). Avoid adjectives, e.g. what is a 'powerful starting point' (abstract) or 'compelling interdisciplinary evidence' but hot air?

We thank the reviewer for this comment. However, we would like to reiterate (as stated in the manuscript) that knockout of Pfs16 in P. falciparum is transmission lethal, i.e. you do not get progression of male gametogenesis. Thus, whilst re-generation of a Pfs16 KO would be interesting in terms of comparing phenotypically with the drug treated parasites, we are not convinced it would add any further evidence of support for or against our conclusion in terms of the ability of the N-4HCS scaffold to target this protein. E.g. we could drug treat a Pfs16 KO but this would not be expected to show gametogenesis irrespective of treatment. Therefore, whilst of academic interest, we believe it is satisfactory to judge our phenotypic work based on published accounts of the Pfs16 KO without having to engage in the costly experiments to regenerate the parasite and work on it side-by-side, especially given the limited resolution it would give towards the overall goal of the work in terms of defining the effect and likely target of this drug class on parasites.

Addressing the second comment, we are happy to alter areas of the paper that may have over-stated the conclusions of the work including the abstract/introduction and discussion.

CROSS-CONSULTATION COMMENTS I think these three reviews are pretty much in line with their overall assessment. I am happy if send as is to authors as it will help them shape a much better paper

Reviewer #1 (Significance (Required)):

The paper shows that very likely a new chemical with some potential for transmission inhibition of malaria parasites for mosquitoes binds to a Plasmodium protein that is specifically expressed in the sexual stages of the parasite.

The paper compares to good papers published in journals like ACS Infectious Diseases or Antimicrobial Agents and Chemotherapy, but I am not sure which of the Review Commons sister journals it would fit to. I am a molecular parasitologist.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): Transmission blocking drugs are of high interest as a strategy to combat malaria but they are difficult to study. For instance it is problematic to raise resistant parasites to find mode of action of transmission blocking drugs and to identify their targets in the cell. In this manuscript Yahiya et al. build on previous work which identified the N-4HCS scaffold, of which DDD01035881 is the lead compound, as an inhibitor P. falciparum male gametocytes. Using PAL to enrich for target proteins Pfs16 was identified and validated as a possible target of DDD01035881. Binding was validated through CESTA. Determination of the phenotype following DDD01035881 treatment was found to partially match the previously published Pfs16 KO phenotype. However curiously no impact was seen in gametocytogenesis despite published evidence of Pfs16 being involved in sexual conversion. The authors speculate as to reasons but a direct experimental comparison with Pfs16 mutant parasites (which likely would have been revealing) is not provided. On the positive side, this analysis of the stage-specific effect of the drug pinpoint the stage inhibited during microgamete development which is a very interesting part of the manuscript.

We thank the reviewer for this positive assessment of our work. Mirroring comments above, our challenge with Pfs16 knockout or mutation is that if we ablate Pfs16 function we cannot assess the effect of drug action. Definition of a mutant that would demonstrate precisely the drug mode of action would require structural resolution of drug bound to target (i.e. to identify which residues to target) – this is a major goal for our research group moving forwards, but likely many years’ work. In general, our core approach here has been one of chemo-proteomic based methods and phenotypic investigation of the novel antimalarial. Further evidence might be forthcoming from molecular genetics/structural biology, but we believe these are beyond the scope of the current work (and our available resources at present). We state future directions in the discussion and can add more to this in any revised manuscript.

This work deepens the understanding of a novel class of transmission blocking drugs with reasonable potency (foremost (-)-DDD01028076, which has low nanomolar activity, the modified versions considerably less). Question on how to achieve serum concentrations for sufficient potency aside, these compounds will in the very least provide experimental tools to study their mode of action and might reveal interesting biology. This work is therefore of interest to the malaria field.

The experimental methodology seems excellent but some of the results raise questions that make definite conclusions difficult and this should be addressed. Overall, this is very solid work but leaves some doubts whether Pfs16 is indeed the (only) target of this class of compounds.

Major comments: 1. The reasons for excluding Etramp10.3 are not convincing. In fact it could be argued it is nearly as good a candidate as Pfs16. Contrary to the author's statements in the results section, etramp10.3 transcription is highly upregulated in gametocytes (see e.g. PMID: 22129310) with a generally very low transcription in asexual stages. It is argued that Etramp10.3 is essential in blood stages because MacKellar et al failed to disrupt the gene and because the PiggyBAC screen predicted it to be essential. However, if this is an argument for exclusion then this would also apply to Pfs16 which is also predicted by the PiggyBAC screen to be essential (likely both are non-essential in blood stages as they are barely expressed but Pfs16 and Etramp10.3 might by chance have not received an insertion in the PiggyBAC screen due to their very small size which may also explain failure of disrupting integration in MacKellar). Given the finding that the drug binds Pfs16 only in late gams it might also be argued that an essential function in asexuals might not be affected if they behave similarly to young gams and hence this criterion is not valid anyway.

Further following this line of thought that ETRAMP10.3 could be a hit equivalent to Pfs16, Figure 2D shows a band below the band considered to be Pfs16. It would not be all surprising if this were ETRAMP10.3 (the size would fit).

We don’t disagree with reviewer 2’s comments that ETRAMP10.3 could be an additional target. Although not traditionally related there is some similarity between these proteins and it may be that at the macroscopic level there is a structural homology between them. As stated elsewhere we are happy to tone down the assertion that Pfs16 is the only drug target candidate, leaving open the possibility of future follow up work that may yet reveal additional targets. This cannot be explored much further without extensive experimentation, which is beyond our current capacity. Given the strong phenotypic effect on gametocytes, whilst ETRAMP may be upregulated, this paper naturally focused its core attention on Pfs16 as a candidate target. We certainly subscribe to the view that absence of evidence is not evidence of absence.

Both, Pfs16 and ETRAMP10.3 can be expected to be very abundant proteins in the parasite periphery in gams. Can the authors exclude that these simply are the first to encounter the N-4HCS photoaffinity probe and that this may have led to their enrichment in the target identification experiments. The biochemical data argues for a specific interaction with Pfs16, but by itself is not that strong. Given the discrepancies of the phenotype with the Pfs16 disruption and the peculiar finding that the drug binds Pfs16 only in later stage gametocytes, it might be a good idea to further caution the conclusion of Pfs16 as the inhibited target.

We don’t necessarily agree that the evidence is not strong (three methods pointing to the same target is by many accounts solid evidence). Additionally, whilst it is true that the N-4HCS photoaffinity probes likely interact with PVM proteins in first instance, it is also worth noting that this doesn’t necessarily deduct from their likelihood to be true targets, but instead fits with the N-4HCS phenotype. We observe the compounds to inhibit microgametogenesis without any prior incubation and to retain this activity even beyond activation of microgametogenesis, specifically during the window in which the PVM remains associated with the parasite. Our phenotypic observations therefore fit with the notion that the molecules target proteins that lie within the PVM and interact with the molecules at first instance. Whilst we understand the concern that PVM proteins may be likely to be enriched given their abundance and localisation, we believe this to support our phenotypic findings.

The phenocopy evidence of the NH compounds with the Pfs16 disruption is based on comparison with published evidence. It would have been much preferred to have a side-by-side comparison with the (or an) actual Pfs16 disruption parasite line. Although the authors stress that the phenotype with DD01035881 fits the phenotype of the targeted gene disruption in the results, this only partially matches the cited publication (PMID: 14698439) which concludes there is an effect on the number of gametocytes produced. The exflagellation phenotype in that publication was classified as preliminary. Although this is discussed, the main results text should be adapted to reflect this and the conclusion that Pfs16 may be the target should be further cautioned.

As stated, we are happy to tone down conclusions in this direction. We also note comments above about Pfs16 disruption.

Minor comments: 4. From the modifications of the compounds it seems the chemical space for further modification to achieve higher potency is limited with this scaffold. Maybe the authors can comment whether they envisage this to be a potential obstacle.

The modification space of the compounds is explored extensively in previous work from our group, which we feel more than adequately addresses this question. See Rueda-Zubiaurre et al (2020) J Med Chem.

Line 67: references are superscript.

We can change this

Line 77: I would recommend replacing 'quiescence' here, a cell that matures is not quiescent.

We can change this

Line 116: consider removing 'interdisciplinary'.

We can change this

Line 120: I would caution here (see major comments) and recommend a less definite proclamation of Pfs16 as a promising new drug target

We can change this along with the general “tone” of the manuscript.

Page 7: compounds 9 is still considered active ("retained micromolar activity"), but in Table 1 this is given as >1000nM. Please add the actual IC50.

We can add this to the final version. The actual IC50 for this compound was 1.7uM. For the SAR study we grouped compounds with IC50 >1uM into discrete groups based on rough IC50 (>1uM, >10uM etc.) hence this fell in the intermediate group.

Line 138- 173: The order in which this is discussed makes it unclear that the work described was done prior to, and guided, the synthesis of compound 1 and probe 2

This can be addressed in a revised manuscript.

Line 194: was the data deposited in a database?

The proteomics data has not been deposited in a database but is accessible in the extended SI.

Line 202: introduction as to the benefits of using a competition + probe condition here could aid reader understanding. The interpretation of this data is complicated by the covalent and reversible binding of the two compounds and the weight of this control is therefore difficult to gage.

We can embellish the description here.

Table 2 and Extended Data Table 1 show different p values and enrichments for the same hits. This is confusing. It would also be useful to label the hits in the scatter plots in Figure 2 for easy identification and comparison to the tables.

We can amend this and label each hit within the scatter plot.

Line 215-218, please correct the data on Etramp10.3 (see major points) and put in perspective to Pfs16 (Etramp10.3 is similarly upregulated in gams where it is highly expressed; PiggyBAC predicts essentiality for Pfs16 and Etramp10.3 in blood stages).

We can discuss this to a limited extent for future exploration of Etramp10.3.

Line 221: the results from the PiggyBAC screen are stated as fact, but what the screen provides is a prediction of the probability of importance for parasite growth. I would replace 'is' with 'is predicted' (even though in the case of Rab1b it seems likely the prediction is correct).

We can change this

Line 233 and elsewhere: define 'reversibility' (binding? activity?).

We can change this

Line 240: clarify what is in the cited paper (see major points).

We can clarify this

Line 297: We utilised in-lysate...... clunky sentence, please rephrase.

We can change this

Line 325: reference is missing the year.

We can change this

Line 343: It is utterly puzzling that binding is specific to Pfs16 in mature gametocytes and I do not find the explanation in the discussion convincing (see point 28 below). Do the authors have another explanation? Could Pfs16 be modified in later gams (or vice versa)?

We believe that Pfs16 is functionally different at different stages of gametocyte development, this is either in terms of its presentation (e.g. perhaps due to complex formation, though this remains elusive) or the functionality of different domains, as per the effect of different truncation mutants. We can address some of these concerns in a revised manuscript.

Line 388: Justification seems odd as a PV protein would be unlikely to directly impact DNA replication. Please rephrase the sentence.

We can change this

Line 405: remove the 'to'

We can change this

Line 411: it would be useful to the reader to state at what IC-value the drug was used in these experiments.

We can state this

Line 431: While the alpha-tubulin staining indicates exflagellation and is similar to the DMSO only control, the staining for the RBC membrane (Glycophorin A) and DNA (DAPI) appear different, yet this is ignored. One interpretation of this could be that while late treatment doesn't block exflagellation, it still impacts other aspects of microgamete development.

We can make mention of this

Line 436: IFA work was done with drug treatment post activation while EM was done post activation but drug treatment prior to activation. Is there a reason for this?

The reviewer is astute to point this out. Limitations with access to the EM facility meant that whilst IFAs were completed for pre-activation treated samples, the post-activation EM became impossible as the EM facility closed during the COVID lockdown. Thus, we do not have a complete set here. However, we do not feel this takes away from the EM observations presented. We can clarify this incompleteness in the revised manuscript.

Line 450: is this really CytB, or was it CytD?

We did indeed used Cytochalasin B here, which whilst less potent than D does still target microfilament formation.

Line 465: Pfs16 localised to vesicles: there is no data showing the dots in the micrograph are vesicles, please rephrase.

We can change this

Page 19 and 20, discussion on stage-specific differences of Pfs16 during gametocytogenesis to explain the difference in binding: without experimental data using H-4HCS in the parasites of the publication cited to explain this (PMID: 21498641), this is very speculative. The cited work used episomal expression of Pfs16 tagged with fluorescent proteins. This would be the first integral PVM protein that is actually inserted into the PV membrane when tagged in that way (usually this results in a PV location), casting some doubt on the findings in that paper. All in all the provided explanation is not very convincing.

We can attempt to clarify this in a revised discussion.

Line 519: if with the conserved part the N-terminus is meant, then this has for other PVM proteins already been shown to be PVM internal, not facing the erythrocyte (show in very early work; PMID: 1852170 but also multiple times after that).

We can clarify this

Line 534: consider replacing 'highly plausible' with something more cautious.

We can change this

Line 550: Given this discussion how stable are N- 4HCS compounds?

We can clarify this.

Table 1: Having all chemical structures in same orientation would be nicer visually. I assume blue indicates modification but this is not stated.

We can change this

Figure 1: Please use different colours or symbols. The dark green crosses and the blue Pfs16 cross are hard to distinguish.

We can change this

Figure 3d: Unclear as to why a difference temperature range is displayed here.

We can clarify this

Figure 3e: Unclear % Inhibition compared to what.

We can clarify this

Figure 5G: What is the white arrow pointing to?

We can clarify this

Figure 5j: Given how the explanation is written this would make more sense between current image 5G and 5J.

We are not sure what the comment relates to here but we can endeavour to clarify this

Figure 6: Erythrocyte membrane colour not stated in legend.

We can change this

Figure 6A: were the exposure times similar? How can so little be left after ~4-5.5 minutes but at later time points there seems to be much more Pfs16 signal left? Maybe amount of signal should be taken into consideration to establish the fate of Pfs16 in the process.

We can endeavour to clarify this

Figure 6B: is the second phenotype (successful but aberrant egress) shown? The only image where WGA is not circular around the parasite is an exact match of Pfs16 which is in dots (image at 7.5-8.5 minutes). The imaging data for this phenotype should be presented more clearly.

We can attempt to clarify this

Reviewer #2 (Significance (Required)):

Nature and significance: a lot of weight has been placed on transmission blocking drugs although there are also a number of problems associated with them (ethics for testing and use etc; drugs acting on asexuals and transmission stages alike might be even more useful). Transmission blocking drugs are difficult to study and this work is therefore important. The experiments are well done, but the conclusions are not fully convincing, leaving some doubts in regard to Pfs16 being the actual target of the class of drugs studied.

Compare to existing published evidence: it is a logic continuation of previous work and this is appropriately highlighted in the manuscript.

Audience: medium interest for malaria researchers; high interest for researchers working on transmission blocking drugs and those studying microgametes.

Your expertise: malaria, P. falciparum, biology of apicomplexans

Reviewer #3 (Evidence, reproducibility and clarity (Required)): The manuscript by Yahiya et al describes an extensive investigation of the mode of action of DDD01028076, which specifically inhibits microgametogenesis in Plasmodium falciparum. The phenotypic characterisation of the MOA uses some very nice imaging to demonstrate the point at which this compound inhibits microgametogenesis. The authors have also attempted to identify the molecular target using chemoproteomics and label-free CETSA techniques. The photoaffinity labelling and pull-down approach suggested the Pfs16 may be preferentially enriched by a PAL probe that is representative of this series. However, the data supporting the validation of this target is not very conclusive, and in some cases argues against Pfs16 being a specific target of DDD01028076. Whilst the presented data makes a significant contribution to the literature regarding a novel drug candidate that targets microgametogenesis, it does not support the author's claims that Pfs16 is the target.

Major Concerns: The strongest evidence for Pfs16 being the target comes from the chemoproteomics pull-down study that found Pfs16 to be the most significantly enriched protein by compound 2 vs DMSO. However, this should be interpreted with caution as it is based on only 3 replicates and omics studies are prone to false-positives. That only 125 proteins were detected also raises questions about the coverage of the proteomics, it is quite possible that the actual target is not detectable using this method, and the Pfs16 appears because it is one of the more abundant proteins during this stage of the lifecycle.

As discussed, we are happy to tone down the conclusions about Pfs16 being an exclusive target for the N-4HCS drug class, however, we feel the reviewer is being unnecessarily negative. There are myriad papers in the literature based on singular proteomics experiments (given their cost, complexity and time -consuming nature) that then facilitate downstream experiments that support findings. We have endeavoured to be as thorough as we could in the work and believe, like others, three replicates of a massive experimental pipeline should be sufficient to make a defined conclusion – whether the additional downstream evidence we have then leaned on is supportive of this (as we judge it to be) is another matter. We agree, proteomics often suffers with low protein abundance. The complexity of growing large quantities of gametocytes is familiar to anyone who has struggled to grow these finicky parasites at a larger scale than 10-25mL dishes. Given the scales we have reached, we believe these might in fact be some of the most comprehensive proteomics studies to date!

Somewhat concerningly, the control with 1 as the competitor did not show significant enrichment of Pfs16, although a trend was observed. More concerning, was the lack of enrichment when using DDD01028076 as the competitor. This result essentially proves that Pfs16 is not the specific target (and the argument about reversibility is unlikely since most drugs are reversible binders, but many have worked with this type of approach). It is surprising that DDD01028076 (ideally the (-) form) wasn't used as the competitor for the proteomics study. This compound has ~100-fold better potency than the probe 2, which should provide much better competition that 1. It would also be more specific than 1, which is an important control considering that (-)-DDD01028076 has activity in the low nanomolar range, whereas 2 acts in the micromolar range. Non-specific interactions are an important consideration to exclude, and whilst 1 is structurally similar, it is not very potent and therefore not the best control to find the target associated with activity.

Whilst we understand the concerns with insignificant enrichment in the competition labelling, we believe the enrichment in the presence of photoaffinity probe 2 over background (i.e. DMSO vs. probe experiments) to be of more value given the design of the experiment. The competition experiments were performed by co-treating gametocytes with photoaffinity probe 2 and parent molecule 1 prior to UV irradiation, to enable irreversible conjugation to protein target(s). However, given that both compounds, probe and parent, theoretically bind to Pfs16 at the PVM in a reversible manner (i.e. losing interaction with even gentle washing), UV irradiation is likely to favour probe-binding irrespective of competition with a marginally more potent parent molecule (in this case, parent molecule 1). This is especially true as treated parasites were very thoroughly washed after irradiation, so should the parent molecule have bound the target protein(s), these drug-target interactions were likely lost during stringent washing. The drug-target interactions with parent molecule 1 wouldn’t have been aided by UV irradiation, as the molecule lacks the functional group required for bioconjugation. So, even if parent molecule-target interactions were more abundant than probe-target interactions, interactions between parent molecule 1 were most likely lost and proteins bound by probe were enriched.

This would have been true with more potent N-4HCS derivatives such and DDD01028076 and (-)-DDD01028076 (where potency is tested in the DGFA, independent of bioconjugation), and here we opted for a structurally similar compound of similar potency to not skew competition solely based on potency.

We can embellish on this in the revised manuscript to make our conclusions from this part clear.

A closer look at the gels in the supplementary data raises many questions that undermine the authors conclusions: - Fig S1a - The lane without probe (2) still identifies Pfs16 (or a protein at that MW) as the most abundant protein. Also, as the Pfs16 band increases, you can see that most other proteins also increase in abundance, so either the loading is inconsistent, or the probe actually causes non-specific enrichment of many proteins. This figure also indicates that the washing protocol is not sufficient to remove non-specific binders. Given the covalent nature of the PAL approach I would think a very thorough washing protocol could be employed.

It is certainly the case that Pfs16 is abundant in gametocytes, a reason behind its early discovery. Thus it is challenging to remove it from background. We still believe the enrichment to be specific, highlighting the comparative work with Pfg377 in Figure 2. Further repetitions with more stringent washing might resolve the background, however, this is beyond our current resources to repeat.

-- Running another negative control in the proteomics using one of the inactive controls from table 1 might help to disambiguate specificity.

We don’t disagree with this though this would involve an entire re-running of the experimental workflow which is not possible.

• Fig S2a - The anti-Pfs16 Western blots show that this protein is actually enriched more in the flow-through than the eluates. This shows that this protein is not specifically enriched by the PAL-CuAAC pull-down, it is just more abundant in the treated samples.

Again, the presence of Pfs16 in the flow-through is unsurprising, given its abundance in stage V gametocytes. The relative abundance in the eluate is not an indication that the binding and subsequent enrichment is not specific, rather this shows the compound does not necessarily bind each and every protein – which is not unexpected. The crucial conclusion to be drawn here is the concentration-dependent enrichment of Pfs16 in the eluate in the presence of probe.

• Fig S2b - The darkest Pfs16 spot is actually the sample with no UV treatment. This is a negative control, so should not enrich the target protein. This sample also has significant signal in replicates A and C.

As we have noted above, it is not unsurprising that modification of the N-4HCS scaffold to yield this probe may introduce a level of irradiation-independent binding, which explains the presence of signal in the UV-independent sample.

• Fig S2c - This blot is very messy and difficult to read, but in general the Pfs16 spots in the IGF don't correlate with the intensities in the anti-Pfs16 western.

These experiments are extremely challenging (something that is perhaps beyond the expertise of the reviewer) and what is presented is the result of substantial optimisation. Loss of AzTB fluorescence in the gel which is subsequently analysed by western blot explains this.

• Fig S2 - This data, and the main figures based on this data, generally don't support the hypothesis that Pfs16 is the specific target. The controls are not as would be expected, and there are no loading controls. Looking at the flow-throughs suggests that there was just more Pfs16 (and possibly total protein) in the treated samples before the enrichment step. The Pfg377 also appears quite variable in the different samples, with replicates B and C not consistent with A.

We do not concur with the reviewer here and their dismissal of what was extremely thorough and well-executed experimtns. These are not like traditional western blots and require substantial optimisation. We refer them to our previous point in reference to the UV controls. With regards to the Pfg377 variability, the experiment itself is inherently variable with such large volumes of parasites. In many cases, for example, the male:female ratio within a mature gametocyte culture can vary and this can contribute to the variability in 377 abundance between replicates.

The other major concern is with the CETSA analysis, which appears to show very minor stabilisation of Pfs16, but the specificity of this target is questionable, and the data has the following inconsistencies. - The supplementary data only shows n=1, yet there are error bars in the main figures. Where did these come from?

The individual western blot replicates can be provided in a revised manuscript if judged important.

• The samples with apparent destabilisation are all near the edge of large western blots, which often doesn't run straight and has no loading controls. We need to see the loading controls.

Given all proteins within a lysate will aggregate with thermal treatment, antibody loading controls are not feasible with these experiments. Each sample is normalised prior to thermal stabilisation (ensuring the same protein quantity is treated in both DMSO and drug, at each temperature) and any protein that is not aggregated is loaded – the nature of CETSA itself is to compare the stabilisation between DMSO and drug.

• The melting temperature of Pfs16 is extremely high at around 85 degrees C. Most plasmodium proteins melt at around 50-60 degrees (Dzekian et al, 2019). Even the cited work on membrane proteins didn't go to those temperatures (Kawatkar et al, 2019) Can this high temperature be explained, and has the CETSA approach been validated at such high temperatures where additional physical and chemical processes may be occurring in the sample?

We agree that this temperature of stabilisation is unusually high and may require further biochemical validation. Without further investigation we cannot say definitively why the melting temperature of Pfs16 is so high, but suspect its size and membrane localisation may play a role.

• The lack of difference between + and - isomers suggests that the very small stabilisation observed here is not specific to drug activity, but is more likely a non-specific binding effect. Additional negative control compounds might help here, but the + isomer is probably the best negative control (albeit the concentrations were not ideal in the presented data).

Please we have already addressed this in the text – refer to line 312 and beyond.

• The very high concentration (100uM) increases the chances of non-specific effects being observed here (especially since the authors claim to see stabilisation at about 10nM). The study should be repeated at lower concentrations (with negative controls) in order to confirm a specific binding effect.

Whilst further replicates with different conditions might be preferable, as discussed extensively here, this would be beyond the scope of what we are able to achieve for a revision.

• The concentration-ranging study was performed at 78.4 degrees, at which temperature very little denaturation of Pfs16 occurs fig S4a (and Fig 3b-c). Therefore, you would not expect to see any drug-induced stabilisation, and it is not plausible that significant stabilisation could occur at this temperature. Therefore, the apparent destabilisation at sub-10nM drug concentrations is highly questionable.

We would have to agree to disagree on this point.

• Stabilisation of Pfs16 did not occur in lysates from younger gametocytes (fig s4g-h), but this is a biophysical assay, so regardless of the function of this protein at different stages, the biophysical interaction between the drug and the protein should be the same regardless of the source of the protein. This data argues against Pfs16 being a specific binding target of Pfs16.

We don’t agree with this statement, since the drug is binding the protein in native lysate – this may be a multi-meric complex (homo or hetero) which only exists at certain stages. As such we disagree with the reviewer that this argues against Pfs16 being the target.

In addition to the above concerns, the fact that this compound doesn't inhibit the earlier functions of Pfs16 in gametocytogenesis, and that it doesn't inhibit P. berghei, also argue against this being the specific target of this drug. Whilst the authors have a valid argument that these findings don't exclude the possibility of stage-specific targeting of Pfs16, we could also argue that all the phenotypic data in figures 4-6 is merely correlative of a drug that acts at the same point in the lifecycle as Pfs16.

We have discussed this in the manuscript and strongly feel the reviewer is being unnecessarily dismissive of a body of work that is coherent. We are happy to tone down the narrative of the paper with Pfs16 being the exclusive target. Structural homology of P. berghei Pfs16 orthologues has never been done but it would not be unprecedented if another target was functionally homologous (an idea we are currently pursuing). Stage specificity is also possible given the nature of Pfs16 (e.g. if it is in a complex). The reviewer appears fixated on a singular entity and unable to imagine a complex scenario where structure or protein-protein interactions might affect drug binding (as it does with other proteins present in complexes, e.g. proteasomal targeting drugs).

Overall, I believe that significant additional studies would be required to identify the target of this compound. Either by repeating the included studies with additional controls and conditions, or by follow-up studies such as genetic manipulation (knock-down or overexpression) or heterologous expression and biophysical binding studies.

Alternatively, the manuscript could be restructured as primarily a report on the phenotypic effect of this compound on microgametogenesis, with the target identification work reported as a hypothesis-generating chemoproteomics study that provides some ideas about possible targets, but requires substantial follow-up to confirm the target (which may be beyond the scope of this report?).

We strongly disagree with this reviewer’s entire dismissal of an extensive body of work. In line with other reviewers comments we accept a need to tone down our conclusions, but do not consent to dropping the majority of the paper in favour of a phenotypic descriptive work.

MINOR COMMENTS The manuscript is very well-written and presented.

Several of the conclusions are overstated (as detailed above) and several statements should be tempered based on this data (e.g. statements linking DDD01028076 effects to Pfs16 function).

We can address the overstatement of conclusions in a revised manuscript.

I find the term 'crosslinking' confusing for the photo-affinity labelling, as crosslinking in proteomics often refers to crosslinking between proteins (not between protein and drug).

This is simple to address – to minimise confusion for readers, we can simply state where photoaffinity labelling and bioconjugation were performed (and not refer to the latter as crosslinking).

The data and terminology around activity (IC50) for compounds in table 1 is a little confusing. Some IC50 values are reported as >1000, while others have precise mean values reported over 1000, and others are >10,000 or >25,000. This is especially confusing where 9 is claimed to have retained activity, but is >1000. If consistent thresholds are not appropriate then perhaps including dose response curves in the supp data might be necessary to explain these?

We can simply provide the provide IC50s for compounds of greater potency. We are also happy to provide the curves but with such a large body of work already, this might be unnecessary.

Reviewer #3 (Significance (Required)):

The work is potentially interesting to Plasmodium biology and drug discovery researchers. The concept of a transmission-blocking drug is quite attractive to this community, so the topic is highly relevant. Keeping in mind that this compound was reported previously, the main novelty is in defining it's window of activity during the microgametogenesis process, and differentiating this from other drugs/compounds that inhibit this process. There is clearly an advance in knowledge presented here.

If Pfs16 were to be confirmed as the target of this series then I think that this study would have much greater impact and attract interest from a broad audience. However, at this stage I don't see strong evidence for this hypothesis, and some of this data casts significant doubt on the likelihood that Pfs16 is the direct target.

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Referee #3

Evidence, reproducibility and clarity

The manuscript by Yahiya et al describes an extensive investigation of the mode of action of DDD01028076, which specifically inhibits microgametogenesis in Plasmodium falciparum. The phenotypic characterisation of the MOA uses some very nice imaging to demonstrate the point at which this compound inhibits microgametogenesis. The authors have also attempted to identify the molecular target using chemoproteomics and label-free CETSA techniques. The photoaffinity labelling and pull-down approach suggested the Pfs16 may be preferentially enriched by a PAL probe that is representative of this series. However, the data supporting the validation of this target is not very conclusive, and in some cases argues against Pfs16 being a specific target of DDD01028076. Whilst the presented data makes a significant contribution to the literature regarding a novel drug candidate that targets microgametogenesis, it does not support the author's claims that Pfs16 is the target.

Major Concerns:

The strongest evidence for Pfs16 being the target comes from the chemoproteomics pull-down study that found Pfs16 to be the most significantly enriched protein by compound 2 vs DMSO. However, this should be interpreted with caution as it is based on only 3 replicates and omics studies are prone to false-positives. That only 125 proteins were detected also raises questions about the coverage of the proteomics, it is quite possible that the actual target is not detectable using this method, and the Pfs16 appears because it is one of the more abundant proteins during this stage of the lifecycle.

Somewhat concerningly, the control with 1 as the competitor did not show significant enrichment of Pfs16, although a trend was observed. More concerning, was the lack of enrichment when using DDD01028076 as the competitor. This result essentially proves that Pfs16 is not the specific target (and the argument about reversibility is unlikely since most drugs are reversible binders, but many have worked with this type of approach). It is surprising that DDD01028076 (ideally the (-) form) wasn't used as the competitor for the proteomics study. This compound has ~100-fold better potency than the probe 2, which should provide much better competition that 1. It would also be more specific than 1, which is an important control considering that (-)-DDD01028076 has activity in the low nanomolar range, whereas 2 acts in the micromolar range. Non-specific interactions are an important consideration to exclude, and whilst 1 is structurally similar, it is not very potent and therefore not the best control to find the target associated with activity.

A closer look at the gels in the supplementary data raises many questions that undermine the authors conclusions:

• Fig S1a - The lane without probe (2) still identifies Pfs16 (or a protein at that MW) as the most abundant protein. Also, as the Pfs16 band increases, you can see that most other proteins also increase in abundance, so either the loading is inconsistent, or the probe actually causes non-specific enrichment of many proteins. This figure also indicates that the washing protocol is not sufficient to remove non-specific binders. Given the covalent nature of the PAL approach I would think a very thorough washing protocol could be employed. -- Running another negative control in the proteomics using one of the inactive controls from table 1 might help to disambiguate specificity.
• Fig S2a - The anti-Pfs16 Western blots show that this protein is actually enriched more in the flow-through than the eluates. This shows that this protein is not specifically enriched by the PAL-CuAAC pull-down, it is just more abundant in the treated samples.
• Fig S2b - The darkest Pfs16 spot is actually the sample with no UV treatment. This is a negative control, so should not enrich the target protein. This sample also has significant signal in replicates A and C.
• Fig S2c - This blot is very messy and difficult to read, but in general the Pfs16 spots in the IGF don't correlate with the intensities in the anti-Pfs16 western.
• Fig S2 - This data, and the main figures based on this data, generally don't support the hypothesis that Pfs16 is the specific target. The controls are not as would be expected, and there are no loading controls. Looking at the flow-throughs suggests that there was just more Pfs16 (and possibly total protein) in the treated samples before the enrichment step. The Pfg377 also appears quite variable in the different samples, with replicates B and C not consistent with A.

The other major concern is with the CETSA analysis, which appears to show very minor stabilisation of Pfs16, but the specificity of this target is questionable, and the data has the following inconsistencies.

• The supplementary data only shows n=1, yet there are error bars in the main figures. Where did these come from?
• The samples with apparent destabilisation are all near the edge of large western blots, which often doesn't run straight and has no loading controls. We need to see the loading controls.
• The melting temperature of Pfs16 is extremely high at around 85 degrees C. Most plasmodium proteins melt at around 50-60 degrees (Dzekian et al, 2019). Even the cited work on membrane proteins didn't go to those temperatures (Kawatkar et al, 2019) Can this high temperature be explained, and has the CETSA approach been validated at such high temperatures where additional physical and chemical processes may be occurring in the sample?
• The lack of difference between + and - isomers suggests that the very small stabilisation observed here is not specific to drug activity, but is more likely a non-specific binding effect. Additional negative control compounds might help here, but the + isomer is probably the best negative control (albeit the concentrations were not ideal in the presented data).
• The very high concentration (100uM) increases the chances of non-specific effects being observed here (especially since the authors claim to see stabilisation at about 10nM). The study should be repeated at lower concentrations (with negative controls) in order to confirm a specific binding effect.
• The concentration-ranging study was performed at 78.4 degrees, at which temperature very little denaturation of Pfs16 occurs fig S4a (and Fig 3b-c). Therefore, you would not expect to see any drug-induced stabilisation, and it is not plausible that significant stabilisation could occur at this temperature. Therefore, the apparent destabilisation at sub-10nM drug concentrations is highly questionable.
• Stabilisation of Pfs16 did not occur in lysates from younger gametocytes (fig s4g-h), but this is a biophysical assay, so regardless of the function of this protein at different stages, the biophysical interaction between the drug and the protein should be the same regardless of the source of the protein. This data argues against Pfs16 being a specific binding target of Pfs16.

In addition to the above concerns, the fact that this compound doesn't inhibit the earlier functions of Pfs16 in gametocytogenesis, and that it doesn't inhibit P. berghei, also argue against this being the specific target of this drug. Whilst the authors have a valid argument that these findings don't exclude the possibility of stage-specific targeting of Pfs16, we could also argue that all the phenotypic data in figures 4-6 is merely correlative of a drug that acts at the same point in the lifecycle as Pfs16.

Overall, I believe that significant additional studies would be required to identify the target of this compound. Either by repeating the included studies with additional controls and conditions, or by follow-up studies such as genetic manipulation (knock-down or overexpression) or heterologous expression and biophysical binding studies. Alternatively, the manuscript could be restructured as primarily a report on the phenotypic effect of this compound on microgametogenesis, with the target identification work reported as a hypothesis-generating chemoproteomics study that provides some ideas about possible targets, but requires substantial follow-up to confirm the target (which may be beyond the scope of this report?).

Minor comments

The manuscript is very well-written and presented.

Several of the conclusions are overstated (as detailed above) and several statements should be tempered based on this data (e.g. statements linking DDD01028076 effects to Pfs16 function).

I find the term 'crosslinking' confusing for the photo-affinity labelling, as crosslinking in proteomics often refers to crosslinking between proteins (not between protein and drug).

The data and terminology around activity (IC50) for compounds in table 1 is a little confusing. Some IC50 values are reported as >1000, while others have precise mean values reported over 1000, and others are >10,000 or >25,000. This is especially confusing where 9 is claimed to have retained activity, but is >1000. If consistent thresholds are not appropriate then perhaps including dose response curves in the supp data might be necessary to explain these?

Significance

The work is potentially interesting to Plasmodium biology and drug discovery researchers. The concept of a transmission-blocking drug is quite attractive to this community, so the topic is highly relevant. Keeping in mind that this compound was reported previously, the main novelty is in defining it's window of activity during the microgametogenesis process, and differentiating this from other drugs/compounds that inhibit this process. There is clearly an advance in knowledge presented here.

If Pfs16 were to be confirmed as the target of this series then I think that this study would have much greater impact and attract interest from a broad audience. However, at this stage I don't see strong evidence for this hypothesis, and some of this data casts significant doubt on the likelihood that Pfs16 is the direct target.

• the dynamic structure of the programming langage
• was the dynamic structure
• of the doument

This IS Intentional Programming

• see : https://indyhub.files.fission.name/p/viewer/web/viewer.html?file=https://indyhub.files.fission.name/p/paper%20-%20simonyi%20-%20death%20of%20computer%20languages%20-%201995%20-%20ip.pdf#annotations:8XPqilEIEe2TdP8XAlWuUA

!- claim : NLS was a nascent Intentional Software System - It was not a language work bench - but a system oriented - a collaboration platform designed to bootstrap itself - into existence and continual improvement and in fact - it was kernel based - and at the heart it had a Command Language Interpreter - that allowed thinking and creating software - as a conversational system - driven by a command language - that was used to articulate the intentions - in terms of other intentions, combinations of intentions ultimately - groundrd in primitiver capabilities - that were implemented in machine code for the target system - migration from one machine to the next - system desigtners and devlopers were able to articulate - ideas and means of realizing those ideas in - self structured forms and used the same capabilities - to organize the embodiment of these intentions - in code that when run exhibited the desired behaviour

https://web.archive.org/web/20161201164608/http://www.theparisreview.org/interviews/6286/adam-phillips-the-art-of-nonfiction-no-7-adam-phillips

Another example to link to: https://hypothes.is/a/yEFMHoCkEeyl34fItJe__w

I knew from the lectures that the works of Aristotle were translated from Greek to Arabic to Latin, but I didn't know that this change happened within a few decades. It's incredible to think about how the "scientific revolution" wasn't called that just because of how much progress was made, but also because of how rapidly it happened. My question is if the "decades" of Aristotle being translated was hindered or helped in any way by the society in which it was being presented. I wonder if Christians reacted it to it differently if it would have taken more or less time to translate and assimilate the works.

Something I cannot help but think about is how will open access ensure change for the entire world, and not just the world of science? Open access doesn’t inspire engagement that is not there, it can encourage it, but I see it as “leading a horse to water, but you cannot force it to drink”. What happens after open access? Will the average person really have that much collaboration or a say on what happens next? It’s more like wishful thinking to me.

this also connects to The Mushroom at the End of the World, where Tsing emphasized the role of the forests and the mushrooms just as much as the stories of the pickers

It's not just a 'rebellion'. In Europe there is a strong establishment regulatory effort, that recognises the geopolitical significance of everything digital/data. So 'rethinking your relationship to tech' is a pretty mainstream discussion for at least 6 yrs.

please stop the 'generation whatitsname' is like 'something'. It is never true. Gen Z hypercognitive and comfortable adopting new tools is just as much nonsense as saying they are lazy and can't concentrate on anything meaningful. Young people always more easily adopt things that are newly introduced when they are in their 10s and 20s, afterwards they will be just as locked in as older people. My grandparents eagerly 'adopted' radio, while their parents likely bemoaned them for not reading the paper anymore. Likewise my parents and TV news, and me with internet. Thinking in 'generations' doesn't have any real value imo. It's shorthand for marketing, and hand waving in every other context.

Viewing the map of the railroad system progressing was something that I found surprising. The size that it became compared to when it was established really shows capitalism as a driving force for innovation and technology. But on the other hand, we also must notice what happened to environment around these tracks as well as what happened to the indigenous people originally living on that land.

I find it incredibly fascinating that we can taste so much flavor when the majority of milk is just made of water, but water and milk taste drastically different. It's insane that the amount/type of calcium can create a completely different taste of cheese. I never thought of how different cheeses will cook differently, and it makes me want to take a closer look at the cheeses I am buying.

From a central idea, Satterwhite expresses themselves across multiple disciplines. This may seem to be fragmented and unfocused. ('all over the place') With a list of current questions one can help soft-focus a range of different information flows, Feynman style, and avoid focusing on 1 type of info. This increases serendipity and combinational creativity. Is what Satterwhite describes the same thing for expression? Having several outlets not as fragmentation, but different probes of expressing the same idea. The idea soft-focuses the expressed across the mediia it is expressed in, not focusing on a single form of expression. Provided one has the skills to do so. Vgl Doctorow exploring in science fiction the same ideas he explores in non-fiction.

I think this is why people say that statistics don't lie, but Staticons do. You can collect so much data and find so many trends that fit your narrative. It's really just confirmation bias for people to fit their narrative and paint whatever narrative fits their agenda

Practically it is a dataset because it stores the image type of data but at the same time its not a data set because its not processed information on a computer.

This analogy was actually quite helpful. The material that humanists work with is not properly, wholly expressible in distinct units. There is a more in-depth value that cannot be captured in a spreadsheet, and so the term data would not be the most suitable description.

I think it's really important that this isn't just a practice that the teachers introduce and encourage students to practice on their own time but that they are meditating 3 times throughout the day.

https://www.reddit.com/r/antinet/comments/ur5xjv/handwritten_cards_to_a_digital_back_up_workflow/

For those who keep a physical pen and paper system who either want to be a bit on the hybrid side, or just have a digital backup "just in case", I thought I'd share a workflow that I outlined back in December that works reasonably well. (Backups or emergency plans for one's notes are important as evidenced by poet Jean Paul's admonition to his wife before setting off on a trip in 1812: "In the event of a fire, the black-bound excerpts are to be saved first.") It's framed as posting to a website/digital garden, but it works just as well for many of the digital text platforms one might have or consider. For those on other platforms (like iOS) there are some useful suggestions in the comments section. Handwriting My Website (or Zettelkasten) with a Digital Amanuensis

Most pople aknowledge the issues in language but ignoe them because that's something we used to do in the past and now we just assume those issues and live with them, therefore it's useless to keep "worrying" about them.

Author Response

Reviewer #1 (Public Review):

This study examines whether the D2 receptor antagonist amisulpride and the mu-opioid receptor antagonist naltrexone bias model-based vs model-free behavior in a well-established two-step task of behavioral control. The authors find that amisulpride enhances model-based choices, which is further supported by computational modeling of the data, revealing an increase in the relative contribution of model-based control of behavior. Naltrexon on the other hand had no reliable effect on model-based behavior.

Overall, this is a very nice study with many strengths, including the task and data analysis. A particular strength of the design is the combination of a between-subject drug administration protocol with two within-subject (baseline vs. drug) sessions. This reduces between-subject variability in baseline model-based vs model-free behavior and enhances the power to detect drug effects.

The introduction could do a better job articulating the rationale for testing the effect of these two specific drugs. Currently, the rationale is that both transmitter systems targeted by these drugs are involved in drug addiction, which is characterized by an imbalance in model-based vs. habitual control of behavior. This appears somewhat indirect.

Blood draws were used to determine serum levels for amisulpride and naltrexone but these data are not included as covariates in the analysis.

We thank the reviewer for the high acclaim of our study, and for the constructive comments to improve it. We acknowledge that the introduction did not motivate the main research goal of the manuscript clearly enough. We have now extended this section and provided further insight into our reasoning behind the study design. Beyond the involvement of opioid and dopamine promoting drugs in addiction, there is abundant evidence from experimental studies showing comparable effects of manipulating receptors of both systems in model-free processes such as reinforcement, and habit formation. Based on this overlap one may predict that both neurotransmitter systems disrupt habit formation in a similar fashion, and that blocking their respective receptors will improve the ability to behave in a model-based manner. However, as we now elaborate in the manuscript, an argument against this could be that disrupting model-free processes might not be enough to promote model-based behaviour, as such behaviour relies heavily on cognitive control. It is therefore especially interesting to compare opioid antagonists, that do not enhance cognitive function, with a D2 antagonist at a dosage that has been shown to increase cognitive control as well as increase the desire to exert cognitive effort.

This is expressed in the following paragraphs of the Introduction (p.2 §3 and p.3 §1):

“Opiates, psychostimulants, and most other drugs of abuse increase the release of dopamine along the mesolimbic pathway (Chiara, 1999; Koob & Bloom, 1988), a circuit that plays a central role in reinforcement learning (Schultz, Dayan, & Montague, 1997). On top of this, the reinforcing properties of addictive drugs also depend on their ability to activate the μ opioid receptors (Becker, Grecksch, & Kraus, 2002; Benjamin, Grant, & Pohorecky, 1993; Le Merrer, Becker, Befort, & Kieffer, 2009). This suggests that both the dopamine and the opioid systems might be particularly relevant in model-free reinforcement learning processes that drive the formation of habitual behaviour. Studies in rodents show that activating receptors of both systems across the striatum increases cue-triggered wanting of rewards (Peciña & Berridge, 2013; Soares-Cunha et al., 2016). Conversely, inhibition of both D1-type and D2-type of dopamine receptors (referred to as D1 and D2 from here on) as well as opioid receptors reduces motivation to obtain or consume rewards (Laurent, Leung, Maidment, & Balleine, 2012; Peciña, 2008; Soares-Cunha et al., 2016). This data raises the hypothesis that the drift towards habitual control is enabled by dopamine and opioid receptors via a common neural pathway. Recent work in humans provides some evidence in this direction, whereby systemic administration of opioid and D2 dopamine receptor antagonists causes a comparable reduction of cue responsivity and reward impulsivity (Weber et al., 2016) and decreases the effort to obtain immediate primary rewards (Korb et al., 2020). This suggests that when allocating control between the model-based and model-free system, dopamine or opioid receptor antagonists might comparatively disrupt model-free behavioural strategies and increase model-based behaviour. Yet, no study in humans has directly investigated this. Furthermore, disrupting habit formation might not in itself lead to increased model-based control, without either increasing the perceived value of applying cognitive control or making it easier to do so.”

We also mention the implications of this direct comparison of the two compounds in the Discussion (p.8 §1):

“Our findings provide initial evidence for a divergent involvement of the dopamine and opioid neurotransmitter systems in the shift between habitual and goal-directed behaviour. The lack of effects of naltrexone on the model-based/model-free trade-off also provides some support for the notion that simply disrupting neurobiological systems that subserve habitual behaviour might not be enough to increase goal-directed behaviour in this task. An increase in the model-based/model-free weight following amisulpride administration advocates for dopamine playing a decisive role in flexibly applying cognitive control to facilitate model-based behavior and highlights the specific functional contribution of the D2 receptor subtype.”

Reviewer #3 (Public Review):

I think this is an interesting study on an important topic. I agree that there is not enough research to understand how the dopaminergic system interfaces with goal-directed planning, and I like the focus on specific types of dopamine receptors. It is interesting that they seem to find a specific effect on just the dopamine antagonist. I also appreciate the clarity with which the authors describe this field of research and their results. However, I also feel that there are several concerns with this paper, both in terms of framing and in terms of the experimental design and analysis. For completeness, I must note that I am not a dopamine expert.

I felt that the introduction of the paper did not sufficiently motivate the focus on the comparison between neurotransmitters systems, and (for the dopaminergic system) the distinction between D1/D2 receptors. Why is the mapping between stability/flexibility and D1/D2 receptors important? How does this relate to model-based control? Why do the authors predict that model-based control would increase when D2 receptors are blocked? If the hypothesis is about contrasting the contribution of D1 and D2 receptors to goal-directed control, why did the authors not use antagonists directly targeting these two systems?

In addition, the predictions that are more explicit, for example, that blocking D2 receptors increases MB control by stabilizing goal-relevant information, are fairly specific. However, the current version of the two-step task is not amenable to testing such a specific hypothesis, because it doesn't allow us to measure the specific components of planning (e.g., maintaining goals, the representation of the structure, prospective reasoning). Moreover, MB control in this version of the two-step task is marked by flexibility, because it requires the agent to be sensitive to switching starting states.

The predictions for the opioid system are also lacking. Why are the authors targeting this system? Why are they comparing the effects of the D2 antagonist with the opioid agonist? Why do the authors predict that amisulpride should have a stronger effect than naltrexone? In my opinion, these predictions were not sufficiently laid out, which made it difficult to appreciate the authors' motivation to run the study.

We thank the reviewer for their critical take on the manuscript and for clearly pointing out the weaknesses in argumentation. In particular, we appreciate the reviewer’s comment on the lack of clarity in describing why the comparison of dopamine and opioid antagonists’ effects on MB/MF behaviour might be particularly interesting and why we focused on D2 and not D1 receptors. We now extended the introduction section to clarify our rationale for comparing these two compounds (p.2-3). In short, apart from the fact that both systems are implicated in addiction, there is also abundant experimental evidence from human and non-human animal studies that the two systems are involved in processes related to forming habitual responses to primary and secondary rewards. This suggests that blocking receptors of either system might comparatively affect the MB/MF trade-off by impairing model-free processes. We therefore proceeded to compare opioid and dopamine antagonists.

As we note, using D1 antagonists would likely be detrimental to cognitive control related processes, and therefore more likely to decrease model-based performance. We therefore chose to compare opioid antagonists to D2 receptor antagonists. Another important reason for comparing the effects of opioid and D2 dopamine antagonists is the reasoning that it is not clear whether blocking model-free processes is in itself enough to promote model-based behaviour, without boosting cognitive control related processes. Given the recent evidence for D2 antagonists increasing cognitive effort (Westbrook et al., 2020) and the proposed role of prefrontal D2 receptors in destabilising prefrontal representations (according to the dual state theory of prefrontal dopamine function proposed by Durstewitz & Seamans, 2008)) we reasoned that D2 receptor blockade might also boost the ability (or willingness) to keep the mapping between spaceships and planets online while making choices.

We incorporated these arguments in the revised Introduction (p.2-3):

“Opiates, psychostimulants, and most other drugs of abuse increase the release of dopamine along the mesolimbic pathway (Chiara, 1999; Koob & Bloom, 1988), a circuit that plays a central role in reinforcement learning (Schultz et al., 1997). On top of this, the reinforcing properties of addictive drugs also depend on their ability to activate the μ opioid receptors (Becker et al., 2002; Benjamin et al., 1993; Le Merrer et al., 2009). This suggests that both the dopamine and the opioid systems might be particularly relevant in model-free reinforcement learning processes that drive the formation of habitual behaviour. Studies in rodents show that activating receptors of both systems across the striatum increases cue-triggered wanting of rewards (Peciña & Berridge, 2013; Soares-Cunha et al., 2016). Conversely, inhibition of both D1-type and D2-type of dopamine receptors (referred to as D1 and D2 from here on) as well as opioid receptors reduces motivation to obtain or consume rewards (Laurent et al., 2012; Peciña, 2008; Soares-Cunha et al., 2016). This data raises the hypothesis that the drift towards habitual control is enabled by dopamine and opioid receptors via a common neural pathway. Recent work in humans provides some evidence in this direction, whereby systemic administration of opioid and D2 dopamine receptor antagonists causes a comparable reduction of cue responsivity and reward impulsivity (Weber et al., 2016) and decreases the effort to obtain immediate primary rewards (Korb et al., 2020). This suggests that when allocating control between the model-based and model-free system, dopamine or opioid receptor antagonists might comparatively disrupt model-free behavioural strategies and increase model-based behaviour. Yet, no study in humans has directly investigated this. Furthermore, disrupting habit formation might not in itself lead to increased model-based control, without either increasing the perceived value of applying cognitive control or making it easier to do so. Crucially, there are important differences in how each of the two neurochemical systems relate to cognitive control that is pivotal for model-based behaviour. Across a wide range of studies using various dosing schemes, opioid receptor antagonists did not have an effect on tasks that require cognitive control, such as working memory (Del Campo, McMurray, Besser, & Grossman, 1992; File & Silverstone, 1981; Volavka, Dornbush, Mallya, & Cho, 1979), sustained attention(Zacny, Coalson, Lichtor, Yajnik, & Thapar, 1994), or mathematical problem-solving (Del Campo et al., 1992) (see (van Steenbergen, Eikemo, & Leknes, 2019) for a review). Dopaminergic circuits, on the other hand, play a central role in higher cognitive functions and goal-directed behaviour (Brozoski, Brown, Rosvold, & Goldman, 1979). In particular, D1 dopamine receptors in the prefrontal cortex enable maintenance of goal-relevant information and working memory(Goldman-Rakic, 1997; Sawaguchi & Goldman-Rakic, 1991; van Schouwenburg, Aarts, & Cools, 2010; Williams & Goldman-Rakic, 1995), while the D2 dopamine receptor activity disrupts prefrontal representations(Durstewitz & Seamans, 2008). In support of this, decreased working memory performance was observed after blocking prefrontal D1, but not prefrontal D2 receptors (Arnsten, 2011; Sawaguchi & Goldman-Rakic, 1991; Seamans & Yang, 2004). In humans, systemic administration of D2 antagonism increased the ability to maintain and manipulate working memory representations (Dodds et al., 2009; Frank & O’Reilly, 2006) and increased the value of applying cognitive effort (Westbrook et al., 2020). This data suggests that blocking D2 receptors, in contrast to blocking opioid receptors, could further facilitate model-based behaviour through enabling or encouraging flexible use of cognitive control.”

Another important point that the reviewer stresses is that the two-step task we use does not allow us to draw any conclusions through which mechanisms amisulpride increases model-based behaviour. Although we base our hypothesis that D2 might promote model-based behaviour (on top of disrupting habit formation) on previous work showing D2 blockade increasing cognitive effort and the ability to manipulate working memory representations, we completely agree that our setup does not give any definite answers about which of these cognitive processes mediated the increase in model-based weights. In the discussion we try to interpret our findings in the context of the dual-state hypothesis framework and within the framework of striatal control of adaptive behaviour (p.8 §3-4), whereby we centre our argumentation around dopaminergic circuits that subserve one or the other mechanism.

We agree with the reviewer that the task requires a high degree of flexible planning and that the dual-state theory might not be enough to account for our effects. We mention this in the Discussion (p. 8 §3):

“The effects of D2 antagonism on model-based/model-free behaviour in our study can be interpreted within this [dual-state] framework to result from increased ability to maintain prefrontal representation of the mapping between the spaceships and the planets online. However, this is difficult to reconcile with the fact that model-based behaviour in dynamic learning paradigms, such as the one used here, also requires flexible updating of action values.”

We also elaborate on the general limitations of drawing inference about the underlying cognitive/computational mechanisms in the Discussion (p. 14 §2):

“Importantly, it should also be acknowledged that the behavioural setup in our study does not allow us to draw definite conclusions about the mechanisms that mediate amisulpride’s effects on model-based or model-free behaviour. For example, it is not clear whether amisulpride increases the perceived benefit of applying cognitive control, or whether it increases the participant’s ability to do so through various possible complementary processes, such as goal maintenance or planning abilities. Future studies should further elucidate the mechanistic contributions of dopamine receptors to the distinct coding and utilisation of task relevant representations (Langdon, Sharpe, Schoenbaum, & Niv, 2018; Stalnaker et al., 2019).”

Related to this, I felt that the introduction was a bit too quiet on the genetic markers. Their discussion in the results was a bit surprising, and it wasn't quite clear why the authors decided to investigate these interaction effects.

We appreciate this comment as we were quite uncertain ourselves on how much weight to give to those data. Previous research had indeed shown profound variability in MB/MF behaviour across genotypes related to baseline dopamine function. The main purpose of the genetic analysis was to control for potential baseline differences and to explore the drug genotype interactions. However, including the serum data as a covariate in analyses, as suggested by the other reviewers, made most results relating to the genetic analysis disappear, even when using less conservative priors that likely understate the variance of posterior distributions of group effects. We have therefore opted to keep coverage of the genetic data to a minimum, but still report the results and make the data available online for future studies.

I found some of the core results confusing. Most importantly, why does amisulpride make people less like to stay after a reward when the first-stage state is the same? When first-stage states repeat, both an MB agent and an MF agent will be more likely to stay after a reward. To me, this kind of behavior doesn't seem particularly model-based. Why does this behavior occur under amisulpride? I was surprised that the authors did not really address it.

We agree that these results have been somewhat difficult to reconcile. However, adding amisulpride serum levels to our analyses now allow us to get a better understanding. It seems that across both serum groups model-based behaviour was increased, however, only in the high serum group did we additionally observe increased exploration. We also note that increased exploration was related to a reduced effect of previous points in the first same state trials, whereas the interaction term (effect of previous points in diff vs. same state trials) was more strongly associated with the model-based weight. In the manuscript this is described in the results section and in the discussion.

The following text is included in the Results (p.6):

“We first observed that the more model-based choices the participants made, the more money they earned (r = 0.65, 95% CI [0.53, 0.76]). This serves as a validity check of the task, which was designed to make cognitive control pay off (literally)45. We then looked at how the model parameters relate to the random slopes from the behavioural analysis of staying behaviour and found that the participant-level (random effect) slope for the effect of previous points on staying behaviour in different vs. same first state trials was most strongly related to ω (d = 0.493, P < 10e-3) and negatively related to the inverse temperature parameter η (d = -0.328, P < 10e-3), and the slope for trials with same first states was mostly related to η (d = 0.822, P < 10e-3), and less so to ω (d = 0.235, P < 10e-3).”

The following text is included in the Discussion (p.8 §2):

“Interestingly, amisulpride also increased choice stochasticity parametrised by the softmax inverse temperature parameter. In a paradigm with two choice options, it cannot be definitively determined whether this indicates higher decision-noise or increased exploration of alternative choices. We can however speculate that increased decision noise would lead to overall detrimental effects on learning in both trial types with same and different consecutive first stage states, which we do not observe in our data. The effect on the choice stochasticity parameter was only present in participants with a higher effective dose75, suggesting that the effect was more likely to be post-synaptic. Similarly, in the same effective dose group, we found some evidence that amisulpride reduces response stickiness indicating increased switching between actions. This is well in line with a prominent model of the cortico-striatal circuitry implicating post-synaptic D2 receptors in exploration/exploitation65 and supported by empirical data. In animal studies, activation of D2 receptors was shown to lead to choice perseverance and more deterministic behaviour, whereas D2 receptor inhibition increases the probability of performing competing actions and increases randomness in action selection76. In humans, a recent neurochemical imaging study showed that D2 receptor availability in the striatum correlated with choice uncertainty parameters across both reinforcement learning and active inference computational modelling frameworks77. Increased choice uncertainty was also observed in a social and non-social learning tasks in a study using 800 mg of sulpiride, a dose that is known to exert post-synaptic effects54,78. We note, however, that the evidence for the difference in exploration between the low and high serum groups was not robust (p=0.066). Furthermore, it has been suggested that increased striatal dopamine is also related to tendency for stochastic, undirected exploration79,80, arising due to overall uncertainty across available options79 or through increasing the opportunity cost of choosing the wrong option68,71. This suggests that the same biological signature that leads to increased cognitive effort expenditure also promotes choice exploration. In line with this, both prior studies that investigated the effect of increasing dopamine availability with L-DOPA on model-based/model-free behaviour observed increase choice exploration as well as increased model-based behaviour (although in one it was only present in individuals with a higher working memory capacity)55,58.”

With regards to the design, it is unfortunate that the order of drug administration is not counterbalanced. As far as I understand, model-based control is always measured without a drug in the first session, and then with the drug (or placebo) in the second. The change between sessions is then tested for all three conditions. Of course, it is possible that the increase in model-based control in the amisulpride condition is only driven by the drug. However, given the lack of counterbalancing, it's also possible that amisulpride increases model-based control only after the experience with the task. That is, if the authors had counterbalanced the drug effect, they may have found that amisulpride had a different effect if it was administered in the first session. That would have changed their interpretation quite a bit! As it stands, they are unable to verify their (admittedly simpler) hypothesis that there is only a main effect.

We thank the reviewer for this comment. Indeed, a full within-subject design would have been statistically more powerful and would have enabled us to exclude the possibility that amisulpride’s effect on model-based behaviour is indirect. We have now included the following paragraph in the discussion that aims to highlight the limitation of not counterbalancing the drug administration (p.10):

“One of the strengths of our design is a baseline measure, and the fact that the participants were all introduced to the task under no administration, thus avoiding potential effects of the treatment on task training. Although this design allowed to reduce between-subjects variability, we cannot completely exclude order effects. Although unlikely, it is possible that the effects of the treatment that we observe come indirectly from the effects of the two drugs on either skill transfer from the previous session, or simply on the effect of the drugs on the part of the experiment that preceded the task. For instance, participants under amisulpride could be less tired from other tasks and therefore more willing to exert effort in the task presented here. Speaking against this is the observation that we found no differences in mood between amisulpride and placebo regardless of low or high serum levels.”

It's sad how these bare bones of interaction are just close enough to caring for him to appreciate them.

I have this habit with short stories to place them all in the 50's/60's unless there are some other context clues other than cars to tell me otherwise. So cellphone just changed my whole perspective, and now my images are all morphing. I'm not sure when this started or why, but it's just the era that I start with and then move forward and backwards from as more information is given.

It's difficult to see anywhere else as a home when we've been in one place all our life.

It's an utility that anyone can use to basically have all the content so that instead of using bookmarks and wonder that cool article content, the title it had. To just search your local archive.

So basically media used it's platform to fantasize war to entice people to want to partake and to make people belive that the issues at hand were larger than they actually were. resulting in more conflict and more of an uproar.

Negative reinforcement with deadlines doesn't work - it doesn't push or provide enough opportunities to learn more. It's safe to assume that students have a natural compulsion to learn, but they're also presented with tempting opportunities; how do you provide a stimulating positive reinforcement cycle that sets students on the right path?

(page 111) I feel like there's a bit of a blind spot in this section--I would disagree with the idea that everyone's sense of self is primarily influenced by others' views of them (like that quote from class about seeing yourself as you think others see you). I think here Johnson may be making the assumption that for everyone it is as simple as accepting your assigned roles and all of the expectations that come along with them (or making the conscious choice to rebel against those roles), but I think it can be argued that the ability to do this comes from some amount of privilege (tying in with what Johnson explains later in this chapter).

For instance, Johnson repeatedly mentions how LGBT people have a lot of expectations placed on them based on their identities, either from the assumption that they are straight, or from the stereotypes associated with LGBT people. But he never addresses something immensely important to this topic: how do these LGBT people figure out their identities if the norm is to assign everyone as straight? Surely that would require some amount of self reflection beyond just what people see externally, but Johnson writes as though these identities are a given. He later mentions how heterosexuals take for granted how they do not have to think carefully about what information is disclosed to others, but does not expand this idea to how people of non-marginalized identities often do not have to question the roles they have been assigned. This isn't an act of rebelling against one's role, such as a student talking back to a professor; this is a direct refutation of the role one has been assigned. I think it's completely fair to say that these situations are still heavily socially influenced, but Johnson doesn't delve into them much beyond the surface level analysis of easily accepted and internalized role expectations.

bless biologist humor

It's more of a social rule, and we're just following the rules

This is a great description of why the animators were able to go on and make their own unique shows now having experience from working on Adventure Time where they were able to add to what Ward had made. It wasn't a show made by one guy there was a collection of people with different ideas. I doubt Ward would have been able to make the songs that Rebecca Sugar added. And from that she was able to then able to pitch her show "Steven Universe" to Cartoon Network wich became it's own classic with even more of her songs.

I stopped traveling heavily mid 2016 when our daughter was born. The pandemic hit right when I was gearing up to do more work outside of Europe again. Like Harold I have reservations about when travel is needed, the reflex to do things on site / in person has changed locally just as much. Am in conversations with WB however to start contributing to their work in Central Asia, maybe sometime next year. Travel is a habit as Bryan Alexander remarked to me, and it is something I do miss. Although I do not miss the tediousness of the travel itself, it's the interaction with professional peers from different context in places that operate differently. Helps me to cut through non-sense excuses at home as well (we can't do this 'cause.... when I just returned from a place where they could with a fraction of the means)

I found this quote from the reading intriguing because it illustrates how women from different cultures can both show respect to others and have respect for themselves without compromising their freewill and autonomy. It's an example of how cultural imperatives don't transfer well across people from different places with different upbringings. To that extent, I would agree with Abu-Lughod that women don't need saving. Not just because they literally don't need saving from their own culture which they are already accustomed to, but also because we all know that war was never really about the women; it was always about fueling whichever news cycle narrative that would best fund the war-machine military industrial complex. In other words, if the Afgahn women were free tomorrow; the narrative would be "save the kids". If the kids were free tomorrow, the new narrative would be "save the future generations".

I found this quote from the reading intriguing because it illustrates how women from different cultures can both show respect to others and have respect for themselves without compromising their freewill and autonomy. It's an example of how cultural imperatives don't transfer well across people from different places with different upbringings. To that extent, I would agree with Abu-Lughod that women don't need saving. Not just because they literally don't need saving from their own culture which they are already accustomed to, but also because we all know that war was never really about the women; it was always about fueling whichever news cycle narrative that would best fund the war-machine military industrial complex. In other words, if the Afgahn women were free tomorrow; the narrative would be "save the kids". If the kids were free tomorrow, the new narrative would be "save the future generations".

I found this quote from the reading intriguing because it illustrates how women from different cultures can both show respect to others and have respect for themselves without compromising their freewill and autonomy. It's an example of how cultural imperatives don't transfer well across people from different places with different upbringings. To that extent, I would agree with Abu-Lughod that women don't need saving. Not just because they literally don't need saving from their own culture which they are already accustomed to, but also because we all know that war was never really about the women; it was always about fueling whichever news cycle narrative that would best fund the war-machine military industrial complex. In other words, if the Afgahn women were free tomorrow; the narrative would be "save the kids". If the kids were free tomorrow, the new narrative would be "save the future generations".

The Ghost Dance was created by American Indians, in response to the unwanted presence/intrusions of white settlers on their territory. As more and more settlers pushed further west, Native populations not only lost the land they had occupied for hundreds of years but the resources they relied upon for just as long. White settlers, in this context, can reasonably be seen as an invasive species to the American landscape. However, rather than out-competing Native populations, colonial settlers dwindled their communities with the disease, forced migrations, and environmental degradation. These circumstances were so drastic to American Indians that they came up with a technique or code, in the Ghost Dance, to help bring about an end to the world they have been forced into. In doing so, they would unlock a new existence free of white settlers who are continually encroaching upon and inflicting themselves on your life. With all this in mind, I would be inclined to agree with Sclonit's assessment of the Ghost Dance from a technological standpoint. It is an organized and systematic practice meant to bring about a specific desired result or experience, in this case, a new world.

First of all, it goes without saying that the phrasing of "the black face" is pretty horrifying. Racism seeps even in what is not said in the film; it's not just "Scarlett loses her temper completely and slaps Prissy's face." Prissy is dehumanized and physically hurt. Many people within the class found issue with the slap for good reason; it's hard for modern audiences to connect and root for a heroine who is physically hurting one of her slaves. Gone With the Wind not only promotes hurtful stereotypes of African Americans, but it glosses over the issue of slavery, making the enslaved characters appear to be happy to be working, showing them as loyal, loving members of the family. While it glosses over slavery for much of the film, this act of violence shows how the threat of violence was both acceptable and utilized when people did not get their way. This is one of the few moments in the film that actually depict an unpleasant moment between the O'Haras and the Black characters.

Oh no! I thought the flag meant post & accidentally reported your thoughtful feedback to moderators instead of replying. Hopefully they figure it out (no obvious way to contact them or "unflag"

Comment Figure 2C → please include indication of statistical significance Figure 3C → please include indication **of statistical significance Figure 6A → please include indication of statistical significance Figure 8B → please include indication of statistical significance Figure S1B → please include indication of statistical significance Figure S3B → please include indication of statistical significance

Response Easy to add

Comment For your overexpression experiments, do the overexpressed proteins have a tag? It would be helpful to have Western blot data showing that the particular proteins are actually being overexpressed. I think the phenotypes that you observe are very compelling so I don’t doubt the conclusions. Western blot data would just provide some additional confirmation that you are actually achieving overexpression of UppS, MraY, and BcrC.

Response The proteins are untagged. For the UppS and BcrC the cell shortening occurs with addition of inducer, , so strong indication expression is occurring. A western would provide information about degree of overexpression, but we don’t think is necessary to support conclusion drawn. Do you think there is an alternative possibility that needs to be excluded? We note that in another preprint (https://www.biorxiv.org/content/10.1101/2022.02.03.479048v1) the authors delete the native uppS in their inducible Phy-uppS strain (Fig S4) and at 100 uM IPTG (10X less than what we used in experiment) the cells have wt growth on LB plates, so we at least know the Phy-uppS is functional and made (or they would die!). We are introducing the uppS deletion into our strain to see if we can identify a concentration of IPTG that doesn’t affect cell growth but still induces shortening.

For MraY, the result is negative, so you are spot on – it is impossible to tell if due to lack of overexpression from data shown. We only know the strain is correctly made from sequencing. We will investigate if there is an antibody or functional fusion available. The reason we were not sure was worth doing is because the MraY reaction is reversible (15131133). This means that without a phenotype, there is no simple way to know the reaction can even be pushed forward even if the overexpression is confirmed (more negative data). We actually overexpressed some other proteins that act downstream (MraY, MurJ, AmJ) and they were also negative for shortening. Probably we should remove the negative data or reword to make the caveats of the negative result clear.

Question Based on your data, there are definitely differences in gene expression when you compare cells grown in media with and without magnesium. Because the majority in cell length increase occurs in such a short time though (the first 10min), I was wondering if you think that some or most of it is not due to gene expression?

Response The shortening is even faster than 10 min (not only statistically significant, but also obvious qualitatively if we mount immediately after adding Mg2+ ). We did not include the first timepoint because original purpose was to check everything was ready with microscope – did not expect shortening so fast! We can definitely add that data in. When we saw, we tried to capture the transition on pads, but going from culture to pad seems to stress the cells too much in the small window where the cool stuff happens. Since growth rate doesn’t appear to be a big factor in those initial divisions, we might be able to grow at lower temp and shift to pads for adjustment period before adding Mg2+. Did not play with it much due to lack of resources atm, but a flowcell setup would probably be best. In short, we think rapid divisions right after transition do not require transcription or translation. It really “smells” more like a biophysical thing.

Question Do you have any hypotheses what is most likely to be affected by magnesium? Do you think if the membrane may be affected?

Response We have a lot of hypotheses, but all speculative. There could be an extracytoplasmic enzyme involved in envelope synthesis is sensitive to Mg2+ availability, and that at lower concentrations, its activity is affected. There is some old literature with membrane preps that suggests PG synthesis requires higher Mg2+ than teichoic acid synthesis. If Und-P is limiting, higher Mg2+ may shift make the pool more available to make the septum. Tingfeng initially hypothesized there might be a receptor/signal mechanism but has not been able to identify one. Und-P seems to be important, but “availability” is not just pool, but how fast (and where!) the flipping across the membrane occurs. If Und-PP needs to be dephosphorylated to Und-P before being flipped back to cytoplasmic side, anything that effects the PP/Pi equilibrium would be predicted to affect the reaction rate, with lower Pi (in periplasm or pseudoperiplasm in case of G+) favoring the dephosphorylation. Cell wall associated Mg2+ could shift equilibrium to be more favorable for a Und-PP phosphatase more closely associated with the divisome. I could go all day… In short, we don’t know enough!

Question Why do you think less magnesium activates this program of less division and more elongation? Additionally why is abundant magnesium activating a program of increased cell division and less elongation? Do you think there is some evolutionary advantage, especially considering how important magnesium is for ATP production?

Response In the window we looked at, the elongation rate is constant (not less or more) and only the division frequency changes. Some bacteria (like Caulobacter and to lesser extent E. coli) clearly elongate and divide simultaneously, so model there is some competition for substrate (like Lipid II) makes sense. Septators like Bacillus seem to delineate the two processes more, though we have found conditions where even Bacillus invaginates during division, so it’s not absolute. Like eukaryotic cells, bacterial undoubtedly have mechanisms not only commit to a round of DNA replication when there is some signal that resources are sufficient. Clearly with some bacteria, this is not the case with cell division. The alternative would be that every cell cycle there is an opportunity to divide if some threshold of something(s) is reached. There is a hypothesis from Mtb literature that it may be GTP, but it’s not at all clear that is sufficient. In yeast, size at cell division is affected by perturbing 1-C pool.

Question Related to this previous question, I also wonder if this magnesium-dependent phenotype would extend to other unicellular organisms, may be protists or algae? That would be a really exciting direction to explore!

Response It’s a great question – lots to do! We didn’t even look at another Gram-positive, but we plan to. It’s trickier to limit Mg2+ in Gram-negatives (see 27471053 – we tried Bsub homolog for those wondering – it’s not responsible for phenotype we see).

Question Regarding the zinc and manganese experiments, why do you think they lead to additional phenotypes compared to magnesium? Do you have any hypotheses?

Response We have hypotheses, but if my (Jen’s @rosh_ba) twitter engagement is any indication, way too speculative for public consumption at present. Need acquire preliminary data/write grant.

Question Regarding your results that Lipid I availability may be a major a problem for the cell division in the absence of magnesium, do you think that is due to effects magnesium has on the enzymes directly, or do you think magnesium affects the substrate availability/conformation by coordinating the phosphate groups? Or something else, may be membrane conformation?

Response Several proteins involved in envelope synthesis (like UppS) are Mg2+ dependent enzymes. But at least for intracellular players, levels of Mg2+ should be more than high enough to support enzyme activity (0.8 – 3.0 mM is Bsub range I recall off top of head). Could have impact extracytoplasmically by lowering pool sponged into the cell wall, but intuition (for what that is worth) is that it is not the coordination of an enzyme with a metal that is impacted rather the equilibrium with other ions like Pi and H+ and their impact on net ATP synthesis. Lots to think about and do, and no simple answers. When Tingfeng started project idea was to find mechanism – didn’t realize we were asking “How does the cell work?” Turned out to be a bit much for a dissertation project :)

General comments:

This study carefully delineates the role of magnesium in cell division versus cell elongation. The results are really important specifically for rod-shaped bacteria and also an important contribution to the broader field of understanding cell shape. Specifically, I love that they are distinguishing between labile and non-labile intracellular magnesium pools, as well as extracellular magnesium! These three pools are really challenging to separate but I commend them on engaging with this topic and using it to provide alternative explanations for their observations!

A major contribution to prior findings on the effects of magnesium is the author’s ability to visualize the number of septa in the elongating cells in the absence of magnesium. This is novel information and I think the field will benefit from the microscopy data shown here.

I completely agree with the authors that we need to be more careful when using rich media such as LB. It is particularly sad that we may be missing really interesting biology because of that! It’s worth moving away from such media or at least being more careful about batch to batch variability. Batch to batch variability is not as well appreciated in microbiology as it is for growing other cell types (for example, mammalian cells and insect cells).

For me, the most exciting finding was that a large part of the cell length changes within the first 10min after adding magnesium. The authors do speculate in the discussion that this is likely happening because of biophysical or enzymatic effects, and I hope they explore this further in the future!

I love how the paper reads like a novel! Congratulations on a very well-written paper!

Kudos to the authors for providing many alternative explanations for their results. It demonstrates critical thinking and an open-mind to finding the truth.

Specific comments:

Figure 2C → please include indication of statistical significance

Figure 3C → please include indication of statistical significance

Figure 6A → please include indication of statistical significance

Figure 8B → please include indication of statistical significance

Figure S1B → please include indication of statistical significance

Figure S3B → please include indication of statistical significance

For your overexpression experiments, do the overexpressed proteins have a tag? It would be helpful to have Western blot data showing that the particular proteins are actually being overexpressed. I think the phenotypes that you observe are very compelling so I don’t doubt the conclusions. Western blot data would just provide some additional confirmation that you are actually achieving overexpression of UppS, MraY, and BcrC.

Questions:

Based on your data, there are definitely differences in gene expression when you compare cells grown in media with and without magnesium. Because the majority in cell length increase occurs in such a short time though (the first 10min), I was wondering if you think that some or most of it is not due to gene expression? Do you have any hypotheses what is most likely to be affected by magnesium? Do you think if the membrane may be affected?

Why do you think less magnesium activates this program of less division and more elongation? Additionally why is abundant magnesium activating a program of increased cell division and less elongation? Do you think there is some evolutionary advantage, especially considering how important magnesium is for ATP production?

Related to this previous question, I also wonder if this magnesium-dependent phenotype would extend to other unicellular organisms, may be protists or algae? That would be a really exciting direction to explore!

Regarding the zinc and manganese experiments, why do you think they lead to additional phenotypes compared to magnesium? Do you have any hypotheses?

Regarding your results that Lipid I availability may be a major a problem for the cell division in the absence of magnesium, do you think that is due to effects magnesium has on the enzymes directly, or do you think magnesium affects the substrate availability/conformation by coordinating the phosphate groups? Or something else, may be membrane conformation?

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Reply to the reviewers

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

This paper provides a detailed step by step protocol of the CUT&RUN technique, which enables high-resolution chromatin mapping and probing, adapted to the malaria parasite Plasmodium falciparum. In particular, Kafsack and colleagues apply the CUT&RUN protocol to infected red blood cells from in-vitro culture and obtain very good quality genome-wide profiles of two histone modifications, H3K4me3 and H3K9me3. The results are congruent with previous ChIP-seq data with a substantial improvement in terms of coverage and chip-to-input noise. The protocol is very detailed and the figures are great.

Major comments: 1. Authors successfully adapted the CUT&RUN protocol in P. falciparum. First, the binding profiles obtained by CUT&RUN for H3K4me3 and H3K9me3 are very similar to those reported by previous ChIP-seq studies. Secondly, by down-sampling 4X and 16X the test samples, authors demonstrate that 1M PE reads of sequencing depth would be enough to obtain accurate profiling of these histone modifications.

Despite this data is convincing, only one region in chr. 8 is shown as an example in figures 2, 3 and 4.

Different regions should be included, at least as supplementary figures, to reinforce their conclusions.

__Response: __We chose that locus on chromosome 8 to provide a gene-level resolution view at a locus encompassing genes in both eu- and heterochromatin states. We have now included Supplementary Figure 1, which shows these tracks for full length chromosomes 4 and 7. Additionally, genome-wide enrichment tracks for all data sets in this study are available for download at NCBI Gene Expression Omnibus under accession number GSE210062.

Related to this, there is evidence of the impact of chromatin structure on ChIP-seq analysis. Specifically, heterochromatin is typically depleted in ChIP input controls because of technical and experimental issues and this can result in a false enrichment of heterochromatic regions in the tested sample. How represented is heterochromatin (i.e. sub-telomeric and telomeric regions) in the test and control samples using the cut&run protocol?

__Response: __The reviewer is correct that chromatin structure may alter accessibility which may bias absolute measurements but since the accessibility biases based to chromatin-structure are identical for both the histone PTM-specific antibody and the isotype control and cancel out in the enrichment score.

How biased is the cut&run sample compare to the ChIP-seq sample?

__Response: __We have included this in Supplementary Figure 1. The H3K4me3 and H3K9me3 enrichment scores are strongly correlated both between CUT&RUN replicates and between CUT&RUN and previously published ChIP-seq results.

In this sense, it would be desirable if authors provide more information about the quality analysis results, for example the chip to input signal ratio and the coverage for heterochromatic (telomeric, centromeric and subtelomeric) regions.

__Response: __We agree that this would be of interest to the reader. For this reason, the full genome-wide enrichment tracks were made available for all datasets in this study. We have added language to draw further attention to this availability.

Additionally, loci typically biased in ChIP-seq samples, i.e. clonally variant gene families in sub-telomeric regions, should be shown as examples.

__Response: __We chose that locus on chromosome 8 to provide a gene-level resolution view at a locus encompassing genes in both eu- and heterochromatin states, including 2 var genes (PF3D7_0808600 and PF3D7_0808700) and two rifin genes (PF3D7_0808800 and PF3D7_0808900). We have now included Supplementary Figure 1, which shows these tracks for full length chromosomes 4 and 7, which also include subtelomeric and non-subtelomeric heterochromatin loci containing these genes. Additionally, genome-wide enrichment tracks for all data sets in this study are available for download at NCBI Gene Expression Omnibus under accession number GSE210062.

1. For P. falciparum WGS a PCR-free library preparation is strongly recommended. We wonder if it would be possible to try to integrate this step in their CUT&RUN protocol.

__Response: __Since such biases are sequence dependent, they would impact raw coverage but cancel out in the enrichment plots since the sequence-based biases are identical in both samples. While PCR-free amplification may be desirable for some applications, we feel this is outside the scope of this study to implement these changes.

It would have been desirable to have tried the CUT&RUN protocol on other type of proteins, different to hPTMs which are highly abundant, for example one of the Pf Api-AP2 transcription factors. Assaying the CUT&RUN protocol on a different type of protein shouldn't be cost/time consuming and would provide evidence of the versatility of the approach.

Response: As indicated by the title, this protocol was optimized specifically for profiling of histone modifications. CUT&RUN has been used in other systems to profile genome-wide binding of other proteins but this was not our aim and outside the scope of this study.

1. The step by step protocol is very detailed, however there are some parts that need to be better explained:

In the section "Binding cells to Concanavalin A-coated beads": it's not mentioned the harvest time and the stage of the parasites used. In addition, several methods are proposed for iRBCs enrichment, but is not mentioned which method was used and the life stage of the parasites. In this part of the protocol authors state "resuspend cells containing 1-5x107 nuclei to a cell density to 1x107 cells/mL".

According to our calculations, to guarantee this nuclei number it would be necessary to enrich in iRBCs and late stages. Otherwise the red blood cells density should be much larger. Could you please clarify this point?

Response: For this study we enriched for trophozoites using a percoll/sorbitol density gradient, which we have now clarified in step 8. However, whether and which enrichment strategy is employed will vary based on the desired parasite stages and experimental design.

In the section "P. falciparum culturing and synchronization of erythrocytic stages" the authors indicate that the method used for synchronization was double-synchronization with sorbitol treatment to achieve a {plus minus} 6 h synchrony. The details provided appear insufficient to replicate the procedure. E.g. it's not explained how the double step synchronization was performed and for how long the culture was incubated after the synchronization.

The number of parasite cells and the life-stage used is mentioned at the end (in the section of expected outcomes). It would be more useful if this information is specified at the beginning together with the most appropriate procedure to get an iRBC culture well synchronised and enriched in late stages.

Response: The stage, synchrony and growth conditions are determined by the scientific question the experimenter is asking, not by the assay. For this reason, we provide the number of infected erythrocytes and nuclei used in our studies so that other experimenters can aim for similar numbers regardless of the stage and synchrony. For this study we used asexual blood-stages at 36±4 hp.i. We have clarified this in step 11.

• With regards to reproducibility, all experiments were done in replicate (3 Rs) and the statistics appear adequate.

Minor comments: - Abstract. A closing bracket is missing.

Response: Corrected - Step 11: Split each sample into 1mL aliquots at ?

Response: Corrected

• The affinity of proteins A/G to IgG antibodies varies based on host species and IgG subtype (see link). This link does not seem to work

Response: Corrected

• Low bind tubes are mentioned several times. Please clarify whether it refers to low bind protein or low bind DNA. Step 77.

Response: The vendor and catalog number for the low-bind tubes are specified in the Reagents, Materials & Equipment list.

Which was the desired sequencing depth per library? It could be mentioned here. It is mentioned later in "Quantification and statistical analysis" that the initial desired depth was of 40M read pairs, but what was the real depth obtained? from the Figure 4 seems to be less than 17M read pairs per sample.

Response: Thank you for catching that error. The target was 10M read pairs per library but since CUT&RUN is so specific the isotype controls release less DNA and the resulting libraries produces fewer clusters than aimed for leading to slight over sequencing of the remaining samples.

• Step 79. Please clarify/justify why 50 bp paired-end reads were chosen as sequence length. Response: After excluding the telomere repeats 100 bp (50+50) are sufficient for uniquely mapping 98.3% of the nuclear. Paired-end sequencing was chosen over single-end because it provides the actual size of each fragment.

In the section "Quantification and statistical analysis", references to Figure 3 and 4 are inverted or do not correspond with the actual figures 3 and 4.

Response: Corrected

• Figure 2. Among the replicates, sample 2 seems to have higher background, could you comment why? Response: It is inherent in biological replicates that one would have the greatest amount of noise but we unfortunately have no further insight into why Sample 2 had a higher elevated background that Samples 1 and 3. Furthermore, even with this slightly higher background the relative enrichment of signal to noise ratio enrichment peaks are readily identifiable.

• Below some suggestions that may help the authors improve the presentation of their data and conclusions:

The limitations and potential shortcomings of the protocol are mentioned along the text (e.g. the use of different antibodies, different targets, weak interactions..), but could be good if they are included in a different section, preferably at the end.

Response: A “Limitations” section was added.

Also in this section it would be good if they develop further (or at least speculate) on the differences in the protocol or things to consider if other type of proteins are assayed (i.e. TFs).

Response: As mentioned above, we have not applied to CUT&RUN to profile chromatin other than Histone PTMs, as this was not the aim of our study. Since chromatin-bound histones always occur within a nucleosomal context, we are hesitant to make claims to the utility of this specific protocol for profiling DNA-binding proteins with smaller DNA-binding footprints. That said, CUT&RUN has been used to great success in other systems to profile a wide range of chromatin-bound proteins. We have included mention of this at the end of the introduction.

Authors should better comment on the potential impact of chromatin structure and DNA sequence (i.e. AT richness) on the biased representation of heterochromatic regions in the data, the level of background and the peak calling analysis.

Response: For the enrichment scores, sequence and accessibility biases cancel out since they are the same for both the PTM-specific antibody and the isotype controls.

The coverage of critical loci, like those belonging to clonally variant gene families, should be calculated and examples of tracks included as supplemental figures.

Response: Gene Expression Omnibus under accession number GSE210062 as indicated in the Quantification & Statistical Analysis and data availability sections.

Authors claim that the CUT&RUN protocol has exceptionally low background and has been successfully used to profile chromatin interactions from very small numbers of cells. But it is not specified how many. That is, which is the standard in other fields and how it compares with the number of cells used here.

Response: As stated in the note following step 10, we did not optimize the minimum number of parasites required in this study since at the 1e7 iRBC required for each sample correspond as little as 1mL of bloodstage culture 2% parasitemia and 5% hematocrit. The down-sampling analysis in figure 3 suggests that the number of input cells can likely be reduced at least 10-fold.

Information about synchronisation, estimation of iRBCs density and nuclear content appears insufficiently described and has been fragmented in different sections so it is difficult to replicate. For example, within the section "Binding cells to Concanavalin A-coated beads" different alternative protocols for iRBCs synchronisation and enrichment are mentioned but it is not clear whether authors actually perform that step. It could be convenient to describe it and include it in the step-by-step protocol.

Response: The stage, synchrony and growth conditions are determined by the scientific question the experimenter is asking, not by the assay. For this reason, we provide the number of infected erythrocytes and nuclei used in our studies so that other experimenters can aim for similar numbers regardless of the stage and synchrony. For this study we used asexual blood-stages at 36±4 hp.i. We have clarified this in step 11.

Significance (Required) The CUT&RUN is a novel technique to profile chromatin modifications genome-wide that has been successfully adapted to P. falciparum by the authors. This technique overcomes important limitations of the traditional ChIP-seq and provides better quality data. First, fewer cells and lower sequencing depths are required which is fundamental for the analysis of certain parasite life stages. Second, the binding step is carried out in-situ using unfixed and intact cells. This allows to avoid crosslinking, which can interfere with target recognition that results in unspecific background, and also avoids the random fragmentation of the chromatin, that can bias in the analysis.

This work is significant since it represents the first CUT&RUN step by step protocol adapted to P. falciparum. The results are important for researchers from the malaria field and parasitologists in general who could eventually leverage this protocol to other Apicomplexa.

Our expertise is on transcriptional regulation, molecular parasitology, genomics and epigenomics, of malaria parasites. We hope the comments above will help the authors to improve the ms. Congratulations on the work.

Reviewer #3 (Evidence, reproducibility and clarity (Required)): ____ In general the paper is very clear and convincing and I have only minor comments for the authors to address.

Introduction The authors state that 'crosslinking presents another challenge as it can interfere with antibody recognition.' Would it be possible to provide a reference to strengthen this statement? Response: Additional references (Baranello et al, O’Neill et al) were added.

In the third paragraph the authors mention that CUT&RUN can be used to profile chromatin interactions from very small numbers of cells. This argument would be strengthened by adding references or examples from mammalian systems, and the authors might mention that the slight modification CUT&TAG has been employed for single cell sequencing. https://doi.org/10.1038/s41587-021-00865-z.

Response: The reference was added.

Figure 2 A label of Relative fold enrichment should be added to the y axis. This applies also to Figure 4. In the legend, it isn't entirely clear from what control the fold enrichment is being generated. Based on the other figures I assume it's the isotype control, and it would be helpful to state that in the legend.

Response: Thank you for the suggestion. We have made these changes.

Figure 3 An HP1 ChIP is included but there is no track for HP1 using CUT&RUN. It isn't entirely clear to me why HP1 is included; is it to make the point that it overlaps with H3K9me3? There is a sentence at the end of the Quantifcation and statistical analysis section that indicates that an HP1 CUT&RUN experiment was performed ('Representative tracks of H3K9me3, H3K4me3, and HP1 produced using CUT&RUN or ChIPseq at chromosome 8 of P. faliciparum are shown in Figure 4), but I don't see an HP1 track for Figure 4 and I don't see CUT&RUN HP1 tracks on Figure 3.

Response: Correct, no HP1 CUT&RUN was performed, we are just trying to show that H3K9me3 CUT&RUN recapitulates ChIP-seq of both H3K9me3 and HP1, which binds to H3K9me3.

Figure 4 The downsampling of reads is a nice demonstration that low numbers of reads are required for the CUT&RUN technique. It might be helpful to include downsampling of ChIP-seq reads within this figure to compare the two techniques more directly.

Response: The reason we included the down-sampling of CUT&RUN sequence reads was to explore whether were over-sequencing our CUT&RUN libraries not to provide a comparison to ChIP-seq. For simplicity we have therefore kept the figure as is.

DNA purification by Phenol/Chloroform extraction In step 38, I noticed that RNAse was not added at this step, as described in the original paper by Skene et al. Can the authors make a brief note about why they omit this reagent?

Response: RNAse A is already present since it was included in the STOP buffer at step 35.

The authors mark the TE buffer in bold, but I don't see a description of its makeup in the buffer section, though possibly I missed it. While this is a pretty standard buffer, it might still be nice to include it for completeness.

Response: TE buffer recipe was added.

Clean-up of PCR amplified library Between step 70 and 71 the authors include a warning to not discard the beads. However, this warning is not included in the Post-ligation Clean-up, which involves much the same procedure. Response: Corrected

Typos and writing It might be helpful to define CUT&RUN in the abstract by spelling out the acronym there. Response: It is defined in the 3rd paragraph of the introduction

I've mostly seen ChIP-seq with a dash between the IP and the seq.

Response: Corrected

Powerful is used twice in consecutive sentences in the first paragraph of the introduction. Consider substituting the words 'important tool' for 'powerful tool.' Response: Corrected

Figure 2 legend. “(purple) in of three biological replicates” should be “(purple) in three biological replicates” Response: Corrected

Figure 3 legend Last sentence should include 'to' between the words 'shown' and 'the'. Response: Corrected

Post-ligation Clean-up “Wash twice with 200µl of 80% Ethanol freshly prepared” should be “Wash twice with 200µl of freshly prepared 80% Ethanol” Response: Corrected

Library PCR amplification “Fragments are PCR amplified using Kapa polymerase, which it is more efficient” should be “Fragments are PCR amplified using Kapa polymerase, which is more efficient” Response: Corrected

Figure 7 legend “Indicated in the tope left panel” Should be “Indicated in the top left panel” Response: Corrected

Expected outcomes “to dismiss any sort of contamination” should be “To dismiss contamination” Response: Corrected

Potential Solution After the sentence 'Incubation buffer is added.' The next letter 'i' should be capitalized in the word Isolate. Response: Corrected

CROSS-CONSULTATION COMMENTS Plasmodium is not my model organism, so I'd defer to Reviewer 2 on the comments regarding additional detail for synchronization and Plasmodium culture conditions. I have nothing further to add, and I'm excited to see what experiments come from the addition of this technique to the parasite field.

Reviewer #3 (Significance (Required)):

This excellent methods paper describes a detailed protocol for the adaptation of the Cleavage Under Targets & Release Using Nuclease (CUT&RUN) technique to Plasmodium falciparum, the causative agent of malaria. CUT&RUN is an alternative to ChIP-seq, and has the advantage that it does not require crosslinking of targets, which can introduce artifacts and cause issues with antibody recognition. CUT&RUN can also be performed with low numbers of cells and has an excellent signal to noise ratio, which the authors demonstrate by downsampling the number of reads used in their analysis. The authors also clearly demonstrate that profiling of histone modifications using CUT&RUN yields comparable results to ChIP-seq. Because it can be difficult to obtain large numbers of cells from Plasmodium cultures, CUT&RUN is especially useful in this important model system. Publication of a detailed protocol will help other Plasmodium researchers answer important questions regarding genomic localization for their targets of interest.

This portion sums up the primary topics of the article; it mentioned all the topics that are present in the article, including the idea of perfectionism being adaptive and it being linked to psychopathology

I think this is a really impactful comparison. Just as it is easy for us to kill a small helpless creature, it's just as easy for God to send you or his enemies to hell.

I like this statement because most people don't even have a second thought about it if they step on worm because it's small and there was no affect to the person that stepped on it. And it could be just as easy for God to send his enemies into hell without a second thought.

I like this sentence here because it's absolutely true. People just thank you all the time, to the point that its almost lost value. Just this morning I swiped my keycard to get into my dorm. I hadn't even open the door yet when I saw somebody coming, so I just let them open the door and come out because it was easier to slide in behind them than actually open the door for myself. Can you guess what happened when they came outside? They thanked me for absolutely no reason. This is why I said that saying thank you has almost lost its entire meaning. But gratitude on the other hand, actually showing someone through your actions and words that you are genuinely thankful is why it's so much more polite than just simply saying thank you.

Need to persevere with theories, they need to be testable but also need the strength to keep tweaking them, not just throw it out if it's wrong, even if sticking to usual expectations does make us prone to wrongness. Sticking to stuff is not the problem, sticking unwavering to stuff and tweaking observations to fit theory rather than theory to fit observations is the problem.

Helpful stuff.

Automatizar páginas web.

Is there a way that the playing field can be leveled so ideas are presented without being attached to a person? I've seen great ideas passed over because the person running the session doesn't like the person who proposed the idea. It's not just extroverts dominating the conversation, it can be political or social.

"What ifs" are some of my favorite ways of brainstorming along with, "yes and." What if this is a bad idea? Yes, and what if it's not a bad idea, just an idea that needs a yes, and?

Yet Chua also knows just how to elicit sighs of envy. The obvious allure is that she gets results. Lulu, you'll be relieved to know, finally learned that piece and was thrilled with herself: "Mommy, look—it's easy!" And that "virtuous circle" of struggle crowned by achievement brought bigger rewards.

Hello u/sscheper,

Let me start by thanking you for introducing me to Zettelkasten. I have been writing notes for a week now and it's great that I'm able to retain more info and relate pieces of knowledge better through this method.

I recently came to notice that there is redundancy in my index entries.

I have two entries for Number Line. I have two branches in my Math category that deals with arithmetic, and so far I have "Addition" and "Subtraction". In those two branches I talk about visualizing ways of doing that, and both of those make use of and underline the term Number Line. So now the two entries in my index are "Number Line (Under Addition)" and "Number Line (Under Subtraction)". In those notes I elaborate how exactly each operation is done on a number line and the insights that can be derived from it. If this continues, I will have Number Line entries for "Multiplication" and "Division". I will also have to point to these entries if I want to link a main note for "Number Line".

Is this alright? Am I underlining appropriately? When do I not underline keyterms? I know that I do these to increase my chances of relating to those notes when I get to reach the concept of Number Lines as I go through the index but I feel like I'm overdoing it, and it's probably bloating it.

I get "Communication (under Info. Theory): '4212/1'" in the beginning because that is one aspect of Communication itself. But for something like the number line, it's very closely associated with arithmetic operations, and maybe I need to rethink how I populate my index.

Presuming, since you're here, that you're creating a more Luhmann-esque inspired zettelkasten as opposed to the commonplace book (and usually more heavily indexed) inspired version, here are some things to think about:<br /> - Aren't your various versions of number line card behind each other or at least very near each other within your system to begin with? (And if not, why not?) If they are, then you can get away with indexing only one and know that the others will automatically be nearby in the tree. <br /> - Rather than indexing each, why not cross-index the cards themselves (if they happen to be far away from each other) so that the link to Number Line (Subtraction) appears on Number Line (Addition) and vice-versa? As long as you can find one, you'll be able to find them all, if necessary.

If you look at Luhmann's online example index, you'll see that each index term only has one or two cross references, in part because future/new ideas close to the first one will naturally be installed close to the first instance. You won't find thousands of index entries in his system for things like "sociology" or "systems theory" because there would be so many that the index term would be useless. Instead, over time, he built huge blocks of cards on these topics and was thus able to focus more on the narrow/niche topics, which is usually where you're going to be doing most of your direct (and interesting) work.

Your case sounds, and I see it with many, is that your thinking process is going from the bottom up, but that you're attempting to wedge it into a top down process and create an artificial hierarchy based on it. Resist this urge. Approaching things after-the-fact, we might place information theory as a sub-category of mathematics with overlaps in physics, engineering, computer science, and even the humanities in areas like sociology, psychology, and anthropology, but where you put your work on it may depend on your approach. If you're a physicist, you'll center it within your physics work and then branch out from there. You'd then have some of the psychology related parts of information theory and communications branching off of your physics work, but who cares if it's there and not in a dramatically separate section with the top level labeled humanities? It's all interdisciplinary anyway, so don't worry and place things closest in your system to where you think they fit for you and your work. If you had five different people studying information theory who were respectively a physicist, a mathematician, a computer scientist, an engineer, and an anthropologist, they could ostensibly have all the same material on their cards, but the branching structures and locations of them all would be dramatically different and unique, if nothing else based on the time ordered way in which they came across all the distinct pieces. This is fine. You're building this for yourself, not for a mass public that will be using the Dewey Decimal System to track it all down—researchers and librarians can do that on behalf of your estate. (Of course, if you're a musician, it bears noting that you'd be totally fine building your information theory section within the area of "bands" as a subsection on "The Bandwagon". 😁)

If you overthink things and attempt to keep them too separate in their own prefigured categorical bins, you might, for example, have "chocolate" filed historically under the Olmec and might have "peanut butter" filed with Marcellus Gilmore Edson under chemistry or pharmacy. If you're a professional pastry chef this could be devastating as it will be much harder for the true "foodie" in your zettelkasten to creatively and more serendipitously link the two together to make peanut butter cups, something which may have otherwise fallen out much more quickly and easily if you'd taken a multi-disciplinary (bottom up) and certainly more natural approach to begin with. (Apologies for the length and potential overreach on your context here, but my two line response expanded because of other lines of thought I've been working on, and it was just easier for me to continue on writing while I had the "muse". Rather than edit it back down, I'll leave it as it may be of potential use to others coming with no context at all. In other words, consider most of this response a selfish one for me and my own slip box than as responsive to the OP.)

@55:10

Sri: [...] you can think about the possibility that we're actually going to do this with structured data but then properly incentivizing people in order to actually moderate and curate the set of facts about the world—

Will: Yeah, so I was gonna mention that, and I'm glad we're on the same wavelength here. What are the economic incentives that would help encourage the adding of correct, factual data to this knowledge graph and dissuade, I guess, spammers? [...]

Sri: Yeah, I think that there needs to be some compelling reason for people to want to add data to the knowledge graph. [...] I think that, "Can we get a knowledge graph that is expansive—as expansive as Wikipedia—that, you know, says all kinds of facts about the entire world?" Yeah, maybe[...]

Will: There are parts of the Web where people do that without financial incentives. I mean people list like every episode of, I dunno, Game of Thrones and annotate every time that people get killed or [...] all sorts of stuff. Fandom is like [a] huge thing and they just put out these... or like the—if you ever played Minecraft and looked at the Minecraft wiki, it's just so (chuckles) so detailed. Like, "Who spends all their time...?" [...]

Sri: The idea of fandom actually is very relevant here, because [...] I have so far been thinking about the idea that the incentives have to be backed by some type of economic value—

Will: Yeah, for a certain class of things [...] There are some things that are very well-tuned to economic incentives and the other stuff is well-tuned to fandom, right?

From the linked wiki:

In Seligman's hypothesis, the dogs do not try to escape because they expect that nothing they do will stop the shock. To change this expectation, experimenters physically picked up the dogs and moved their legs, replicating the actions the dogs would need to take in order to escape from the electrified grid. This had to be done at least twice before the dogs would start willfully jumping over the barrier on their own. In contrast, threats, rewards, and observed demonstrations had no effect on the "helpless" Group 3 dogs.

There are a lot of neuroses a person can acquire around locus of control. If you look at the wiki for that one, they'll sort of make it sound like "strong" locus of control means believing that everything is under your own control, and within a certain (very American) mindset that is obviously Right And Good. Yet... that can lead to a lot of flailing or self-blame over things that were never really up to you.

("You"? It's me. I am the person with an inappropriately internal locus of control. I have very hubristic intestinal bacteria or something: in my gut, I foolishly feel that if I were to Just Buckle Down, I could solve a good chunk of the world's problems, certainly all of my problems... this also thus means that anything I ever come across that's wrong is, conversely, My Fault.)

My mother told me about Seligman's experiments, in reference to someone who has a very external locus of control. The thing that she pointed out was that you really can't pressure the dog out of its learned helplessness – but the dog really can't will itself out, either. It is persistent external support that makes it possible to learn the actions you can take that make a difference. Even if you understand on a cognitive level – t's training wheels that keep a bike upright so you can get a feel for moving forward in the way that'll let you balance later on your own.

I have not yet lost someone in the way that Peter has. I think it's the kind of thing that you can't understand till you've been through it. But I have seen learned helplessness before – so if there's one tiny corner of this metaphor I'd dog-ear (sorry not sorry), it's that importance of someone physically moving the dogs' legs to show them the motions of getting out. Not even once, but more than once.

Probably what that looks like is very situational. It's hard to recognize learned helplessness, so it's probably even harder to figure out what the support is that you'd need. I won't presume to advise Peter because

a. I would cosign that he is a "smart guy" b. meanwhile I am a lumpy goblin who just ate more ice cream than I meant to because it was warming in my hands and continuing to eat was easier than figuring out whether I should put it back c. he does emphasize asking for help, "over and over and over again"

So just generally, then, to anyone else who may be vibing with the sentiment here but may not have grokked why the help is so important... As someone who's worked from the other direction to not see every problem in the world as an Insufficient Application of Maya's Will, I can only note that, you know, maybe there's some way that the people who love you can help move your paws as you're figuring it out.

it's important because it's difficult to devise a plausible societal explanation for how such a statistic would arise without invoking some other biological effect it has (verboten). there is some plausibility in saying, "look, white people just don't like blacks, so they've excluded them from privileged positions, and being poor leads to criminal behavior." there's not a similar explanation for a gene that has no phenotypical expression that can implicate "society"

I could see why they would make some students take the course, but I don't see why it wouldn't be an admission requirement for everyone. It doesn't seem fair that way. I understand that they were just conducting a survey, but I feel like it would be better to make everyone have the same requirements so it's not more or less stress on the students. Also, if they were all required to do the course for an admission requirement, then they could all ask each other for help or make a study group.

I mean it would be counter intuitive for them to get you off the app.

This is both the end of Goodrum's anecdote, but also a wonderful conclusion to the article. The ideal "perfect" woman is something that is generally unachievable for many people to the point that is may just simply not exist. There is a call for action; stop striving for something else that society wants and start celebrating what we have with ourselves now instead of comparing ourselves to others.