not a very diverse group of people, could be missing factors in the results

This entire process is how they ended up with 14 families that explored most or all possible info and theories

being aware and sensitive to nuances and complexities in data. They started this way so that other themes could still be revealed

They had a software program perform coding to pick which interviews would be analyzed based off the amount of info given related to the topic, emerging questions from people analyzing data, and provisional theories

probably to make them feel more comfortable, since this subject can be sensitive

not the most reliable method, could be more randomized

The interviews had to be carefully picked and studies because the original study at the time of the interviews had a wider focus than this study

work in applied sciences (including behavioral and social sciences) they took data from people who live near them (nonwestern data, but still applies to how western families work)

Recent article, later found out their data was from 2015, meaning not much research about the individualization theory was done between then and now, so we can still use this to deepen our limited knowledge

Grounded theory techniques- qualitative research methods used to develop theories directly from data (you don't start with a hypothesis)

Constructivist grounded theory- generating theories by gathering data from participants instead of using preexisting theories (useful when little or no theories exist for a topic)

gathered data this way so they can find unexpected themes

Interviews weren't done in real time, they found an already existing database

Mostly theory, not enough direct proof

That's why they interviewed families in the Netherlands

Theorists believed individualization made extended family relationships weaker and more fragile because their relationships changed once people's circumstances or emotional needs changed

Before the 19th century, extended family lived together and depended on each other. Then during the modernization process individuals stopped being dependent on extended family to live and people started focusing more on spouses and children (Nuclear family)

Detraditionalization- the process of individuals gradual detachment to traditional institutions (Church, trade unions, political parties, and family)

Individualization has become an umbrella term, making it harder to define

There is change, but not for the reasons theorized???

Method: They interviewed 14 families and analyzed the narratives from 3 generations. (this included narratives from children) (42 people total)

the individualization theory has not been thoroughly observed directly (not focused on extended family)

Individualization Theory- The theory that the increase of individual choice (along with families shifting from an instrumental to an emotional function) have cause extended family bonds to become unstable

use synonyms if needed

Teir Two Sources: official reports from government agencies or major institutions; studies that submit statistics within communities? Magazines, books from non-academic presses that cite their sources written bb y journalists

similar to state legislative updates that are published annually for the courts/attorney's (reference to my own paper)

keep audience in mind when writing essay as well

based off research question through formal resources

The first lines of the stanza stuck out to me, as a contrast from the final lines of the previous stanza where there is “a damp gust bringing rain.” Imagery of water abounds here, a break from the water/rock section found earlier, where the word “water” is hyper-present, but water itself is missing, creating a desert, or a waste land. In this passage the wind is “damp” and brings “rain,” water seeping into the imagery of the poem in reality. The imagery appears to continue in this passage, with the language of “sunken” and the mention of “Ganga,” another word for the river Ganges. However, upon closer inspection, the images here evoke the barren land of before, with rain clouds “distant,” leaves still “limp,” a landscape “waiting for rain.” The storm is on the horizon, anticipation plaguing the landscape, “the jungle crouched” waiting to pounce, to soak up the water that has not appeared yet, but is closer than ever. In her annotation on this section, specifically the line “bringing rain,” Jeannie argues “The irony of waiting (p)ages for something so abrupt simulates the imbalanced power dynamic between gods and men.” The gods, in her interpretation, are in control of the rain, refusing that the water be distributed in the hands of the men, until finally they break the dam and the rain flows. The gods toy with the humans to an even greater degree, explaining the discontinuous nature of the timeline. They give rain, then take it away, leaving a landscape “sunken” and “limp,” still in the state of anticipation. I find an interesting connection between this and the Anniversary Papers, where Kittredge writes “the Brahmans are never wear of inculcating the duty of free-handedness, and deem it more blessed to give than to receive.” In TWL, that attitude remains, the people and the land giving all they have, waiting to receive water. The gods don’t give rain, allowing the land to wait, anticipation building, blessing them, as they are giving and not receiving. This implies a double standard, where the land and the people must follow the rules of the gods, but the gods are not held to those same standards, a potential reflection of the elitism that destroyed the waste land as a public space.

After reading Weston's Perilous Chapel (Chapter XIII), Eliot's inclusion of an empty chapel takes on new significance. Weston explores the recurring motif of the "Perilous Chapel" within Grail legend. She goes on to tell numerous stories of knights encountering a mysterious chapel on a voyage. For instance, Here, Eliot references an "empty chapel" as the speaker travels through a desolate landscape of rock and an absence of water (therefore connecting to a loss in fertility). in other words, "What the Thunder Said" is a further exploration of a physical wasteland. This dark mood and sense of ruin in taken beyond the land and into a manmade space, the chapel. It is described as "empty" without any windows to let in sunlight. It is also the home of the wind, highlighting a lack of spirit of divinity as a force from nature sweeps through the space. The lack of windows could also symbolize a disconnect between nature and man as a key gateway between the two is absent. This is followed up by a reference to "dry bones", a powerful image of ultimate death. This image comes in stark contrast to the chapel stories explored by Weston. For instance, PErceval rides through a storm before encountering the chapel. Here, the building has been cursed by the black hand and it is up to Pereval to first, fight off the force, and second, bless the chapel by spreading holy water over the walls. I interpreted this as a haunting force of death. Therefore, I believe Weston and Eliot explore the similar themes of death and decay in different means. In other words, Eliot ties the glum setting of the wasteland to the absence of a higher power in the empty chapel

The note for this line roughly translates to: "Already half Europe, at all events half Eastern Europe, is on the road to Chaos. In a state of drunken illusion she is reeling into the abyss and, as she reels, she sings a drunken hymn such as Dmitri Karamazov sang. The insulted citizen laughs that song to scorn, the saint and seer hear it with tears" (Hesse).

In the framework of TWL, the saint and seer can indeed be read as Tiresias-- the narrator, and the ultimate observer of the collective human tragedy. He alone views the fall of humanity with profound compassion and insight: while others "laugh at the song to scorn", he mourns it.

What fascinates me the most in these two lines is the description of the lamentation as "maternal." It intensifies the emotional and spiritual dimension of grief, for there is nothing more sacred and compelling than the cry of a mother for her child: Mary's witnessing and mourning of Jesus's crucifixion, the blight on earth caused by Demeter's lamentation of her daughter Persephone's abduction by Hades (again, creating A WASTE LAND), Achilles' mother grieving his prophesied death in the Trojan War, and Constance's emotional regression after the capture and death of her son in the Shakespearean play King John. In Hesse's analysis, Europe is now distilled into the archetype of the bereaved mother, mourning the cultural and spiritual fall of her people. Within Eliot’s poem, Tiresias’s lament thus acquires a maternal resonance. Having lived as both man and woman---and, according to myth, having even borne children---Tiresias embodies the universal capacity for empathy and suffering. His tears are not only those of a prophet who witnesses and forsees the suffering of his fellow species, but are the sacred image of a profoundly empathetic mother.

El cuerpo femenino casi siempre se ha entendido a través de un esquema visual patriarcal (solo existe para fines reproductivos, estéticos y / o médicos) y lp que queda fuera de esta mirada se vuelve inexistente. Esto no es casual es totalmente político ya que hay una relación entre lo que no se representa y lo que no se puede experimentar. El que haya sido invisibilizado durante tanto tiempo es una forma de controlar el placer y de negar la anatomía propia del cuerpo. Esto igual funciona como una metáfora para los sistemas de poder en general en donde todo lo que sale de la norma se tapa, se borra o se vuelve una amenaza. Desprogramar entonces sería una forma de reconfigurar nuestro conocimiento a través de lo que se nos ha negado o escondido.

En una sociedad que mide todo en términos de productividad el hacer algo simplemente por placer es un acto de resistencia. Desprogramar el clímax también sería una forma de desarmar la manera en la que el capitalismo entiende el cuerpo humano (como una máquina funcional). Así se podría habitar un cuerpo que existe sin objetivos externos.

Esto implica que las respuestas a nuestras preguntas estén muy condicionadas. Un buen ejemplo es el SEM (Search Engine Marketing): el orden y visibilidad de los resultados no dependen simplemente de la relevancia, sino del presupuesto que puede invertir cada página. Así, "nuestras respuestas" ya llega mediado por intereses económicos, no por nuestra curiosidad o libertad de exploración.

Publishing companies often require authors to assign all copyright of their works to the publisher as a requirement for publication. This is not always necessary. There are ways to negotiate retention of some author rights. Most publishers support author sharing policies. This allows faculty to retain certain rights to their works, such as sharing their works openly on an institutional repositiory, like Purdue e-Pubs. For more information on copyrights, visit the Purdue University Copyright Office (https://lib.purdue.edu/uco/).

Author Rights and Sharing Policy Support

Why?

*dividing (spelling)

effect sizes geven hoeveel groepen verschillen en confidence intevals geven de range

zorgt voor nauwkeurigheid en consistentie als je twee steekproeven vergelijkt waarvan de sample size verschilt

populatiegemiddelen onder de ho dus deze zijn vaak 0

observed difference is het verschil tussen de steekproef gemiddelen. deze steekproeven hebben we namelijk geobserveerd. het verwachte verschil si het verschil tussen populatiegemiddelden

wanneer de geobserveerde verschillen in gemiddelen groter zijn dan de standaard error dan kan er of geen effect zijn of wel omdat het twee verschillende populaties zijn. dit zijn de twee opties maar stel het verschil tussen de geobserveerde verschillen en de se is heel groot dan is de kans dat het om t2 gaat veel groter

dit komt omdat je niet met de volledige variable werkt dus gebruik je minder informatie en heb je minder power

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Reply to the reviewers

__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __

This study explores chromatin organization around trans-splicing acceptor sites (TASs) in the trypanosomatid parasites Trypanosoma cruzi, T. brucei and Leishmania major. By systematically re-analyzing MNase-seq and MNase-ChIP-seq datasets, the authors conclude that TASs are protected by an MNase-sensitive complex that is, at least in part, histone-based, and that single-copy and multi-copy genes display differential chromatin accessibility. Altogether, the data suggest a common chromatin landscape at TASs and imply that chromatin may modulate transcript maturation, adding a new regulatory layer to an unusual gene-expression system.

I value integrative studies of this kind and appreciate the careful, consistent data analysis the authors implemented to extract novel insights. That said, several aspects require clarification or revision before the conclusions can be robustly supported. My main concerns are listed below, organized by topic/result section.

TAS prediction * Why were TAS predictions derived only from insect-stage RNA-seq data? Restricting TAS calls to one life stage risks biasing predictions toward transcripts that are highly expressed in that stage and may reduce annotation accuracy for lowly expressed or stage-specific genes. Please justify this choice and, if possible, evaluate TAS robustness using additional transcriptomes or explicitly state the limitation.

TAS predictions derived only from insect-stage RNA-seq data because in a previous study it was shown that there are no significant differences between stages in the 5’UTR procesing in T. cruzi life stages (https://doi.org/10.3389/fgene.2020.00166) We are not testing an additional transcriptome here, because the robustness of the software was already probed in the original article were UTRme was described (Radio S, 2018 doi:10.3389/fgene.2018.00671).

Results - "There is a distinctive average nucleosome arrangement at the TASs in TriTryps": * You state that "In the case of L. major the samples are less digested." However, Supplementary Fig. S1 suggests that replicate 1 of L. major is less digested than the T. brucei samples, while replicate 2 of L. major looks similarly digested. Please clarify which replicates you reference and correct the statement if needed.

The reviewer has a good point. We made our statement based on the value of the maximum peak of the sequenced DNA molecules, which in general is a good indicative of the extension of the digestion achieved by the sample (Cole H, NAR, 2011).

As the reviewer correctly points, we should have also considered the length of the DNA molecules in each percentile. However, in this case both, T. brucei’s and L major’s samples were gel purified before sequencing and it is hard to know exactly what fragments were left behind in each case. Therefore, it is better not to over conclude on that regard.

We have now comment on this in the main manuscript, and we have clarified in the figure legends which data set we used in each case.

* It appears you plot one replicate in Fig. 1b and the other in Suppl. Fig. S2. Please indicate explicitly which replicate is in each plot. For T. brucei, the NDR upstream of the TAS is clearer in Suppl. Fig. S2 while the TAS protection is less prominent; based on your digestion argument, this should correspond to the more-digested replicate. Please confirm.

The replicates used for the construction of each figure are explicitly indicated in Table S1. Although we have detailed in the table the original publication, the project and accession number for each data set, the reviewer is correct that in this case it was still not completely clear to which length distribution heatmap was each sample associated with. To avoid this confusion, we have now added the accession number for each data set to the figure legends and also clarified in Table S1. Regarding the reviewer’s comment on the correspondence between the observed TAS protection and the extent of samples digestion, he/she is correct that for a more digested sample we would expect a clearer NDR. In this case, the difference in the extent of digestion between these two samples is minor, as observed the length of the main peak in the length distribution histogram for sequenced DNA molecules is the same. These two samples GSM5363006, represented in Fig1 b, and GSM5363007, represented in S2, belong to the same original paper (Maree et al 2017), and both were gel purified before sequencing. Therefore, any difference between them could not only be the result of a minor difference in the digestion level achieved in each experiment but could be also biased by the fragments included or not during gel purification. Therefore, I would not over conclude about TAS protection from this comparison. We have now included a brief comment on this, in the figure discussion

* The protected region around the TAS appears centered on the TAS in T. brucei but upstream in L. major. This is an interesting difference. If it is technical (different digestion or TAS prediction offset), explain why; if likely biological, discuss possible mechanisms and implications.

We appreciate the reviewer suggestion. We cannot assure if it is due to technical or biological reasons, but there is evidence that L. major ‘s genome has a different dinucleotide content and it might have an impact on nucleosome assembly. We have now added a comment about this observation in the final discussion of the manuscript.

Results - "An MNase sensitive complex occupies the TASs in T. brucei": * The definition of "MNase activity" and the ordering of samples into Low/Intermediate/High digestion are unclear. Did you infer digestion levels from fragment distributions rather than from controlled experimental timepoints? In Suppl. Fig. S3a it is not obvious how "Low digestion" was defined; that sample's fragment distribution appears intermediate. Please provide objective metrics (e.g., median fragment length, fraction 120-180 bp) used to classify digestion levels.

As the reviewer suggests, the ideal experiment would be to perform a time course of MNase reaction with all the samples in parallel, or to work with a fixed time point adding increasing amounts of MNase. However, even when making controlled experimental timepoints, you need to check the length distribution histogram of sequenced DNA molecules to be sure which level of digestion you have achieved.

In this particular case, we used public available data sets to make this analysis. We made an arbitrary definition of low, intermediate and high level of digestion, not as an absolute level of digestion, but as a comparative output among the tested samples. We based our definition on the comparison of __the main peak in length distribution heatmaps because this parameter is the best metric to estimate the level of digestion of a given sample. It represents the percentage of the total DNA sequenced that contains the predominant length in the sample tested. __Hence, we considered:

low digestion: when the main peak is longer than the expected protection for a nucleosome (longer than 150 bp). We expect this sample to contain additional longer bands that correspond to less digested material.

intermediate digestion, when the main peak is the expected for the nucleosome core-protection (˜146-150bp).

high digestion, when the main peak is shorter than that (shorter than 146 bp). This case, is normally accompanied by a bigger dispersion in fragment sizes.

To do this analysis, we chose samples that render different MNase protection of the TAS when plotting all the sequenced DNA molecules relative to this point and we used this protection as a predictor of the extent of sample digestion (Figure 2). To corroborate our hypothesis, that the degree of TAS protection was indeed related to the extent of the MNase digestion of a given sample, we looked at the length distribution histogram of the sequenced DNA molecules in each case. It is the best measurement of the extent of the digestion achieved, especially, when sequencing the whole sample without any gel purification and representing all the reads in the analysis as we did. The only caveat is with the sample called “intermediate digestion 1” that belongs to the original work of Mareé 2017, since only this data set was gel purified.

Whether the sample used in Figure 1 (from Mareé 2017) is also from the same lab and is an MNase-seq. Strictly speaking, there is no methodological difference between MNase-seq and the input of a native MNase-ChIP-seq, since the input does not undergo the IP.

* Several fragment distributions show a sharp cutoff at ~100-125 bp. Was this due to gel purification or bioinformatic filtering? State this clearly in Methods. If gel purification occurred, that can explain why some datasets preserve the MNase-sensitive region.

The sharp cutoff is neither due to gel purification or bioinformatic filtering, it is just due to the length of the paired-end read used in each case. In earlier works the most common was to sequence only 50bp, with the improvement of technologies it went up to 75,100 or 125 bp. We have now clarified in Table S1 the length of the paired-reads used in each case when possible.

* Please reconcile cases where samples labeled as more-digested contain a larger proportion of >200 bp fragments than supposedly less-digested samples; this ordering affects the inference that digestion level determines the loss/preservation of TAS protection. Based on the distributions I see, "Intermediate digestion 1" appears most consistent with an expected MNase curve - please confirm and correct the manuscript accordingly.

As explained above, it's a common observation in MNase digestion of chromatin that more extensive digestion can still result in a broad range of fragment sizes, including some longer fragments. This seemingly counter-intuitive result is primarily due to the non-uniform accessibility of chromatin and the sequence preference of the MNase enzyme, which has a preference for AT reach sequences.

The rationale of this is as follows: when you digest chromatin with MNase and the objective is to map nucleosomes genome-wide, the ideal situation would be to get the whole material contained in the mononucleosome band. Given that MNase is less efficient to digest protected DNA but, if the reaction proceeds further, it always ends up destroying part of it, the result is always far from perfect. The better situation we can get, is to obtain samples were ˜80% of the material is contained in the mononucloesome band. __And here comes the main point: __even in the best scenario, you always get some additional longer bands, such as those for di or tri nucleosomes. If you keep digesting, you will get less than 80 % in the nucleosome band and, those remaining DNA fragments that use to contain di and tri nucleosomes start getting digested as well, originating a bigger dispersion in fragments sizes. How do we explain persistence of Long Fragments? The longest fragments (di-, tri-nucleosomes) that persist in a highly digested sample are the ones that were originally most highly protected by proteins or higher-order structure, or by containing a poor AT sequence content, making their linker DNA extremely resistant to initial cleavage. Once the majority of the genome is fragmented, these few resistant longer fragments become a more visible component of the remaining population, contributing to a broader size dispersion. Hence, you end up observing a bigger dispersion in length distributions in the final material. Bottom line, it is not a good practice to work with under or over digested samples. Our main point, is to emphasize that especially when comparing samples, it important to compare those with comparable levels of digestion. Otherwise, a different sampling of the genome will be represented in the remaining sequenced DNA.

Results - "The MNase sensitive complexes protecting the TASs in T. brucei and T. cruzi are at least partly composed of histones": * The evidence that histones are part of the MNase-sensitive complex relies on H3 MNase-ChIP signal in subnucleosomal fragment bins. This seems to conflict with the observation (Fig. 1) that fragments protecting TASs are often nucleosome-sized. Please reconcile these points: are H3 signals confined to subnucleosomal fragments flanking the TAS while the TAS itself is depleted of H3? Provide plots that compare MNase-seq and H3 ChIP signals stratified by consistent fragment-size bins to clarify this.

What we learned from other eukaryotic organisms that were deeply studied, such as yeast, is that NDRs are normally generated at regulatory points in the genome. In this sense, yeast tRNA genes have a complex with a bootprint smaller than a nucleosome formed by TFIIIC-TFIIB (Nagarajavel, doi: 10.1093/nar/gkt611). On the other hand, many promotor regions have an MNase-sensitive complex with a nucleosome-size footprint, but it does not contain histones (Chereji, et al 2017, doi:10.1016/j.molcel.2016.12.009). The reviewer is right that from Figure 1 and S2 we could observe that the footprint of whatever occupies the TAS region, especially in T. brucei, is nucleosome-size. However, it only shows the size, but it doesn’t prove the nature of its components. Nevertheless, those are only MNase-seq data sets. Since it does not include a precipitation with specific antibodies, we cannot confirm the protecting complex is made up by histones. In parallel, a complementary study by Wedel 2017, from Siegel’s lab, shows that using a properly digested sample and further immunoprecipitating with a-H3 antibody, the TAS is not protected by nucleosomes at least not when analyzing nucleosome size-DNA molecules. Besides, Briggs et. al 2018 (doi: 10.1093/nar/gky928) showed that at least at intergenic regions H3 occupancy goes down while R-loops accumulation increases. We have now added a supplemental figure associated to Figure 3 (new Suplemental 5) replotting R-loops and MNase-ChIP-seq for H3 relative to our predicted TAS showing this anti-correlation and how it partly correlates with MNase protection as well. As a control we show that Rpb9 trends resembles H3 as Siegel’s lab have shown in Wedel 2018.

* Please indicate which datasets are used for each panel in Suppl. Fig. S4 (e.g., Wedel et al., Maree et al.), and avoid calling data from different labs "replicates" unless they are true replicates.

In most of our analysis we used real replicated experiments. Such is the case MNase-seq data used in Figure 1, with the corresponding replicate experiments used in Figure S2; T. cruzi MNase-ChIP-seq data used in Figure 3b and 4a with the respective replicate used in Figures S4 and S5 (now S6 in the revised manuscript). The only case in which we used experiments coming from two different laboratories, is in the case of MNase-ChIP-seq for H3 from T. brucei. Unfortunately, there are only two public data sets coming each of them from different laboratories. The samples used in Fig 3 (from Siegel’s lab) whether the IP from H3 represented in S4 and S5 (S6 n the updated version) comes from another lab (Patterton’s). To be more rigorous, we now call them data 1 and 2 when comparing these particular case.

The reviewer is right that in this particular case one is native chromatin (Pattertons’) while the other one is crosslinked (Siegel’s). We have now clarified it in the main text that unfortunately we do not count on a replicate but even under both condition the result remains the same, and this is compatible with my own experience, were crosslinking does not affect the global nucleosome patterns (compared nucleosome organization from crosslinked chromatin MNAse-seq inputs Chereji, Mol Cell, 2017 doi: 10.1016/j.molcel.2016.12.009 and native MNase-seq from Ocampo, NAR, 2016 doi: 10.1093/nar/gkw068).

* Several datasets show a sharp lower bound on fragment size in the subnucleosomal range (e.g., ~80-100 bp). Is this a filtering artifact or a gel-size selection? Clarify in Methods and, if this is an artifact, consider replotting after removing the cutoff.

We have only filtered adapter dimmer or overrepresented sequences when needed. In Figures 2 and S3 we represented all the sequenced reads. In other figures when we sort fragments sizes in silico, such as nucleosome range, dinucleosome or subnucleosome size, we make a note in the figure legends. What the reviewer points is related to the length of the sequence DNA fragment in each experiment. As we explained above, the older data-sets were performed with 50 bp paired-end reads, the newer ones are 75, 100 or 125bp. This is information is now clarified in Table S1.

__Results - "The TASs of single and multi-copy genes are differentially protected by nucleosomes": __

__ __* Please include T. brucei RNA-seq data in Suppl. Fig. S5b as you did for T. cruzi.

We have shown chromatin organization for T. brucei in S5b to show that there is a similar trend. Unfortunately, we did not get a robust list of multi-copy genes for T. brucei as we did get for T. cruzi, therefore we do not want to over conclude showing the RNA-seq for these subsets of genes. The limitation is related to the fact that UTRme restrict the search and is extremely strict when calling sites at repetitive regions.

* Discuss how low or absent expression of multigene families affects TAS annotation (which relies on RNA-seq) and whether annotation inaccuracies could bias the observed chromatin differences.

The mapping of occurrence and annotations that belong to repetitive regions has great complexity. UTRme is specially designed to avoid overcalling those sites. In other words, there is a chance that we could be underestimating the number of predicted TASs at multi-copy genes. Regarding the impact on chromatin analysis, we cannot rule out that it might have an impact, but the observation favors our conclusion, since even when some TASs at multi-copy genes can remain elusive, we observe more nucleosome density at those places.

* The statement that multi-copy genes show an "oscillation" between AT and GC dinucleotides is not clearly supported: the multi-copy average appears noisier and is based on fewer loci. Please tone down this claim or provide statistical support that the pattern is periodic rather than noisy.

We have fixed this now in the preliminary revised version

* How were multi-copy genes defined in T. brucei? Include the classification method in Methods.

This classification was done the same way it was explained for T. cruzi

Genomes and annotations: * If transcriptomic data for the Y strain was used for T. cruzi, please explain why a Y strain genome was not used (e.g., Wang et al. 2021 GCA_015033655.1), or justify the choice. For T. brucei, consider the more recent Lister 427 assembly (Tb427_2018) from TriTrypDB. Use strain-matched genomes and transcriptomes when possible, or discuss limitations.

The most appropriate way to analyze high throughput data, is to aline it to the same genome were the experiments were conducted. This was clearly illustrated in a previous publication from our group were we explained how should be analyzed data from the hybrid CL Brener strain. A common practice in the past was to use only Esmeraldo-like genome for simplicity, but this resulted in output artifacts. Therefore, we aligned it to CL Brener genome, and then focused the main analysis on the Esmeraldo haplotype (Beati Plos ONE, 2023). Ideally, we should have counted on transcriptomic data for the same strain (CL Brener or Esmeraldo). Since this was not the case at that moment, we used data from Y strain that belongs to the same DTU with Esmeraldo.

In the case of T. brucei, when we started our analysis and the software code for UTRme was written, the previous version of the genome was available. Upon 2018 version came up, we checked chromatin parameters and observed that it did not change the main observations. Therefore, we continue working with our previous setups.

Reproducibility and broader integration: * Please share the full analysis pipeline (ideally on GitHub/Zenodo) so the results are reproducible from raw reads to plots.

We are preparing a full pipeline in GitHub. We will make it available before manuscript full revision

* As an optional but helpful expansion, consider including additional datasets (other life stages, BSF MNase-seq, ATAC-seq, DRIP-seq) where available to strengthen comparative claims.

We are now including a new suplemental figure including DRIP-seq and Rp9 ChIP-seq (revised S5). Additionally, we added a new panel c to figure 4, representing FAIRE-seq data for T. cruzi fore single and multi-copy genes

We are working on ATAC-seq analysis and BSF MNase-seq

Optional analyses that would strengthen the study: * Stratify single-copy genes by expression (high / medium / low) and examine average nucleosome occupancy at TASs for each group; a correlation between expression and NDR depth would strengthen the functional link to maturation.

We have now included a panel in suplemental figure 5 (now revised S6), showing the concordance for chromatin organization of stratified genes by RNA-seq levels relative to TAS.

__Minor / editorial comments: __ * In the Introduction, the sentence "transcription is initiated from dispersed promoters and in general they coincide with divergent strand switch regions" should be qualified: such initiation sites also include single transcription start regions.

We have clarified this in the preliminary revised version

* Define the dotted line in length distribution plots (if it is not the median, please clarify) and consider placing it at 147 bp across plots to ease comparison.

The dotted line is just to indicate where the maximum peak is located. It is now clarified in figure legends.

* In Suppl. Fig. 4b "Replicate2" the x-axis ticks are misaligned with labels - please fix.

We have now fixed the figure. Thanks for noticing this mistake.

* Typo in the Introduction: "remodellingremodeling" → "remodeling

Thanks for noticing this mistake, it is fixed in the current version of the manuscript

**Referee cross-commenting** Comment 1: I think Reviewer #2 and Reviewer #3 missed that they authors of this manuscript do cite and consider the results from Wedel at al. 2017. They even re-analysed their data (e.g. Figure 3a). I second Reviewer #2 comment indicating that the inclusion of a schematic figure to help readers visualize and better understand the findings would be an important addition.

Comment 2: I agree with Reviewer #3 that the use of different MNase digestion procedures in the different datasets have to be considered. On the other hand, I don't think there is a problem with figure 1 showing an MNase-protected TAS for T. brucei as it is based on MNase-seq data and reproduces the reported results (Maree et al. 2017). What the Siegel lab did in Wedel et al. 2017 was MNase-ChIPseq of H3 showing nucleosome depletion at TAS, but both results are not necessary contradictory: There could still be something else (which does not contain H3) sitting on the TAS protecting it from MNase digestion.

Reviewer #1 (Significance (Required)):

This study provides a systematic comparative analysis of chromatin landscapes at trans-splicing acceptor sites (TASs) in trypanosomatids, an area that has been relatively underexplored. By re-analyzing and harmonizing existing MNase-seq and MNase-ChIP-seq datasets, the authors highlight conserved and divergent features of nucleosome occupancy around TASs and propose that chromatin contributes to the fidelity of transcript maturation. The significance lies in three aspects: 1. Conceptual advance: It broadens our understanding of gene regulation in organisms where transcription initiation is unusual and largely constitutive, suggesting that chromatin can still modulate post-transcriptional processes such as trans-splicing. 2. Integrative perspective: Bringing together data from T. cruzi, T. brucei and L. major provides a comparative framework that may inspire further mechanistic studies across kinetoplastids. 3. Hypothesis generation: The findings open testable avenues about the role of chromatin in coordinating transcript maturation, the contribution of DNA sequence composition, and potential interactions with R-loops or RNA-binding proteins. Researchers in parasitology, chromatin biology, and RNA processing will find it a useful resource and a stimulus for targeted experimental follow-up.

My expertise is in gene regulation in eukaryotic parasites, with a focus on bioinformatic analysis of high-throughput sequencing data

__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

Siri et al. perform a comparative analysis using publicly available MNase-seq data from three trypanosomatids (T. brucei, T. cruzi, and Leishmania), showing that a similar chromatin profile is observed at TAS (trans-splicing acceptor site) regions. The original studies had already demonstrated that the nucleosome profile at TAS differs from the rest of the genome; however, this work fills an important gap in the literature by providing the most reliable cross-species comparison of nucleosome profiles among the tritryps. To achieve this, the authors applied the same computational analysis pipeline and carefully evaluated MNase digestion levels, which are known to influence nucleosome profiling outcomes.

In my view, the main conclusion is that the profiles are indeed similar-even when comparing T. brucei and T. cruzi. This was not clear in previous studies (and even appeared contradictory, reporting nucleosome depletion versus enrichment) largely due to differences in chromatin digestion across these organisms. The manuscript could be improved with some clarifications and adjustments:

1. The authors state from the beginning that available MNase data indicate altered nucleosome occupancy around the TAS. However, they could also emphasize that the conclusions across the different trypanosomatids are inconsistent and even contradictory: NDR in T. cruzi versus protection-in different locations-in T. brucei and Leishmania.

We start our manuscript by referring to the first MNase-seq data sets publicly available for each TriTryp and we point that one of the main observations, in each of them, is the occurrence of a change in nucleosome density or occupancy at intergenic regions. In T. cruzi, in a previous publication from our group, we stablished that this intergenic drop in nucleosome density occurs near the trans-splicing acceptor site. In this work, we extend our study to the other members of TriTryps: T. brucei and L. major.

In T. brucei the papers from Patterton’s lab and Siegel’s lab came out almost simultaneously in 2017. Hence, they do not comment on each other’s work. The first one claims the presence of a well-positioned nucleosome at the TAS by using MNase-seq, while the second one, shows an NDR at the TAS by using MNase-ChIP-seq. However, we do not think they are contradictory, or they have inconsistency. We brought them together along the manuscript because we think these works can provide complementary information.

On one hand, we infer data from Pattertons lab is slightly less digested than the sample from Siegel’s lab. Therefore, we discuss that this moderate digestion must be the reason why they managed to detect an MNase protecting complex sitting at the TAS (Figure 1). On the other hand, Sigel’s lab includes an additional step by performing MNase-ChIP-seq, showing that when analyzing nucleosome size fragments, histones are not detected at the TAS. Here, we go further in this analysis on figure 3, showing that only when looking at subnucleosome-size fragments, we are able to detect histone H3. And this is also true for T. cruzi.

By integrating every analysis in this work and the previous ones, we propose that TASs are protected by an MNase-sensitive complex (probed in Figure 2). This complex most likely is only partly formed by histones, since only when analyzing sub-nucleosomes size DNA molecules we can detect histone H3 (Figure 3). To be absolutely sure that the complex is not entirely made up by histones, future studies should perform an MNse-ChIP-seq with less digested samples. However, it was previously shown that R-loops are enriched at those intergenic NDRs (Briggs, 2018 doi: 10.1093/nar/gky928) and that R-loops have plenty of interacting proteins (Girasol, 2023 10.1093/nar/gkad836). Therefore, most likely, this MNase-sensitive complexed have a hybrid nature made up by H3 and some other regulatory molecules, possibly involved in trans-splicing. We have now added a new figure S5 showing R-loop co-localization with the NDR.

Regarding the comparison between different organisms, after explaining the sensitivity to MNase of the TAS protecting complex, we discuss that when comparing equally digested samples T. cruzi and T. brucei display a similar chromatin landscape with a mild NDR at the TAS (See T. cruzi represented in Figure 1 compared to T. brucei represented in Intermediate digestion 2 in Figure 2, intermediate digestion in the revised manuscript). Unfortunately, we cannot make a good comparison with L. major, since we do not count on a similar level of digestion.

Another point that requires clarification concerns what the authors mean in the introduction and discussion when they write that trypanosomes have "...poorly organized chromatin with nucleosomes that are not strikingly positioned or phased." On the other hand, they also cite evidence of organization: "...well-positioned nucleosome at the spliced-out region.. in Leishmania (ref 34)"; "...a well-positioned nucleosome at the TASs for internal genes (ref37)"; "...a nucleosome depletion was observed upstream of every gene (ref 35)." Aren't these examples of organized chromatin with at least a few phased nucleosomes? In addition, in ref 37, figure 4 shows at least two (possibly three to four) nucleosomes that appear phased. In my opinion, the authors should first define more precisely what they mean by "poorly organized chromatin" and clarify that this interpretation does not contradict the findings highlighted in the cited literature.

For a better understanding of nucleosome positioning and phasing I recommend the review: Clark 2010 doi:10.1080/073911010010524945, Figure 4. Briefly, in a cell population there are different alternative positions that a given nucleosome can adopt. However, some are more favorable. When talking about favorable positions, we refer to the coordinates in the genome that are most likely covered by a nucleosome and are predominant in the cell population. Additionally, nucleosomes could be phased or not. This refers not only the position in the genome, but to the distance relative to a given point. In yeast, or in highly transcribed genes of more complex eukaryotes, nucleosomes are regularly spaced and phased relative to the transcription start site (TSS) or to the +1 nucleosome (Ocampo, NAR, 2016, doi:10.1093/nar/gkw068). In trypanosomes, nucleosomes have some regular distribution when making a browser inspection but, given that they are not properly phased with respect to any point, it is almost impossible to make a spacing estimation from paired-end data. This is also consistent with a chromatin that is transcribed in an almost constitutive manner.

As the reviewer mention, we do site evidence of organization. We think the original observations are correct, but we do not fully agree with some of the original statements. In this manuscript our aim is to take the best we learned from their original works and to make a constructive contribution adding to the original discussions. In this regard, in trypanosomes there are some conserved patterns in the chromatin landscape, but their nucleosomes are far from being well-positioned or phased. For a better understanding, compare the variations observed in the y axis when representing av. nucleosome occupancy in yeast with those observed in trypanosomes and you will see that the troughs and peaks are much more prominent in yeast than the ones observed in any TryTryp member.

Following the reviewer’s suggestion we have now clarified this in the main text

The paper would also benefit from the inclusion of a schematic figure to help readers visualize and better understand the findings. What is the biological impact of having nucleosomes, di-nucleosomes, or sub-nucleosomes at TAS? This is not obvious to readers outside the chromatin field. For example, the following statement is not intuitive: "We observed that, when analyzing nucleosome-size (120-180 bp) DNA molecules or longer fragments (180-300 bp), the TASs of either T. cruzi or T. brucei are mostly nucleosome-depleted. However, when representing fragments smaller than a nucleosome-size (50-120 bp) some histone protection is unmasked (Fig. 3 and Fig. S4). This observation suggests that the MNase sensitive complex sitting at the TASs is at least partly composed of histones." Please clarify.

We appreciate the reviewer’s suggestion to make a schematic figure. We are working on this and will be added to the manuscript upon final revision.

Regarding the biological impact of having mono, di or subnucleosome fragments, it is important to unveil the fragment size of the protected DNA to infer the nature of the protecting complex. In the case of tRNA genes in yeast, at pol III promoters they found footprints smaller than a nucleosome size that ended up being TFIIB-TFIIC (Nagarajavel, doi: 10.1093/nar/gkt611). Therefore, detecting something smaller than a nucleosome might suggest the binding of trans-acting factors different than histones or involving histones in a mixed complex. These mixed complexes are also observed, and that is the case of the centromeric nucleosome which has a very peculiar composition (Ocampo and Clark, Cells Reports, 2015). On the other hand, if instead we detect bigger fragments, it could be indicative of the presence of bigger protecting molecules or that those regions are part of higher order chromatin organization still inaccessible for MNase linker digestions.

Here we show on 2Dplots, that complex or components protecting the TAS have nucleosome size, but we cannot assure they are entirely made up by histones, since, only when looking at subnucleosome-size fragments, we are able to detect histone H3. We have now added part of this explanation to the discussion.

By integrating every analysis in this work and the previous ones, we propose that the TAS is protected by an MNase-sensitive complex (Figure 2). This complex most likely is only partly formed by histones, since only when analyzing sub-nucleosomes size DNA molecules we can detect histone H3 (Figure 3). As explained above, to be absolutely sure that the complex is not entirely made up by histones, future studies should perform an MNse-ChIP-seq with less digested samples. However, it was previously shown that R-loops are enriched at those intergenic NDRs (Briggs 2018) and that R-loops have plenty of interacting proteins (Girasol, 2023). Therefore, most likely, this MNase-sensitive complexed have a hybrid nature made up by H3 and some other regulatory molecules. We have now added a new S5 figure showing R-loop co-localization.

Some references are missing or incorrect:

we will make a thorough revision

"In trypanosomes, there are no canonical promoter regions." - please check Cordon-Obras et al. (Navarro's group). Thank you for the appropiate suggestion.

We have now added this reference

Please, cite the study by Wedel et al. (Siegel's group), which also performed MNase-seq analysis in T. brucei.

We understand that reviewer number 2# missed that we cited this reference and that we did used the raw data from the manuscript of Wedel et. al 2017 form Siegel’s group. We used the MNase-ChIP-seq data set of histone H3 in our analysis for Figures 3, S4b and S5b (S6c in the revised version), also detailed in table S1. To be even more explicit we have now included the accession number of each data set in the figure legend.

Figure-specific comments: Fig. S3: Why does the number of larger fragments increase with greater MNase digestion? Shouldn't the opposite be expected?

This a good observation. As we also explained to reviewer#1:

It's a common observation in MNase digestion of chromatin that more extensive digestion can still result in a broad range of fragment sizes, including some longer fragments. This seemingly counter-intuitive result is primarily due to the non-uniform accessibility of chromatin and the sequence preference of the MNase enzyme.

The rationale of this is as follows: when you digest chromatin with MNase and the objective is to map nucleosomes genome-wide, the ideal situation would to get the whole material contained in the mononucleosome band. Given that MNase is less efficient to digest protected DNA but, if the reaction proceeds further, it always ends up destroying part of it, the result is always far from perfect. The better situation we can get, is to obtain samples were ˜80% of the material is contained in the mononucloesome band. __And here comes the main point: __even in the best scenario, you always have some additional longer bands, such as those for di or tri nucleosomes. If you keep digesting, you will get less than 80 % in the nucleosome band and, those remaining DNA fragments that use to contain di and tri nucleosomes start getting digested as well originating a bigger dispersion in fragments sizes. How do we explain persistence of Long Fragments? The longest fragments (di-, tri-nucleosomes) that persist in a highly digested sample are the ones that were originally most highly protected by proteins or higher-order structure, making their linker DNA extremely resistant to initial cleavage. Once the majority of the genome is fragmented, these few resistant longer fragments become a more visible component of the remaining population, contributing to a broader size dispersion. Hence, there you end up having a bigger dispersion in length distributions in the final material. Bottom line, it is not a good practice to work with under or overdigested samples. Our main point is to emphasize that especially when comparing samples, it important to compare those with comparable levels of digestion. Otherwise, a different sampling of the genome will be represented in the remaining sequenced DNA Fig. S5B: Why not use MNase conditions under which T. cruzi and T. brucei display comparable profiles at TAS? This would facilitate interpretation.

The reviewer made a reasonable observation. The reason why we used MNase-ChIP_seq instead of just MNase to test occupancy at TAS at the subsets of genes, is because we intended to be more certain if we were talking about the presence of histones or something else. By using IP for histone H3 we can see that at multi-copy genes this protein is present when looking at nucleosome-size fragments. Additionally, as shown in figure S4b, length distribution histograms are also similar for the compared IPs.

Minor points:

There are several typos throughout the manuscript.

Thanks for the observation. We will check carefully.

Methods: "Dinucelotide frecuency calculation."

We will add a code in GitHub

Reviewer #2 (Significance (Required)):

In my view, the main conclusion is that the profiles are indeed similar-even when comparing T. brucei and T. cruzi. This was not clear in previous studies (and even appeared contradictory, reporting nucleosome depletion versus enrichment) largely due to differences in chromatin digestion across these organisms. Audience: basic science and specialized readers.

Expertise: epigenetics and gene expression in trypanosomatids.

__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

The authors analysed publicly accessible MNase-seq data in TriTryps parasites, focusing on the chromatin structure around trans-splicing acceptor sites (TASs), which are vital for processing gene transcripts. They describe a mild nucleosome depletion at the TAS of T. cruzi and L. major, whereas a histone-containing complex protects the TASs of T. brucei. In the subsequent analysis of T. brucei, they suggest that a Mnase-sensitive complex is localised at the TASs. For single-copy versus multi-copy genes, the authors show different di-nucleotide patterns and chromatin structures. Accordingly, they propose this difference could be a novel mechanism to ensure the accuracy of trans-splicing in these parasites.

Before providing an in- depth review of the manuscript, I note that some missing information would have helped in assessing the study more thoroughly; however, in the light of the available information, I provide the following comments for consideration.

The numbering of the figures, including the figure legends, is missing in the PDF file. This is essential for assessing the provided information.

We apologized for not including the figure numbers in the main text, although they are located in the right place when called in the text. The omission was unwillingly made when figure legends were moved to the bottom of the main text. This is now fixed in the updated version of the manuscript.

The publicly available Mnase- seq data are manyfold, with multiple datasets available for T. cruzi, for example. It is unclear from the manuscript which dataset was used for which figure. This must be clarified.

This was detailed in Table S1. We have now replaced the table by an improved version, and we have also included the accession number of each data set used in the figure legends.

Why do the authors start in figure 1 with the description of an MNase- protected TAS for T.brucei, given that it has been clearly shown by the Siegel lab that there is a nucleosome depletion similar to other parasites?

We did not want to ignore the paper from Patterton’s lab because it was the first one to map nucleosomes genome-wide in T. brucei and the main finding of that paper claimed the existence of a well-positioned nucleosome at intergenic regions, what we though constitutes a point worth to be discussed. While Patterton’s work use MNase-seq from gel-purified samples and provides replicated experiments sequenced in really good depth; Siegel’s lab uses MNase-ChIP-seq of histone H3 but performs only one experiment and its input was not sequenced. So, each work has its own caveats and provides different information that together contributes to make a more comprehensive study. We think that bringing up both data sets to the discussion, as we have done in Figures 1 and 3, helps us and the community working in the field to enrich the discussion.

If the authors re- analyse the data, they should compare their pipeline to those used in the other studies, highlighting differences and potential improvements.

We are working on this point. We will provide a more detail description in the final revision.

Since many figures resemble those in already published studies, there seems little reason to repeat and compare without a detailed comparison of the pipelines and their differences.

Following the reviewer advice, we are now working on highlighting the main differences that justify analyzing the data the way we did and will be added in the finally revised method section.

At a first glance, some of the figures might look similar when looking at the original manuscripts comparing with ours. However, with a careful and detailed reading of our manuscripts you can notice that we have added several analyses that allow to unveil information that was not disclosed before.

First, we perform a systematic comparison analyzing every data set the same way from beginning to end, being the main difference with previous studies the thorough and precise prediction of TAS for the three organisms. Second, we represent the average chromatin organization relative to those predicted TASs for TriTryps and discuss their global patterns. Third, by representing the average chromatin into heatmaps, we show for the very first time, that those average nucleosome landscape are not just an average, they keep a similar organization in most of the genome. These was not done in any of the previous manuscripts except for our own (Beati, PLOS One 2023). Additionally, we introduce the discussion of how the extension of MNase reaction can affect the output of these experiments and we show 2D-plots and length distribution heatmaps to discuss this point (a point completely ignored in all the chromatin literature for trypanosomes). Furthermore, we made a far-reaching analysis by considering the contributions of each publish work even when addressed by different techniques. Finally, we discuss our findings in the context of a topic of current interest in the field, such as TriTryp’s genome compartmentalization.

Several previous Mnase- seq analysis studies addressing chromatin accessibility emphasized the importance of using varying degrees of chromatin digestion, from low to high digestion (30496478, 38959309, 27151365).

The reviewer is correct, and this point is exactly what we intended to illustrate in figure number 2. We appreciate he/she suggests these references that we are now citing in the final discussion. Just to clarify, using varying degrees of chromatin digestion is useful to make conclusions about a given organism but when comparing samples, strains, histone marks, etc. It is extremely important to do it upon selection of similar digested samples.

No information on the extent of DNA hydrolysis is provided in the original Mnase- seq studies. This key information can not be inferred from the length distribution of the sequenced reads.

The reviewer is correct that “No information on the extent of DNA hydrolysis is provided in the original Mnase-seq studies” and this is another reason why our analysis is so important to be published and discussed by the scientific community working in trypanosomes. We disagree with the reviewer in the second statement, since the level of digestion of a sequenced sample is actually tested by representing the length distribution of the total DNA sequenced. It is true that before sequencing you can, and should, check the level of digestion of the purified samples in an agarose gel and/or in a bioanalyzer. It could be also tested after library preparation, but before sequencing, expecting to observe the samples sizes incremented in size by the addition of the library adapters. But, the final test of success when working with MNase digested samples is to analyze length of DNA molecules by representing the histograms with length distribution of the sequenced DNA molecules. Remarkably, on occasions different samples might look very similar when run in a gel, but they render different length distribution histograms and this is because the nucleosome core could be intact but they might have suffered a differential trimming of the linker DNA associated to it or even be chewed inside (see Cole Hope 2011, section 5.2, doi: 10.1016/B978-0-12-391938-0.00006-9, for a detailed explanation).

As the input material are selected, in part gel- purified mono- nucleosomal DNA bands. Furthermore the datasets are not directly comparable, as some use native MNase, while others employ MNase after crosslinking; some involve short digestion times at 37 {degree sign} C, while others involve longer digestion at lower temperatures. Combining these datasets to support the idea of an MNase- sensitive complex at the TAS of T. brucei therefore may not be appropriate, and additional experiments using consistent methodologies would strengthen the study's conclusions.

In my opinion, describing an MNase- sensitive complex based solely on these data is not feasible. It requires specifically designed experiments using a consistent method and well- defined MNase digestion kinetics.

As the reviewer suggests, the ideal experiment would be to perform a time course of MNase reaction with all the samples in parallel, or to work with a fix time point adding increasing amounts of MNase. However, the information obtained from the detail analysis of the length distribution histogram of sequenced DNA molecules the best test of the real outcome. In fact, those samples with different digestion levels were probably not generated on purpose.

The only data sets that were gel purified are those from Mareé 2017 (Patterton’s lab), used in Figures 1, S1 and S2 and those from L. major shown in Fig 1. It was a common practice during those years, then we learned that is not necessary to gel purify, since we can sort fragment sizes later in silico when needed.

As we explained to reviewer #1, to avoid this conflict, we decided to remove this data from figures 2 and S3. In summary, the 3 remaining samples comes from the same lab, and belong to the same publication (Mareé 2022). These sample are the inputs of native MNase ChIp-seq, obtain the same way, totally comparable among each other.

Reviewer #3 (Significance (Required)):

Due to the lack of controlled MNase digestion, use of heterogeneous datasets, and absence of benchmarking against previous studies, the conclusions regarding MNase-sensitive complexes and their functional significance remain speculative. With standardized MNase digestion and clearly annotated datasets, this study could provide a valuable contribution to understanding chromatin regulation in TriTryps parasites.

As we have explained in the previous point our conclusions are valid since we do not compare in any figure samples coming from different treatments. The only exception to this comment could be in figure 3 when talking about MNase-ChIP-seq. We have now added a clear and explicit comment in the section and the discussion that despite having subtle differences in experimental procedures we arrive to the same results. This is the case for T. cruzi IP, run from crosslinked chromatin, compared to T. brucei’s IP, run from native chromatin.

Along the years it was observed in the chromatin field that nucleosomes are so tightly bound to DNA that crosslinking is not necessary. However, it is still a common practice specially when performing IPs. In our own hands, we did not observe any difference at the global level neither in T. cruzi or in my previous work with yeast.

...

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Referee #3

Evidence, reproducibility and clarity

The authors analysed publicly accessible MNase-seq data in TriTryps parasites, focusing on the chromatin structure around trans-splicing acceptor sites (TASs), which are vital for processing gene transcripts. They describe a mild nucleosome depletion at the TAS of T. cruzi and L. major, whereas a histone-containing complex protects the TASs of T. brucei. In the subsequent analysis of T. brucei, they suggest that a Mnase-sensitive complex is localised at the TASs. For single-copy versus multi-copy genes, the authors show different di-nucleotide patterns and chromatin structures. Accordingly, they propose this difference could be a novel mechanism to ensure the accuracy of trans-splicing in these parasites.

Before providing an in- depth review of the manuscript, I note that some missing information would have helped in assessing the study more thoroughly; however, in the light of the available information, I provide the following comments for consideration.

The numbering of the figures, including the figure legends, is missing in the PDF file. This is essential for assessing the provided information. The publicly available Mnase- seq data are manyfold, with multiple datasets available for T. cruzi, for example. It is unclear from the manuscript which dataset was used for which figure. This must be clarified. Why do the authors start in figure 1 with the description of an MNase- protected TAS for T.brucei, given that it has been clearly shown by the Siegel lab that there is a nucleosome depletion similar to other parasites? If the authors re- analyse the data, they should compare their pipeline to those used in the other studies, highlighting differences and potential improvements. Since many figures resemble those in already published studies, there seems little reason to repeat and compare without a detailed comparison of the pipelines and their differences. Several previous Mnase- seq analysis studies addressing chromatin accessibility emphasised the importance of using varying degrees of chromatin digestion, from low to high digestion (30496478, 38959309, 27151365). No information on the extent of DNA hydrolysis is provided in the original Mnase- seq studies. This key information can not be inferred from the length distribution of the sequenced reads. As the input material are selected, in part gel- purified mono- nucleosomal DNA bands. Furthermore the datasets are not directly comparable, as some use native MNase, while others employ MNase after crosslinking; some involve short digestion times at 37 {degree sign} C, while others involve longer digestion at lower temperatures. Combining these datasets to support the idea of an MNase- sensitive complex at the TAS of T. brucei therefore may not be appropriate, and additional experiments using consistent methodologies would strengthen the study's conclusions. In my opinion, describing an MNase- sensitive complex based solely on these data is not feasible. It requires specifically designed experiments using a consistent method and well- defined MNase digestion kinetics.

Significance

Due to the lack of controlled MNase digestion, use of heterogeneous datasets, and absence of benchmarking against previous studies, the conclusions regarding MNase-sensitive complexes and their functional significance remain speculative. With standardized MNase digestion and clearly annotated datasets, this study could provide a valuable contribution to understanding chromatin regulation in TriTryps parasites.

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Referee #2

Evidence, reproducibility and clarity

Siri et al. perform a comparative analysis using publicly available MNase-seq data from three trypanosomatids (T. brucei, T. cruzi, and Leishmania), showing that a similar chromatin profile is observed at TAS (trans-splicing acceptor site) regions. The original studies had already demonstrated that the nucleosome profile at TAS differs from the rest of the genome; however, this work fills an important gap in the literature by providing the most reliable cross-species comparison of nucleosome profiles among the tritryps. To achieve this, the authors applied the same computational analysis pipeline and carefully evaluated MNase digestion levels, which are known to influence nucleosome profiling outcomes.

In my view, the main conclusion is that the profiles are indeed similar-even when comparing T. brucei and T. cruzi. This was not clear in previous studies (and even appeared contradictory, reporting nucleosome depletion versus enrichment) largely due to differences in chromatin digestion across these organisms. The manuscript could be improved with some clarifications and adjustments:

1. The authors state from the beginning that available MNase data indicate altered nucleosome occupancy around the TAS. However, they could also emphasize that the conclusions across the different trypanosomatids are inconsistent and even contradictory: NDR in T. cruzi versus protection-in different locations-in T. brucei and Leishmania.
2. Another point that requires clarification concerns what the authors mean in the introduction and discussion when they write that trypanosomes have "...poorly organized chromatin with nucleosomes that are not strikingly positioned or phased." On the other hand, they also cite evidence of organization: "...well-positioned nucleosome at the spliced-out region.. in Leishmania (ref 34)"; "...a well-positioned nucleosome at the TASs for internal genes (ref37)"; "...a nucleosome depletion was observed upstream of every gene (ref 35)." Aren't these examples of organized chromatin with at least a few phased nucleosomes? In addition, in ref 37, figure 4 shows at least two (possibly three to four) nucleosomes that appear phased. In my opinion, the authors should first define more precisely what they mean by "poorly organized chromatin" and clarify that this interpretation does not contradict the findings highlighted in the cited literature.
3. The paper would also benefit from the inclusion of a schematic figure to help readers visualize and better understand the findings. What is the biological impact of having nucleosomes, di-nucleosomes, or sub-nucleosomes at TAS? This is not obvious to readers outside the chromatin field. For example, the following statement is not intuitive: "We observed that, when analyzing nucleosome-size (120-180 bp) DNA molecules or longer fragments (180-300 bp), the TASs of either T. cruzi or T. brucei are mostly nucleosome-depleted. However, when representing fragments smaller than a nucleosome-size (50-120 bp) some histone protection is unmasked (Fig. 3 and Fig. S4). This observation suggests that the MNase sensitive complex sitting at the TASs is at least partly composed of histones." Please clarify. Some references are missing or incorrect:

"In trypanosomes, there are no canonical promoter regions." - please check Cordon-Obras et al. (Navarro's group).

Please, cite the study by Wedel et al. (Siegel's group), which also performed MNase-seq analysis in T. brucei.

Figure-specific comments:

Fig. S3: Why does the number of larger fragments increase with greater MNase digestion? Shouldn't the opposite be expected?

Fig. S5B: Why not use MNase conditions under which T. cruzi and T. brucei display comparable profiles at TAS? This would facilitate interpretation.

Minor points:

There are several typos throughout the manuscript.

Methods: "Dinucelotide frecuency calculation."

Significance

In my view, the main conclusion is that the profiles are indeed similar-even when comparing T. brucei and T. cruzi. This was not clear in previous studies (and even appeared contradictory, reporting nucleosome depletion versus enrichment) largely due to differences in chromatin digestion across these organisms.

Audience: basic science and specialized readers.

Expertise: epigenetics and gene expression in trypanosomatids.

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Referee #1

Evidence, reproducibility and clarity

This study explores chromatin organization around trans-splicing acceptor sites (TASs) in the trypanosomatid parasites Trypanosoma cruzi, T. brucei and Leishmania major. By systematically re-analyzing MNase-seq and MNase-ChIP-seq datasets, the authors conclude that TASs are protected by an MNase-sensitive complex that is, at least in part, histone-based, and that single-copy and multi-copy genes display differential chromatin accessibility. Altogether, the data suggest a common chromatin landscape at TASs and imply that chromatin may modulate transcript maturation, adding a new regulatory layer to an unusual gene-expression system.

I value integrative studies of this kind and appreciate the careful, consistent data analysis the authors implemented to extract novel insights. That said, several aspects require clarification or revision before the conclusions can be robustly supported. My main concerns are listed below, organized by topic/result section.

TAS prediction:

• Why were TAS predictions derived only from insect-stage RNA-seq data? Restricting TAS calls to one life stage risks biasing predictions toward transcripts that are highly expressed in that stage and may reduce annotation accuracy for lowly expressed or stage-specific genes. Please justify this choice and, if possible, evaluate TAS robustness using additional transcriptomes or explicitly state the limitation.

Results

• "There is a distinctive average nucleosome arrangement at the TASs in TriTryps":
• You state that "In the case of L. major the samples are less digested." However, Supplementary Fig. S1 suggests that replicate 1 of L. major is less digested than the T. brucei samples, while replicate 2 of L. major looks similarly digested. Please clarify which replicates you reference and correct the statement if needed.
• It appears you plot one replicate in Fig. 1b and the other in Suppl. Fig. S2. Please indicate explicitly which replicate is in each plot. For T. brucei, the NDR upstream of the TAS is clearer in Suppl. Fig. S2 while the TAS protection is less prominent; based on your digestion argument, this should correspond to the more-digested replicate. Please confirm. The protected region around the TAS appears centered on the TAS in T. brucei but upstream in L. major. This is an interesting difference. If it is technical (different digestion or TAS prediction offset), explain why; if likely biological, discuss possible mechanisms and implications.

Results

• "An MNase sensitive complex occupies the TASs in T. brucei":
• The definition of "MNase activity" and the ordering of samples into Low/Intermediate/High digestion are unclear. Did you infer digestion levels from fragment distributions rather than from controlled experimental timepoints? In Suppl. Fig. S3a it is not obvious how "Low digestion" was defined; that sample's fragment distribution appears intermediate. Please provide objective metrics (e.g., median fragment length, fraction 120-180 bp) used to classify digestion levels.
• Several fragment distributions show a sharp cutoff at ~100-125 bp. Was this due to gel purification or bioinformatic filtering? State this clearly in Methods. If gel purification occurred, that can explain why some datasets preserve the MNase-sensitive region.
• Please reconcile cases where samples labeled as more-digested contain a larger proportion of >200 bp fragments than supposedly less-digested samples; this ordering affects the inference that digestion level determines the loss/preservation of TAS protection. Based on the distributions I see, "Intermediate digestion 1" appears most consistent with an expected MNase curve - please confirm and correct the manuscript accordingly. Results - "The MNase sensitive complexes protecting the TASs in T. brucei and T. cruzi are at least partly composed of histones":
• The evidence that histones are part of the MNase-sensitive complex relies on H3 MNase-ChIP signal in subnucleosomal fragment bins. This seems to conflict with the observation (Fig. 1) that fragments protecting TASs are often nucleosome-sized. Please reconcile these points: are H3 signals confined to subnucleosomal fragments flanking the TAS while the TAS itself is depleted of H3? Provide plots that compare MNase-seq and H3 ChIP signals stratified by consistent fragment-size bins to clarify this.
• Please indicate which datasets are used for each panel in Suppl. Fig. S4 (e.g., Wedel et al., Maree et al.), and avoid calling data from different labs "replicates" unless they are true replicates.
• Several datasets show a sharp lower bound on fragment size in the subnucleosomal range (e.g., ~80-100 bp). Is this a filtering artifact or a gel-size selection? Clarify in Methods and, if this is an artifact, consider replotting after removing the cutoff. Results - "The TASs of single and multi-copy genes are differentially protected by nucleosomes":
• Please include T. brucei RNA-seq data in Suppl. Fig. S5b as you did for T. cruzi.
• Discuss how low or absent expression of multigene families affects TAS annotation (which relies on RNA-seq) and whether annotation inaccuracies could bias the observed chromatin differences.
• The statement that multi-copy genes show an "oscillation" between AT and GC dinucleotides is not clearly supported: the multi-copy average appears noisier and is based on fewer loci. Please tone down this claim or provide statistical support that the pattern is periodic rather than noisy.
• How were multi-copy genes defined in T. brucei? Include the classification method in Methods.

Genomes and annotations:

• If transcriptomic data for the Y strain was used for T. cruzi, please explain why a Y strain genome was not used (e.g., Wang et al. 2021 GCA_015033655.1), or justify the choice. For T. brucei, consider the more recent Lister 427 assembly (Tb427_2018) from TriTrypDB. Use strain-matched genomes and transcriptomes when possible, or discuss limitations.

Reproducibility and broader integration:

• Please share the full analysis pipeline (ideally on GitHub/Zenodo) so the results are reproducible from raw reads to plots.
• As an optional but helpful expansion, consider including additional datasets (other life stages, BSF MNase-seq, ATAC-seq, DRIP-seq) where available to strengthen comparative claims. Optional analyses that would strengthen the study:
• Stratify single-copy genes by expression (high / medium / low) and examine average nucleosome occupancy at TASs for each group; a correlation between expression and NDR depth would strengthen the functional link to maturation.

Minor / editorial comments:

• In the Introduction, the sentence "transcription is initiated from dispersed promoters and in general they coincide with divergent strand switch regions" should be qualified: such initiation sites also include single transcription start regions.
• Define the dotted line in length distribution plots (if it is not the median, please clarify) and consider placing it at 147 bp across plots to ease comparison.
• In Suppl. Fig. 4b "Replicate2" the x-axis ticks are misaligned with labels - please fix.
• Typo in the Introduction: "remodellingremodeling" → "remodeling."

Referee cross-commenting

Comment 1: I think Reviewer #2 and Reviewer #3 missed that they authors of this manuscript do cite and consider the results from Wedel at al. 2017. They even re-analysed their data (e.g. Figure 3a). I second Reviewer #2 comment indicating that the inclusion of a schematic figure to help readers visualize and better understand the findings would be an important addition.

Comment 2: I agree with Reviewer #3 that the use of different MNase digestion procedures in the different datasets have to be considered. On the other hand, I don't think there is a problem with figure 1 showing an MNase-protected TAS for T. brucei as it is based on MNase-seq data and reproduces the reported results (Maree et al. 2017). What the Siegel lab did in Wedel et al. 2017 was MNase-ChIPseq of H3 showing nucleosome depletion at TAS, but both results are not necessary contradictory: There could still be something else (which does not contain H3) sitting on the TAS protecting it from MNase digestion.

Significance

This study provides a systematic comparative analysis of chromatin landscapes at trans-splicing acceptor sites (TASs) in trypanosomatids, an area that has been relatively underexplored. By re-analyzing and harmonizing existing MNase-seq and MNase-ChIP-seq datasets, the authors highlight conserved and divergent features of nucleosome occupancy around TASs and propose that chromatin contributes to the fidelity of transcript maturation.

The significance lies in three aspects:

1. Conceptual advance: It broadens our understanding of gene regulation in organisms where transcription initiation is unusual and largely constitutive, suggesting that chromatin can still modulate post-transcriptional processes such as trans-splicing.
2. Integrative perspective: Bringing together data from T. cruzi, T. brucei and L. major provides a comparative framework that may inspire further mechanistic studies across kinetoplastids.
3. Hypothesis generation: The findings open testable avenues about the role of chromatin in coordinating transcript maturation, the contribution of DNA sequence composition, and potential interactions with R-loops or RNA-binding proteins. Researchers in parasitology, chromatin biology, and RNA processing will find it a useful resource and a stimulus for targeted experimental follow-up.

My expertise is in gene regulation in eukaryotic parasites, with a focus on bioinformatic analysis of high-throughput sequencing data

https://en.wikipedia.org/wiki/Dutton%27s_Books

Honestly this is a very interesting argument. We can all sit here hundreds of years later and say that this was all wrong but then the world we know would not exist. This brings up so many unanswerable questions about morality, history, and philosophy. What is even the point of discussing such things? The only reason I see for judging the morality of historical actions is so that we can make better decisions in the present. So yes, the vast majority of Americans benefit from this genocide, but that does not make criticism of it irrelevant. What it does mean is that we should take no satisfaction or moral victory from criticizing history. As for Jackson's point in his own time, it isn't really asking this question. The point is actually to argue that this genocide has been and will continue to be worth it. Jackson is saying that if he was sent back 150 years he would do the same thing as the forefathers. It is worth noting that this argument reframes the conflict, shifting attention from the possibility of better treatment in the future to justifying past sins. It thus implicitly argues that destroying the natives is fine.

Sounds like a french name

noisy, energetic, and cheerful; rowdy

Tessie was chosen to get stoned by the lottery

A veces pienso que la escritura es como una memoria que no se cansa. Las personas olvidan, cambian o se van, pero lo que queda escrito sigue ahí esperando a ser leído, eso me hace pensar que de cierta forma escribir es una manera de vencer al olvido. Sara Sarria.

Esta parte del texto muestra que Aristóteles era visto como alguien que sabía todo lo que se podía saber en su época, pero ese “todo” era muy limitado. En su tiempo, el conocimiento estaba restringido porque la escritura y el contacto con otros pueblos eran escasos, por eso, aunque fuera muy sabio, solo conocía lo que existía dentro de su pequeño mundo, la idea final resalta que el conocimiento siempre depende del contexto y que incluso los más sabios ignoran cosas que ni siquiera saben que existen. Allison Marentes Reyes

Me pareció un texto muy interesante porque reflexiona sobre cómo ha cambiado nuestra relación con el conocimiento a lo largo del tiempo. En la época de Aristóteles, era posible que una persona conociera casi todo lo que se sabía en su entorno, ya que el mundo que se conocía era limitado y la información circulaba de manera más sencilla. Hoy en día, vivimos rodeados de muchísima información a la que podemos acceder fácilmente gracias al internet y la tecnología. Aun así, tener tanta información también tiene su parte negativa, porque no siempre sabemos qué es cierto o qué vale la pena creer. Por eso, lo difícil ahora no es saberlo todo, sino aprender a entender bien lo que encontramos y usarlo de la mejor manera. Stephania Parra

En este párrafo se reconoce que las redes sociales transformaron nuestra manera de relacionarnos con la información, permitiendo que más voces puedan ser escuchadas, aunque a costa de perder una sensación compartida de realidad. Personalmente, considero que este cambio no puede juzgarse de forma completamente negativa ni positiva. Si bien es cierto que las redes han fragmentado la percepción colectiva y fomentado la desinformación, también democratizaron el acceso a la expresión y al debate público. En conclusión , su valor depende del uso que las personas hagan de ellas: son un reflejo de nuestras dinámicas sociales más que una causa directa de su deterioro.

La crítica me parece muy cierta porque la inteligencia artificial, aunque ayuda bastante, también puede hacer que dejemos de pensar por nosotros mismos. Si usamos un chatbot para todo, terminamos repitiendo lo que nos da sin entenderlo, y eso nos quita la capacidad de aprender o de resolver problemas por nuestra cuenta. El problema no es la herramienta, sino cuando dejamos que piense por nosotros. El texto invita a reflexionar sobre cómo la tecnología puede afectar nuestra forma de aprender y crear. No se trata de rechazar la inteligencia artificial, sino de usar su potencial sin perder el pensamiento crítico ni la creatividad humana, porque al final lo que nos diferencia es justamente nuestra manera de cuestionar y comprender lo que hacemos.

Laura Ximena Bolivar Roman

El texto plantea una preocupación real sobre cómo la inteligencia artificial está cambiando la forma de programar. Hoy en día muchas personas usan chatbots para generar código sin entender realmente cómo funciona, y eso puede ser peligroso a largo plazo. Los programadores con experiencia todavía corrigen esos errores, pero llegará un momento en que esas personas ya no estén. Entonces, ¿quién quedará con el conocimiento? Más que una crítica a la tecnología, el texto nos hace pensar en la importancia de no perder las habilidades humanas detrás del progreso digital. Daniel Camargo

Lo que me pareció más importante es cómo el texto muestra que a veces pensamos en innovación solo como avances tecnológicos súper complejos, como nuevo hardware mucho mas potente y eficiente o máquinas "futuristas" como robots animatronicos, pero se nos olvida que lo digital, como las redes sociales, también es una forma de innovación que cambia profundamente cómo vivimos, esas innovaciones digitales entran más fácil en nuestra cultura, se vuelven parte de lo cotidiano sin que nos demos mucha cuenta, y terminan transformando nuestras costumbres más rápido que muchos avances tecnológicos que parecen más “grandes”.

Jose Sebastian Mosquera Dediego

mo estudiante de arquitectura, este texto me resulta especialmente relevante porque plantea una reflexión que también atraviesa nuestro campo: hasta qué punto delegar en la tecnología puede limitar nuestras propias capacidades. Así como Sócrates temía que la escritura atrofiara la memoria, hoy podríamos decir que la inteligencia artificial puede atrofiar nuestra forma de pensar y crear. En arquitectura, depender completamente de herramientas digitales o de IA para diseñar puede hacernos perder el criterio, la intuición y la sensibilidad espacial que se construyen con la experiencia y el pensamiento crítico. El ensayo de Pablo Medina Uribe invita a usar la tecnología como apoyo, no como sustituto del aprendizaje ni del proceso creativo. JULIANA MORA

Estoy de acuerdo con esta parte del texto, es inevitable pensar que la inteligencia artificial va a cambiar muchos aspectos en nuestras vidas, y uno de ellos es nuestra habilidad de pensar criticamente, debido a que esta misma herramienta hará todo por nosotros sin tener que esforzarnos en pensar.

Estoy de acuerdo con esta afirmación porque aunque la IA ofrece muchas ventajas su uso excesivo puede generar una fuerte dependencia que afecte nuestras capacidades. Si dejamos que la IA piense, escriba, decida y solucione todo por nosotros poco a poco perderemos la habilidad de razonar, analizar y crear por nuestra propia cuenta "la historia demuestra que las herramientas deben complementar al ser humano, no remplazarlo". Ana Sofia Chacón Casasbuenas

A pesar de que Sócrates no confiaba en la escritura, esta termino siendo parte esencial de la vida humana. Con el tiempo, casi todas las sociedades la adoptaron y gracias a ella hoy podemos guardar y compartir conocimiento, comunicarnos rápidamente y hacernos la vida mas fácil.

Me parece muy importante esta frase, es mas valioso tener la fortuna de pensar por nosotros mismos. Siempre he pensado que la IA nunca podrá reemplazarnos, porque es incapaz de crear algo, sirve como motor de búsqueda, crea algo a partir de miles de cosas que ya existen, si, es una gran ventaja y por eso le es tan fácil "crear", pero nada como un cerebro humano investigando, haciendo lluvias de ideas, sacando inspiración de vivencias propias, sintiendo algo con tanta fuerza que pueda sacar una maravillosa pieza artística, grafica o escrita, la IA nunca podrá reemplazar esto. Si bien es posible que algún día aprendan a crear de ceros, nunca será un cerebro humano lleno de recuerdos, sentimientos, motivaciones y razones para pensar. Además recordemos que la IA siempre va a necesitar de un humano que le diga que hacer.

The attempt to replicate the actions of the powerful. Just like Haiti.

Greed is so powerful it made the French, English, and Spanish get along

This is a very particular kind of conquest, as the goal is to make it land for use by average people. It is also the only contemporary example I can think of where the imperialist and the conquered are in adjacent territories.

I didn't know this.

This is more traditional colonialism

This is very interesting because it isn't settler colonialism but rather the imposition of a one sided trading relationship through military force.

I think it's important to keep in mid technology is a big part of society now and even in school, but it is important to limit the use of AI with it.

Most students believe it should be limited and 10 percent of that thinks it should be banned.

This quote shows that some people do have succus with Ai and its a important part of the future

This quote shows how some people might be scared that AI might take over there lives and the world soon in a age of new Technology

This quote shows how much people don't know much about Ai and that they need to know because the world is changing

This quote shows that a bunch of students heavily rely on ChatGPT

Having it do your whole assignment you would spend more time trying to make it seem like AI didn't write it then you would just writing the assignment out.

I think its good to use AI when u need ur writing in a certain format or need punctuation. Or even to give a summary of an article and its key points. I think those are good way to use AI for coursework but having it do the whole assignment is not helping.

This quote shows how fear of being flagged online can stop students from using AI resources.

This is an excellent goal, and becoming culturally proficient as a coach is a must in order to be effective.

TEXT: The meaning of this text is saying that space between physical and spirituality are permeable. Then it goes on to bring up music in heaven and wondering if there is similarity here on earth, if there is no boundary between earth and heaven. The font of the next part is in a different font signifying the importance of it, it says "Reach down, hands of angels. Reach across, hands of humans", I feel like this text shows the permeable between heave and earth almost as if the angels will guide humans. Then it goes on to prove this when it says "which were sent from heaven, to cut the ropes that bind me?" signifying that the angles are being portrayed as saviors to humans. The text is written in a ancient typewriter font, tells us that the thin places are something that has been around.

COLOR:I feel like the image being black and white signifies that further. These colors contrast each other simply signifies timeless.

TEXT: The meaning of this text is saying that space between physical and spirituality are permeable. Then it goes on to bring up music in heaven and wondering if their is similarity here on earth, if their is no boundary between earth and heaven. The font of the next part is in a different font signifying the importance of it, it says "Reach down, hands of angels. Reach across, hands of humans", I feel like this text shows the permeable between heave and earth almost as if the angels will guide humans. Then it goes on to prove this when it says "which were sent from heaven, to cut the ropes that bind me?" signifying that the angles are being portrayed as saviors to humans. The text is written in a ancient typewriter font, tells us that the thin places are something that has been around.

COLOR:I feel like the image being black and white signifies that further. These colors contrast each other simply signifies timeless.

image: the image is of a person tied up and what seems to be angel maybe coming down to untie it and rescue it from what looks like the illuminati symbol in a cloud.

color: the entire picture is grey which could represent the colorless life the person feels since their binded but once their unbinded maybe color will come back into their life

Some Cherokees did in fact sell their land and move, which essentially weakened everyone else.

I like how they establish their ethos here. They sort of speak for all mothers here, calling on the authority of those who raised the warriors that are now in charge. Its clear that there is still a remnant of the former respect for women.

They are now at the point where they have to justify their inclusion on the matter.

They seem to believe the rhetoric about them. Perhaps they actually were flourishing as farmers idk.

This is a very cool debate to see. The mass scale, strictly defined individualism and liberalism vs. a smaller, more intimate and natural form of economy and governance based on solidarity and common belief.

True. Monroe did push for assimilation. Its very interesting that that call him "our father the president".

This is very interesting. I wonder why they believed in Monroe like this.

I never knew that this is what caused this event.

Jackson

Its so interesting how this happens. I guess ultimately no matter how successful you were as a non-white person, the shape of society was dictated by white people, and this is what success meant.

larger natural gas consumption as a challenge towards chicago

point towards nuclear power.

Why compare Denmark to Chicago

advice via Nashville Typewriter, a repair person. <br /> https://reddit.com/r/typewriters/comments/1o4qxvn/chasing_problems_stuck_keys/

**Autopoietic/Meta/Design-0rigo-folder * for Gyuri's 📓diary

/hyperpost/🌐/🎭/gyuri/📓/0/index.html

♖🌐.🌌💬.🚧.🤝🌐 HyperPost Development ColabSpace -

create/curate slef-documenting information pertaining to all development by the indy.player/actor 🎭/gyuri/ within the Web Hosted directory for the Peergos account: ♖/hyperpost part of on going 👨‍💻development ♒stream

drawing hands autopoiesis

text edit: Increase the reach of your scholarly work by publishing Open Access

I think this is very interesting and also accurate, although my personal experiences have been a bit different. I did not feel as if race was a huge factor in school until I cam to college, likely because I went to a predominately hispanic school with most of the instructors also being hispanic. But now at university, I see how race can play a huge role in the education system and how there are structures which go along racial lines, at times unrecognized.

basically saying that school is the only thing most people collectively do and they do for a long time.

Racial inequality has been so deeply engraved into people of color that now you're forced to compete not only against the oppressive system set up by the white elites but now youre placed into competiotn with other people of color

Some children can be aware while others are oblivious of the racism that is occurring because of its normalization in society. Those affected, like in this instance, are forced to accept that it is reality even when they specifically have done nothing to provoke except be born their race. Those inflicting the dance at times aren't aware of what theyre doing, especially children who are likely just copying the behavior of their parents. This is why there needs to be an emphasis on education to ensure all students are aware of their actions and history which took place.

I totally agree with this. Many people started to discriminate others based on their environment for friends that they make with. Because of the fact that you are immersed in an environment with lots of hatred to another race, it is barely possible for you not to discriminate that same race. Also, children stepped into their youth, they might get in many different new things such as smoking or vaping, and they think that copying others behavior might make themselves look cool. And I guess copying other people to discriminate another group of people also makes certain people feel like it’s a cool behavior. Hence, the problem lies with how to hinder children from copying inappropriate behaviors at their youth. And my answer to that question is school should educate them in advance by telling them what is wrong and what is right.

Speaking of standardized test, it is definitely most of the Chinese students strength. However, I would like to share phenomenon I see in my high school. I attended the top tier international school back in China, so most of the kids are very smart and hard-working. Therefore, many of the students who are in our school achieved a 1550+ SAT standardized score. For those that usually perform academic excellence in school subject, people would not doubt the authenticity of their score. However, for those who did not perform that well on school subjects, but did perform very well on the standardized testing would be doubted. When I was in the bathroom, I heard people saying a person must have been cheated on SAT because she is not good at school subjects, and that Asians are prone to be cheating. The point I want to make here is that there are rumors saying Asians love cheating, but this is not true, because it depends on individual‘s values. When I hear people of my same race targeted at our own race and play around with the typical stereotype as a joke, I got very angry. This is the same case as for many Black people. I watched a movie regarding slavery, and a line in the movie that stand out the most to me is “ we as the blacks must stop using the N word ourselves before anyone else start to respect us”. This should applies to all different races of people in order to win other peoples respect

The emotional and social burden carried by many racialized individuals to endure or downplay discrimination in order to be accepted in unfortunate. It highlights how racism doesn’t only operate through open hostility but also through subtle dynamics of power and belonging. The desire for social inclusion can force individuals to internalize or minimize harm, showing how oppression is often sustained through everyday interpersonal interactions rather than just overt acts of hatred.

At first, what seems like an isolated or neutral event can later be recognized as part of a larger pattern of racial targeting. This delayed recognition shows how systemic racism works not just through direct acts, but through its ability to be hidden and passive.

early racists experiences can deeply affect a childs self esteem and sense of belonging in turn causing kids to internalize their feelings of inadequacy

(Buying a Typewriter<br /> by [[Joe Van Cleave]] and [[Bob Marshall]] of Typewriter Muse on YouTube<br /> accessed on 2025-10-16T09:27:44

Bob Marshall prefers "extreme collector" to "hoarder" when it comes to typewriter collecting.

In July 20222, Bob Marshall was charging $60/hour for typewriter service.

Very good game

not daily budget, but project budget. Daily budget we create by sitting with production team beforehand.

Client fills out the form answering all the questions

identifies the person.

HMRC / BFI often raises questions. There for please consider rewrite it, like something below.

100% approval with or without queries. or All claims were approved without material queries

I prefer the detailed breakdown given in this website https://farm-cove-proposal.vercel.app/

Is this the on-set accountant? If yes, why only 1-2 days? If not, what would he/she do?

I think it's "from BFI certification to HMRC submission" not from BFI certification through HMRC submission.

I can't understand what this means? Is it referring to any supplier specific information or the invoices or the legitimacy of the supplier? How can a person be documented?

text: the text seems like something a preacher would say or something you could possible even find in a bible so the text is trying to portray or give the sense of a "holy" message.

color: the whole page beside the door and text is gold and when i think of gold i think of how the streets in heaven are gold streets like it says in the bible so i think the color is trying to show that this is a religious poster

Carbon Paper Duplication<br /> by [[OrcaType]] on YouTube<br /> accessed on 2025-10-16T09:17:15

Benefits of wax carbon paper over film-based carbon paper:<br /> - darker printing<br /> - better shelf-life

Benefits of film-based carbon paper over wax carbon paper:<br /> - crumple resistant<br /> - smudge resistant<br /> - sharper text over generations

https://www.youtube.com/watch?v=1vy2ht2t4pI

https://pmc.ncbi.nlm.nih.gov/articles/PMC9545458/

We included 69 patients with EoE variants. Endoscopic abnormalities were found in 53.6%. We identified three histological subtypes: EoE‐like esophagitis (36/69, 52.2%), lymphocytic esophagitis (14/69, 20.3%), and non‐specific esophagitis (19/69, 27.5%). Immunohistochemistry revealed—in contrast to EoE—no significant increase in inflammatory cell infiltrates compared with GERD and healthy controls, except for lymphocytes in lymphocytic esophagitis.

Could emphasize that since K is squared in the energy that for |k| > 0 the degeneracy is 2(2J+1) while it is 2J+1 for K = 0.

Should be rewritten to make clear degeneracy due to space frame of Mj quantum number.

the head word will determine the rest of the sentence structure

sometimes its not the obvious answer

The noun can change its class depending on how the sentence is written

Sigma

These two examples no longer work

When I see stuff like this, it enrages me beyond anything else because how are you gonna kill and rob and take from an entire race of people. Then say you found everything then NOT give them citizenship of their own land.

this gotta be the stupidest sentence i've ever read. The "Founders" took what they "discovered" and they killed the people that were here already MY ANCESTORS

Having context, previous learning and applying new knowledge is all crucial when growing.

加邦神父算是民主派

read

here

hope

counting

read

stop

power of theatre here

read. futility

repetition. the same stories, the attempt for justice

read

why antigone

then what

read here to hated you

This explains how detached gen Z is from the other generations

Form of coping mechanism.

Gen Z slang.

This paragraph in particular displays the emotion behind the Gen Z stare.

Reflects the struggles behind the Gen-Z stare.

this is explaining how gen z aren't showing good morals or smiling they just have a plain and serious face

I wager TikTok was the first to point this out because it's a Gen-Z orientated platform. The introduction also describes what the Gen-Z stare means, which I appreciate.

this talks about how recently people have noticed that Gen z have been less likely to smile and most likely to be emotinal

BUT not to the extent that Trazodone does!

read. sound the alarm. anakhita

Michaela

Luiza

Elizabeth, biases

end. then S2P pipelline PP

Read for a page. Avoidance.

Read then look at design

The struggle with Asian American identity is conflicting with the fact that assimilation does not equal acceptance. Even when individuals adapt to dominant cultural norms, systemic racism continues to mark them as outsiders. Racial identity is often shaped not by self-definition, but by how society chooses to perceive and categorize people.

this whole paragraph aims to show how a longstanding “white racial frame” sustains systemic racism and ranks racial groups, placing whites at the top and others, including Asian Americans, in lower positions.

Rather than being harmless descriptions, these so-called “observations” reflect how racism is packaged as common sense. By presenting stereotypes as factual traits, the media helped cement racial hierarchies and justify exclusionary actions. This tactic made prejudice seem rational and inevitable, allowing systemic discrimination to thrive under the guise of neutrality.

When Asian American history is left untaught, it silences entire communities and their struggles, reinforcing a false narrative that their experiences are peripheral or insignificant. The absence of these stories in classrooms actively shapes how people understand race and power, allowing stereotypes to fill the gaps where real history should be.

Rather than offering protection, it masks discrimination by suggesting Asian Americans don’t face real hardship. This narrative not only silences individual experiences with prejudice and mental health struggles but also reinforces existing racial hierarchies by setting Asian Americans apart from other marginalized groups. Ultimately, the label creates an illusion of privilege while deepening invisibility.

The author finds herself living through the very statistics she studies. The blurred personal and professional boundaries underscores how systemic issues like racism and mental health disparities directly impact the researcher's own life and community.

i think there should be time dedicated to connecting a teacher and student on a deeper level because it then gives the student reason to care in the classroom and the teacher a reason to teach properly.

although it wont let me highlight it this whole idea that schools take away your culture and language is so true. as a kid i had a stutter and instead of saying it was because i was a kid my school said it was because i was learning spanish and english at the same time. as such my parents worried i wouldnt have an adulthood in which white society accepted me stopped teaching me spanish.

The Dexamethasone Suppression Test measures whether your body can suppress cortisol after a synthetic glucocorticoid — and failure to suppress indicates stress-system overactivity, typical in melancholic depression.

i believe myself to be very lucky here. although i come from an extremely low income community i had the ability to go to a free charter school in which as opposed to history i was taught ethnic studies in which my teacher taught about the ways that white historians altered history to make them look more sophisticated and nicer

I think to how in school there were so many comprehension tests that ti a point it became less of a whos learning the most and more of a who can remember the most dates and names. i was learning nothing and just remembering things. without the ability to comprehend a writing reading is fruitless

set dressing to better represent the time period this was taking place in as this was a big deal back then to look ladylike.

evenwichtige aanpak

I feel like i can connect to this in a deeper level because although i have alot of education under my belt and tons of experience in various areas my speech has never been the most eloquent and thus ive been oftentimes seen as less intelligent than i truly am and can do nothing but act as though the belittling doesn't impact me

As a poli sci major ive gone through study after study that highlights inequalities at a systematic level and it’s in a way amazing to see the lengths that these inequalities can go in order to make sure communities of color are less likely to grow than white communities

Racial awareness begins with lived experience, school incidents often serve as those "events".

I think this line caputres how external percetion drives internal identity work.

Tatum roots racial identity in the universal adolescent search for self, showing how race becomes part of that process.

Agregar análisis

Propuesta y generación de índices y agregar una subsección de descriptivos

En vez de esto debería ir el esquema y más grande

Esto se va a la siguiente sección

Esto debería terminar acá

Este párrafo arriba de la tabla. Debería ser conceptualización en vez de esquema.

Tiene que estrucutrar el documento. Cómo está organizado: COntar que se parte hablando de la primera experiencia de medición de cohesión para américa latina, cómo se conceptualiza la cohesión horizontal; para luego plantear la emdición de cohesión de otro documento. Finalmente, medición de cohesión social longitudinal con elsoc.

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When a prokaryotic cell reproduces, its circular chromosome replicates, and the cell divides in a process called binary fission (Figure 2.5). Replication usually begins at a specific place on the circular chromosome, called the origin of replication. The origins of the two newly replicated chromosomes move away from each other and toward opposite ends of the cell.

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Edmund Quincy was an author, editor, and member of the Anti-Slavery Society. He edited several abolitionist journals such as The Abolitionist and The Liberator. The inclusion of his letter at the beginning of Brown's narrative lends credibility to the text, just like William Lloyd Garrison's letter at the beginning of Frederick Douglass's Narrative of the Life of Frederick Douglass.

Learn practical strategies to design AI systems that avoid bias and ensure fairness. Discover techniques like diverse data, transparent algorithms, and robust evaluation pipelines to build ethical AI.

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Appeler un taxi ? :)

eLife Assessment

This useful study attempts to place an ancient maize sample from Bolivia, dated to the end of the Incan empire, in genetic and geographical context. The analyses show that this sample is most closely related to ancient Peruvian maize, but the data remain inadequate to determine the direction of dispersal and the extent of Inca influence over the genetic make up of the analyzed sample. There are additional deficiencies in the statistical analyses and selection inferences. The topic of the study would appeal to researchers studying maize dispersal and adaptation.

Reviewer #1 (Public review):

Summary:

In this manuscript, authors describe a good quality ancient maize genome from 15th century Boliva and try to link the genome characteristics to Inca influence. Overall, the revised manuscript is still below the standard in the field. While dating of the sample and the authentication of ancient DNA has been evidenced robustly, the downstream genetic analyses do not support the conclusion that genomic changes can be attributed to Inca influence. There is more story telling than story testing in this manuscript, analyses are not robust and possibly of very narrow interest.

Strengths:

Technical data related to the maize sample are robust. Radiocarbon dating strongly evidenced sample age, estimated to around 1474 AD. Authentication of ancient DNA has been done robustly. Spontaneous C-to-T substations which are present in all ancient DNA are visible in reported sample with the expected pattern. Despite low fraction of C-to-T at the 1st base, this number could be consistent with cool and dry climate in which the sample was preserved. The distribution of DNA fragment sizes is consistent with expectations for sample of this age.

Weaknesses:

(1) The geographic placement of the sample based on genetic data is not robust. To make use of the method correctly, it would be necessary to validate that genetic samples in this region follow the assumption of the 'isolation-by-distance' with dense sampling, which has not been done. Without this important information, we do not know if genetic similarity is influenced by demographic events and/or selection. The analysis is not a robust evidence of sample connectivity.

(2) The conclusion that Ancient Andean maize is genetically similar to European varieties and hence share similar evolutionary history is not well supported. PCA plot in Fig. 4 merely represents sample similarity based on two components (jointly responsible for about 20% of variation explained). Contrary to authors' conclusion, the direct test of similarity using outgroup f3 statistic does not support that European varieties are particularly closely related to ancient Andean maize. These levels of shared drift could be due ancient Andean maize relationship with other related groups, such as ancient or modern Brazil. A relationship test between multiple populations would be necessary to show significant direct relationship between ancient Andean maize and European maize.

(3) The conclusion that selection detected in aBM sample is due to Inca influence has no support. Firstly, selection signature can be due to environmental or any other factors. To disentangle those, authors would need to generate the data for a large number of samples from similar cultural context and from a wide-ranging environmental context followed by a formal statistical test. Secondly, allele frequency increase can be attributed to selection or demographic processes, and alone is not a sufficient evidence for selection. Presented XP-EHH method seems unsuitable for single individual. Overall, methods used in this paper raise some concerns: i) how accurate are allele-frequency tests of selection when only single individual is used as a proxy for a whole population, ii) the significance threshold has been arbitrary fixed to an absolute number based on other studies, but the standard is to use, for example, top fifth percentile.

In sum, this manuscript presents new data that seem to be of high quality, but the analyses are frequently inappropriate and/or over-interpreted.

Reviewer #2 (Public review):

I am glad to see a revised version of the manuscript. The authors have successfully handled some of my comments, but others require additional attention. In particular, the dataset seems quite robust and valuable to publish, and the descriptive analysis of its position relative to other modern and ancient genomes is generally sound. The selection analyses remain unsupported, and should be removed from the paper. In addition, I agree with the other reviewers and reiterate my comment that the Locator analysis is not robust.

As I said in my original review, the XP-EHH method is not applicable to pseudohaploid variant calls in a single individual. This method is simply not appropriate to apply to the data at hand, as the method relies on knowledge of diploid genotypes, usually phased, and the results from this test are not robust. It is possible that the XP-EHH method could be extended to this data type or genotype likelihoods with extensive validation and conditioning on a large reference panel, but in general haplotype-based approaches have not been extensible to low-coverage pseudohaplotype datasets. At any rate, any off-the-shelf implementation is inappropriate and unsupported. I am sorry to be this negative about this analysis, but it cannot be used as presented, the results from using it in this way would be spurious by definition.

In addition, identifying GO terms without statistical assessment of enrichment is not a robust analysis, nor is selecting genes with a high proportion of rare alleles without extensive additional contextualization based on the expectations of neutrality and deviations potentially tied to selection. For this reason, the two genes linked with height traits have no support here as genuinely being targets of selection. It is a frustrating reality for us in the ancient DNA field that small numbers of highly degraded genomes offer extremely limited scope for selection analyses, but that's the unfortunate state of play, and is the situation here.

My other major critique remains the application of the Locator method. As Reviewer 1 notes, this method must be built on a densely sampled dataset with strong isolation by distance, which is not done here. The authors explained their approach with more detail in their response, but it is fundamentally inappropriate for this dataset. It does not add anything more than the f3 analysis, and creates a falsely precise inference of genetic-geographic origins that is not supported.

Per authors' response to my previous recommendation 6, it is not advisable to re-map the reads after damage masking, and doing this with a conservative hard-masking approach will lead to a high mismatch rate and significant loss of reads in BWA. This could also exacerbate reference sequence bias which is already a major challenge for ancient DNA (see Gunther et al 2019 PLoS Genet). The correct approach is to map reads, mask or rescale for damage, and then proceed with the modified alignment file. In response to Reviewer 3's comment 3, the authors also refer to a "0 mismatch alignment" strategy. This is not concordant with the damage analysis, and if they truly do not allow mismatches this would be very inadvisable, as it would allow an extreme reference sequence bias.

Author response:

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

Reviewer #1 (Public review): 

Summary: 

In this manuscript, the authors describe a good-quality ancient maize genome from 15th-century Bolivia and try to link the genome characteristics to Inca influence. Overall, the manuscript is below the standard in the field. In particular, the geographic origin of the sample and its archaeological context is not well evidenced. While dating of the sample and the authentication of ancient DNA have been evidenced robustly, the downstream genetic analyses do not support the conclusion that genomic changes can be attributed to Inca influence. Furthermore, sections of the manuscript are written incoherently and with logical mistakes. In its current form, this paper is not robust and possibly of very narrow interest. 

Strengths: 

Technical data related to the maize sample are robust. Radiocarbon dating strongly evidenced the sample age, estimated to be around 1474 AD. Authentication of ancient DNA has been done robustly. Spontaneous C-to-T substitutions, which are present in all ancient DNA, are visible in the reported sample with the expected pattern. Despite a low fraction of C-to-T at the 1st base, this number could be consistent with the cool and dry climate in which the sample was preserved. The distribution of DNA fragment sizes is consistent with expectations for a sample of this age. 

Weaknesses: 

Thank you for all your thoughtful comments. See below for comments on each.

(1) Archaeological context for the maize sample is weakly supported by speculation about the origin and has unreasonable claims weighing on it. Perhaps those findings would be more convincing if the authors were to present evidence that supports their conclusions: i) a map of all known tombs near La Paz, ii) evidence supporting the stone tomb origins of this assemblage, and iii) evidence supporting non-Inca provenance of the tomb. 

We believe we are clear about what information we have about context.  First, the intake records from the MSU Museum from 1890 are not as detailed as we would like, but we cannot enhance them. The mummified girl and her accoutrements, including the maize, came from a stone tower or chullpa south of La Paz, in what is now Bolivia. We do not know which stone chullpa, so a map would be of limited use.  The mortuary group is identified as Inca, but as we note the accoutrements do not appear of high status, so it is possible that she is not an elite.  Mud tombs are normally attributed to the local population, and stone towers to Inca or elites. We have clarified at multiple places in the text that the maize is from the period of Inca incursion in this part of Bolivia and have modified text to reflect greater uncertainty of Inca or local origin, but that selection for environmentally favorable characteristics had taken place.  Regardless, there are three 15th c CE or AD AMS ages on the maize, a cucurbita rind, and a camelid fiber.  The maize is almost certainly mid to late 15th century CE.

(2) Dismissal of the admixture in the reported samples is not evidenced correctly. Population f3 statistic with an outgroup is indeed one of the most robust metrics for sample relatedness; however, it should not be used as a test of admixture. For an admixture test, the population f3 statistic should be used in the form: i) target population, ii) one possible parental population, iii) another possible parental population. This is typically done iteratively with all combinations of possible parental populations. Even in such a form, the population f3 statistic is not very sensitive to admixture in cases of strong genetic drift, and instead population f4 statistic (with an outgroup) is a recommended test for admixture. 

We have removed “Our admixture f3-statistics test results suggest aBM is not admixed” in our revised manuscript. Since our goal here is to identify which group(s) has(have) the highest relatedness with aBM, so population f3 statistic with an outgroup is the most robust metric to do the test and to support our conclusion here.

(3) The geographic placement of the sample based on genetic data is not robust. To make use of the method correctly, it would be necessary to validate that genetic samples in this region follow the assumption of the 'isolation-by-distance' with dense sampling, which has not been done. Additionally, the authors posit that "This suggests that aBM might not only be genetically related to the archaeological maize from ancient Peru, but also in the possible geographic location." The method used to infer the location is based on pure genetic estimation. The above conclusion is not supported by this method, and it directly contradicts the authors' suggestion that the sample comes from Bolivia.  

We understood that it is necessary to validate the assumption of the 'isolation-by-distance' with dense sampling. But we did not do it because: 1) the ancient maize age ranges from ~5000BP to ~100BP and they were found in very different countries at different times. 2) isolation-by-distance is a population genetic concept and it's often used to test whether populations that are geographically farther apart are also more genetically different. Considering we only have 17 ancient samples in total our sample size is not sufficient for a big population test.

For "It directly contradicts the authors' suggestion that the sample comes from Bolivia.”, as we described in our manuscript that “Given the provenience of the aBM and its age, it is possible the samples were local or alternatively were introduced into western highland Bolivia from the Inca core area – modern Peru.” The sample recording file did show the aBM sample was found in Bolivia, but we do not know where aBM originally came from before it was found in Bolivia. To answer this question, we used locator.py to predict the potential geographic location that aBM may have originally come from, and our results showed that the predicted location is inside of modern Peru and is also very close to archaeological Peruvian maize.  

Therefore, our conclusion that "This suggests that aBM might not only be genetically related to the archaeological maize from ancient Peru, but also in the possible geographic location” does not contradict that the sample was found Bolivia.

(4) The conclusion that Ancient Andean maize is genetically similar to European varieties and hence shares a similar evolutionary history is not well supported. The PCA plot in Figure 4 merely represents sample similarity based on two components (jointly responsible for about 20% of the variation explained), and European samples could be very distant based on other components. Indeed, the direct test using the outgroup f3 statistic does not support that European varieties are particularly closely related to ancient Andean maize. Perhaps these are more closely related to Brazil? We do not know, as this has not been measured. 

Our conclusion is “We also found that a few types of maize from Europe have a much closer distance to the archaeological maize cluster compared to other modern maize, which indicates maize from Europe might expectedly share certain traits or evolutionary characteristics with ancient maize. It is also consistent with the historical fact that maize spread to Europe after Christopher Columbus's late 15th century voyages to the Americas. But as shown, maize also has diversity inside the European maize cluster. It is possible that European farmers and merchants may have favored different phenotypic traits, and the subsequent spread of specific varieties followed the new global geopolitical maps of the Colonial era”.

We understood your concerns that two components only explain about 20% of the variation. But as you can see from the Figure 2b in Grzybowski, M.W. et al., 2023 publication, it described that “the first principal component (PC1) of variation for genetic marker data roughly corresponded to the division between domesticated maize and maize wild relatives is only 1.3%”. It shows this is quite common in maize, especially when the datasets include landraces, hybrids, and wild relatives. For our maize dataset, we have archaeological maize data ranging from ~5,000BP to ~100BP, and we also have modern maize, which makes the genetic structure of our data more complicated. Therefore, we think our two components are currently the best explanation currently possible. We also included PCA plot based on component 1 and 3 in Fig4_PCA13.pdf. It does not show that the European samples are very distant.

For “Perhaps these are more closely related to Brazil?”, thank you for this very good question, but we apologize that we cannot answer this question from our current study because our study focuses on identifying the location where aBM originally came from, establishing and explaining patterns of genetic variability of maize, with a specific focus on maize strains that are related to our current aBM. Thus, we will not explore the story between maize from Brazil and European maize in our current study.

(5) The conclusion that long branches in the phylogenetic tree are due to selection under local adaptation has no evidence. Long branches could be the result of missing data, nucleotide misincorporations, genetic drift, or simply due to the inability of phylogenetic trees to model complex population-level relationships such as admixture or incomplete lineage sorting. Additionally, captions to Figure S3, do not explain colour-coding.  

We have removed “aBM tends to have long branches compare to tropicalis maize, which can be explained by adaption for specific local environment by time.” in our revised manuscript.

We have added the color-coding information under Fig. S3 in our revised manuscript.

(6) The conclusion that selection detected in aBM sample is due to Inca influence has no support. Firstly, selection signature can be due to environmental or other factors. To disentangle those, the authors would need to generate the data for a large number of samples from similar cultural contexts and from a wide-ranging environmental context, followed by a formal statistical test. Secondly, allele frequency increase can be attributed to selection or demographic processes, and alone is not sufficient evidence for selection. The presented XP-EHH method seems more suitable. Overall, methods used in this paper raise some concerns: i) how accurate are allele-frequency tests of selection when only single individual is used as a proxy for a whole population, ii) the significance threshold has been arbitrary fixed to an absolute number based on other studies, but the standard is to use, for example, top fifth percentile. Finally, linking selection to particular GO terms is not strong evidence, as correlation does not imply causation, and links are unclear anyway. 

In sum, this manuscript presents new data that seems to be of high quality, but the analyses are frequently inappropriate and/or over-interpreted. 

Regarding your suggestion that “from similar cultural contexts and from a wide-ranging environmental context, followed by a formal statistical test”, we apologize that this cannot be done in our current study because we could not find other archaeological maize samples/datasets that are from similar cultural contexts.

For “Secondly, allele frequency increase can be attributed to selection or demographic processes, and alone is not sufficient evidence for selection.” Yes, we agree, and that’s why we said it “inferred” the conclusion instead of “indicated”. Furthermore, we revised the whole manuscript following all reviewers’ comments and reorganized and reduced the part on selection on aBM.

For “The presented XP-EHH method seems more suitable”, we do not think XP-EHH is the best method that could be used here because we only have one aBM sample, but XP-EHH is more suitable for a population analysis.

For “Finally, linking selection to particular GO terms is not strong evidence, as correlation does not imply causation, and links are unclear anyway.”, as we described in our manuscript, our results “inferred” instead of “indicated” the conclusion.

Reviewer #2 (Public review): 

Summary: 

The manuscript presents valuable new datasets from two ancient maize seeds that contribute to our growing understanding of the maize evolution and biodiversity landscape in pre-colonial South America. Some of the analyses are robust, but the selection elements are not supported. 

Strengths: 

The data collection is robust, and the data appear to be of sufficiently high quality to carry out some interesting analytical procedures. The central finding that aBM maize is closely related to maize from the core Inca region is well supported, although the directionality of dispersal is not supported. 

Weaknesses: 

Thank you for your comments and suggestions. See below for responses and explanations.

The selection results are not justified, see examples in the detailed comments below. 

(1) The manuscript mentions cultural and natural selection (line 76), but then only gives a couple of examples of selecting for culinary/use traits. There are many examples of selection to tolerate diverse environments that could be relevant for this discussion, if desired. 

We have added related examples with references supported in our revised manuscript.  

(2) I would be extremely cautious about interpreting the observations of a Spanish colonizer (lines 95-99) without very significant caveats. Indigenous agriculture and food ways would have been far more nuanced than what could be captured in this context, and the genocidal activities of the Europeans would have impacted food production activities to a degree, and any contemporaneous accounts need to be understood through that lens.  

We agree with the first part of this comment and have softened our use of this particular textual material such that it is far less central to interpretation.While of interest, we cannot evaluate the impact of colonial European activities or observational bias for purposes of this analysis.

(3) The f3 stats presented in Figure 2 are not set up to test any specific admixture scenarios, so it is unsupported to conclude that the aBM maize is not admixed on this basis (lines 201-202). The original f3 publication (Patterson et al, 2012) describes some scenarios where f3 characteristics associate with admixture, but in general, there are many caveats to this approach, and it's not the ideal tool for admixture testing, compared with e.g., f4 and D (abba-baba) statistics.  

You make an important point that f3 stats is not the ideal tool for admixture testing. Since our study goal here is to identify which group(s) has(have) the highest relatedness with aBM, the population f3 statistic with an outgroup is the most robust metrics with which to do the test and to support our conclusion here. We have removed the “Our admixture f3-statistics test results suggest aBM is not admixed” in our revised manuscript.

(4) I'm a little bit skeptical that the Locator method adds value here, given the small training sample size and the wide geographic spread and genetic diversity of the ancient samples that include Central America. The paper describing that method (Battey et al 2020 eLife) uses much larger datasets, and while the authors do not specifically advise on sample sizes, they caution about small sample size issues. We have already seen that the ancient Peruvian maize has the most shared drift with aBM maize on the basis of the f3 stats, and the Locator analysis seems to just be reiterating that. I would advise against putting any additional weight on the Locator results as far as geographic origins, and personally I would skip this analysis in this case.  

As we described in our manuscript, we have 17 archaeological samples in total. Please find more detailed information from the “geographical location prediction” section.

We cannot add more ancient samples because they are all that we could find from all previous publications. We may still want to keep this analysis because f3 stats indicates the genome similarity, but the purpose of locator.py analysis is indicating the predicted location of origin of a genetic sample by comparing it to a set of samples of known geographic origin. 

(5) The overlap in PCA should not be used to confirm that aBM is authentically ancient, because with proper data handling, PCA placement should be agnostic to modern/ancient status (see lines 224-226). It is somewhat unexpected that the ancient Tehuacan maize (with a major teosinte genomic component) falls near the ancient South American maize, but this could be an artifact of sampling throughout the PCA and the lack of teosinte samples that might attract that individual.  

We have removed “which supports the authenticity of aBM as archaeological maize” in our revised manuscript. The PCA was only applied for all maize samples, so we did not include any teosinte samples in the analysis.

(6) What has been established (lines 250-251) is genetic similarity to the Inca core area, not necessarily the directionality. Might aBM have been part of a cultural region supplying maize to the Inca core region, for example? Without a specific test of dispersal directionality, which I don't think is possible with the data at hand, this is somewhat speculative. 

We added this and re-wrote this part in our revised manuscript.

(7) Singleton SNPs are not a typical criterion for identifying selection; this method needs some citations supporting the exact approach and validation against neutral expectations (line 278). Without Datasets S2 and S3, which are not included with this submission, it is difficult to assess this result further. However, it is very unexpected that ~18,000 out of ~49,000 SNPs would be unique to the aBM lineage. This most likely reflects some data artifact (unaccounted damage, paralogs not treated for high coverage, which are extremely prevalent in maize, etc). I'm confused about unique SNPs in this context. How can they be unique to the aBM lineage if the SNPs used overlap the Grzybowski set? The GO results do not include any details of the exact method used or a statistical assessment of the results. It is not clear if the GO terms noted are statistically enriched.  

We have added references 53 and 54 in our revised manuscript, and we also uploaded the Datasets S2 and S3.

For “I'm confused about unique SNPs in this context. How can they be unique to the aBM lineage if the SNPs used overlap the Grzybowski set?”, as we described in our materials and method part that “To achieve potential unique selection on aBM, we calculated the allele frequency for each SNPs between aBM and other archaeological maize, resulting in allele frequency data for 49,896 SNPs. Of these,18,668 SNPs were unique to aBM.”  Thus, the unique SNPs for aBM came from the comparison between aBM with other archaeological maize, and we did not use any modern maize data from the Grzybowski set.

For “The GO results do not include any details of the exact method used or a statistical assessment of the results. It is not clear if the GO terms noted are statistically enriched.” We did not do GO Term enrichment, so there are no statistical assessments for the results. What we have done was we retained the GO Terms information for each gene by checking their biological process from MaizeGDB, after that, we summarized the results in Dataset S4.

(8) The use of XP-EHH with pseudo haplotype variant calls is not viable (line 293). It is not clear what exact implementation of XP-EHH was used, but this method generally relies on phased or sometimes unphased diploid genotype calls to observe shared haplotypes, and some minimum population size to derive statistical power. No implementation of XP-EHH to my knowledge is appropriate for application to this kind of dataset. 

We used the same XP-EHH as this publication “Sabeti, P.C. et al. Genome-wide detection and characterization of positive selection in human populations. Nature 449, 913-918 (2007).” Specifically in our analysis, the SNP information of modern maize was compared with ancient maize. The code is available in https://doi.org/10.5061/dryad.w6m905qtd.

XP-EHH is a statistical method used in population genetics to detect recent positive selection in one population compared to another, and it often applied in modern large maize populations in previous research. In our study, we wanted to detect recent positive selection in modern maize compared to ancient maize, thus, we applied XP-EHH here. Although the population size of ancient maize is not big, it is the best method that we can apply for our dataset here to detect recent selection on modern maize.

Reviewer #3 (Public review): 

Summary: 

The authors seek to place archaeological maize samples (2 kernels) from Bolivia into genetic and geographical context and to assess signatures of selection. The kernels were dated to the end of the Incan empire, just prior to European colonization. Genetic data and analyses were used to characterize the distance from other ancient and modern maize samples and to predict the origin of the sample, which was discovered in a tomb near La Paz, Bolivia. Given the conquest of this region by the Incan empire, it is possible that the sample could be genetically similar to populations of maize in Peru, the center of the Incan empire. Signatures of selection in the sample could help reveal various environmental variables and cultural preferences that shaped maize genetic diversity in this region at that time. 

Strengths: 

The authors have generated substantial genetic data from these archaeological samples and have assembled a data set of published archaeological and modern maize samples that should help to place these samples in context. The samples are dated to an interesting time in the history of South America during a period of expansion of the Incan empire and just prior to European colonization. Much could be learned from even this small set of samples. 

Weaknesses: 

Many thanks for your comments and suggestions.  We have addressed these below and provided further explanation.

(1) Sample preparation and sequencing: 

Details of the quality of the samples, including the percentage of endogenous DNA are missing from the methods. The low percentage of mapped reads suggests endogenous DNA was low, and this would be useful to characterize more fully. Morphological assessment of the samples and comparison to morphological data from other maize varieties is also missing. It appears that the two kernels were ground separately and that DNA was isolated separately, but data were ultimately pooled across these genetically distinct individuals for analysis. Pooling would violate assumptions of downstream analysis, which included genetic comparison to single archaeological and modern individuals. 

We did not do the morphological assessment of the samples and comparison to morphological data from other maize varieties because we only have 2 aBM kernels, and we do not have other archaeological samples that could be used to do comparison.

For “It appears that the two kernels were ground separately and that DNA was isolated separately, but data were ultimately pooled across these genetically distinct individuals for analysis”, as you can see from our Materials and Methods section that “Whole kernels were crushed in a mortar and pestle”, these two kernels were ground together before sequenced. 

While morphological assessment of the sample would be interesting, most morphological data reported for maize are from microremains (starch, phytoliths, pollen) and this is beyond the scope of our study. Most studies of macrobotanical remains do not appear to focus solely on individual kernels, but instead on (or in combination with) cob and ear shape, which were not available in the assemblage.

(2) Genetic comparison to other samples: 

The authors did not meaningfully address the varying ages of the other archaeological samples and modern maize when comparing the genetic distance of their samples. The archaeological samples were as old as >5000 BP to as young as 70 BP and therefore have experienced varying extents of genetic drift from ancestral allele frequencies. For this reason, age should explicitly be included in their analysis of genetic relatedness. 

We have changed related part in our revised manuscript.

(3) Assessment of selection in their ancient Bolivian sample: 

This analysis relied on the identification of alleles that were unique to the ancient sample and inferred selection based on a large number of unique SNPs in two genes related to internode length. This could be a technical artifact due to poor alignment of sequence data, evidence supporting pseudogenization, or within an expected range of genetic differentiation based on population structure and the age of the samples. More rigor is needed to indicate that these genetic patterns are consistent with selection. This analysis may also be affected by the pooling of the Bolivian archaeological samples.  

We do not think it is because of poor alignment of sequence data since we used BWA v0.7.17 with disabled seed (-l 1024) and 0 mismatch alignment. Therefore, there are no SNPs that could come from poor alignment. Please see our detailed methods description here “For the archaeological maize samples, adapters were removed and paired reads were merged using AdapterRemoval60 with parameters --minquality 20 --minlength 30. All 5՛ thymine and 3՛ adenine residues within 5nt of the two ends were hard-masked, where deamination was most concentrated. Reads were then mapped to soft-masked B73 v5 reference genome using BWA v0.7.17 with disabled seed (-l 1024 -o 0 -E 3) and a quality control threshold (-q 20) based on the recommended parameter61 to improve ancient DNA mapping”.

For “More rigor is needed to indicate that these genetic patterns are consistent with selection”, Could you please be more specific about which method or approach we should use here? For example, methods from specific publications that could be referenced? Or which specific tool could be used?

“This analysis may also be affected by the pooling of the Bolivian archaeological samples.” As we could not prove these two seeds came from two different individual plants, we do not think this analysis was affected by the pooling of the Bolivian archaeological samples.

(4) Evidence of selection in modern vs. ancient maize: In this analysis, samples were pooled into modern and ancient samples and compared using the XP-EHH statistic. One gene related to ovule development was identified as being targeted by selection, likely during modern improvement. Once again, ancient samples span many millennia and both South, Central, and North America. These, and the modern samples included, do not represent meaningfully cohesive populations, likely explaining the extremely small number of loci differentiating the groups. This analysis is also complicated by the pooling of the Bolivian archaeological samples. 

Yes, it is possible that ovule development might be a modern improvement. We re-wrote this part in our revised manuscript.

Reviewer #1 (Recommendations for the authors): 

My suggestion is to address the comments that outline why the methods used or results obtained are not sufficient to support your conclusions. Overall, I suggest limiting the narrative of Inca influence and framing it as speculation in the discussion section. Presenting conclusions of Inca influence in the title and abstract is not appropriate, given the very questionable evidence. 

We agree and have changed the title to “Fifteenth century CE Bolivian maize reveals genetic affinities with ancient Peruvian maize”.

Reviewer #2 (Recommendations for the authors): 

(1) Line 74: Mexicana is another subspecies of teosinte; the distinction is between ssp. mexicana and ssp. parviglumis (Balsas teosinte), not mexicana and teosinte. 

We have corrected this in our revised manuscript.

(2) Line 100-102: This is a bit confusing, it cannot have been a symbol of empire "since its first introduction", since its introduction long predates the formation of imperial politics in the region. Reference 17 only treats the late precolonial Inca context, while ref 22 (which cites maize cultivation at 2450 BC, not 3000 BC) makes no reference to ritual/feasting contexts; it simply documents early phytolith evidence for maize cultivation. As such, this statement is not supported by the references offered.

lines 100-102. This point is well taken and was poor prose on our part.  We have modified this discussion to reflect both the confusing statement and we have corrected our mistake in age for reference 22. associated prose has been modified accordingly.

We have corrected them as “Indeed, in the Andes, previous research showed that under the Inca empire, maize was fulfilled multiple contextual roles. In some cases, it operated as a sacred crop” and “…since its first introduction to the region around 2500 BC”.

(3) Line 161: IntCal is likely not the appropriate calibration curve for this region; dates should probably be calibrated using SHCal.  

We greatly appreciate this important (and correct) observation. We have completely recalibrated the maize AMS result based on the southern hemisphere calibration curve, discussed the new calibrations, and have also invoked two other AMS dates also subjected to the southern hemisphere calibration on associated material for comparison.We are confident in a 15th century AD/CE age for the maize, most likely mid- to late 15th century.  

(4) Lines 167-169: The increase of G and A residues shown in Supplementary Figure S1a is just before the 5' end of the read within the reference genome context, and is related to fragmentation bias - a different process from postmortem deamination. Deamination leads to 5' C->T and 3' G->A, resulting in increased T at 5' ends and increased A at 3' ends, and the diagnostic damage curve. The reduction of C/T just before reads begin is not a result of deamination. 

We have removed the “Both features are indicative of postmortem deamination patterns” in our revised manuscript.

(5) Lines 187-196 This section presents a lot of important external information establishing hypotheses, and needs some references.  

We have added the related references here.

(6) Line 421: This makes it sound like damage masking was done BEFORE read mapping. However, this conflicts with the previous paragraph about map Damage, and Supplementary Figure 1 still shows a slight but perceptible damage curve, which is impossible if all terminal Ts and As are hard-masked. This should be reconciled.  

The Supplementary Figure 1 shows the raw ancient maize DNA sample before damage masking. Specifically, Step1: We used map Damage to check/estimate if the damage exists, and we made the Supplementary Figure 1. Step 2: Then we used our own code hard-masked the damage bases and did read mapping.

The purpose of Supplementary Figure 1 is to show the authenticity of aBM as archaeological maize. Therefore, it should show a slight but perceptible damage curve.

(7) Line 460: PCA method is not given (just the LD pruning and the plotting).  

The merged dataset of SNPs for archaeological and modern maize was used for PCA analysis by using “plink –pca”.

(8) "tropicalis" maize is not common usage, it is not clear to me what this refers to. 

We have changed all “tropicalis maize” as “tropical maize” in our revised manuscript.

(9) The Figure 4 color palette is not accessible for colorblind/color-deficient vision.  

We have changed the color of Figure 4. Please find the new colors in our upload Figure 4.

(10) Datasets S2 and S3 are not included with this submission. 

Thank you for letting us know and your suggestion. We have included Datasets S2 and S3 here.

This makes me think about how silence can be a political act. By refusing to engage, the UDC tries to preserve its version of history through absence rather than argument. Can a society truly move forward if those who created harmful narratives refuse to speak or take accountability?

This transformation raises questions about who “owns” history and how public spaces can evolve when the people demand new meanings. When citizens transform monuments like this, are they erasing history or creating a more honest version of it?