should be written: for any input 'r', there is only one output 'A'. Comma castatstrophe. Use commas to set off extra information.

The original problem doesn't name inputs as 'q' and 'r', nor output 'n'.

a link here where I could open these up in a different browser tab would be useful here. I hate to have to leave my place in this online book and have to figure out where I left off when I get back. Better yet, it's an online text, why not just put the information here too. The original problem doesn't name inputs as 'q' and 'r', nor output 'n'.

A good exercise here would be to come up with the equation of the graphed function. (x-1)^2=f(x) f(-1)=4 (-1,4), f(3)=4 (3,4)

This information should have been included in the beginning table examples.

This information is clearly wrong.

Species - Typical Short‑Term Memory - Long‑Term Memory

Puppy Hours to days Months to years

Adult Dog Hours to days Months to years

Cat Hours to days Months to years

Goldfish Minutes to hours Months to years

Betta Fish Minutes to hours Weeks to months

This information is clearly wrong.

Species - Typical Short‑Term Memory - <br /> Long‑Term Memory

Puppy Hours to days <br /> Months to years

Adult Dog Hours to days <br /> Months to years

Cat Hours to days <br /> Months to years

Goldfish Minutes to hours <br /> Months to years

Betta Fish Minutes to hours <br /> Weeks to months

A better description of how one would determine whether something is a function based on two variables that appear to be dependent on each other would make sense here. It would seem that they are functions of each other when written this way. Reference to the following example is not a factor in this statement.

Why am I finding h(4) in the solution?

We could include here that f(a) is contained within the output of f(a+h) and substitute it to show that it =f(a)+h^2+2ah+3h

we can apply algebra and find that for (a+4)(a-1), a=1, -4

It is unclear whether height could be a function of the age.

Franz Kafka- German Language Jewish Czech writer known for "Metamorphosis","The Trial", "The Castle"

Known for writing "Bastard Out of Carolina", Short story collection "Trash", and "Two or Three Things I know for Sure"

wrote "Pilgram at Tinker Creek","An American Childhood", and "The Writing Life"

it is very helpful to provide reasoning to questions, or background, before asking them. so, students can understand the question and create answers more thoughtfully.

When put in a group, students can see other standpoints to the same question. And it is enlightening to see where your peers stand on the matter.

the curriculum is based on the student's personal interest, besides textbooks.

Kids that are seen as misbehaved are incredibly intelligent because they are questioning authority.

I understand why some teachers may be stuck in this thought process. Because they are so used to the traditional way of teaching, it may be a little out of the ordinary to switch something up suddenly.

I am incredibly fond of this strategy.

rather than trying to make your questions too important, make sure the questions the kids are asking get special attention.

This is such a restricted way to "teach" if even.

What I take from this is that it's easier to demand attention rather than grabbing people's attention. Although it is much more beneficial to try and grab peoples attention.

It's okay to not know what questions to ask as a teacher. But even if you don't know the questions to ask, it is a good learning experience for both the students and the teacher.

Recently, I've experienced professors "eliciting" questions, and I realized it makes students think harder .

Instead of taking your time creating new ideas, you're stuck trying to figure out ideas that were already created.

I agree with this, but sometimes it's necessary to know the right answer.

what does this mean? lol.

spiraling upwards. as in it gets better and better the more you figure out ways to ask and answer questions.

it is okay for questions to be more open ended, especially if you want whoever you're asking the question to, to think more critically.

Ive experienced "fun" questions and then I've experienced these types of questions... depends on my mood to tell you which one fits me more.

testing

This paper was super cool, we are now seeing certain species have increased competition advantage as human impacts increase. It interesting to hear about the raccoon dog an animal i'm not as familiar with and how this convergent evolution could be a scary sign.

Synanthropic species would be super cool area of study. I wonder how subspecies relationships work among more humanized animals vs wild animals?

This is super interesting to think about and the context it gives in invasive species. I wonder if there is a correlation between generalized strategies across invasive species. As rare ecosystems are threatened by climate change will the spread of generalized plants and animals into less diverse ecosystems mean they are even harder to manage.

This line was my favorite line to read. The way sounds so sexual, but it really isn't. Because obviously, you can't plunge one's tongue to another's heart. But the way this is about knowing someone on more than just a surface level. In fact, it is about knowing someone so deeply and intimately that the only way it can be described is by this imagery.

I believe this line is speaking to things like work, getting promoted, alcohol, food, sex, and the constant want of needing more things. These things seem normal, but the narrator wants us to take a deeper look these things are not 'us' just simply a category we try to fit into.

I like this line but it also confused me. Based off of the previous line I wonder if this is the narrator being angry with his clothing? Because in the context of the previous line he mentions a disguise and then being naked.

I really found this part interesting because to be it feels as though once the reader learns nature, they will possess poetry within. The earth is poetry but don't take the writers words for it--go out there and find it.

Cox-1 is constitiutionally expressed. Cox-2 is for inflammation.

Cox-2 inhibitors reduce gastric side effects, increase MACE, similar kidney outcomes.

The Increased MACE may be a class effect (except aspirin), and should be avoided post PCI and post bypass/ and in elderly at risk.

Anchoring what this paper is about This is a subject I am familiar with but it never hurts to review and maybe learn something new.

eLife Assessment

This study presents a valuable finding on impacts of the common driver mutations APC, KRAS G12D, and TP53 in murine colorectal organoids. In particular, the authors examine how the order of APC and TP53 acquisition influences tumor phenotype. The evidence supporting the claims of the authors is solid. The work will be of interest to scientists working in the field of breast cancer.

Reviewer #2 (Public review):

Summary:

This study addresses an important and timely question in colorectal cancer biology by systematically examining the effects of the common driver mutations APC, KRAS G12D, and TP53 in murine colorectal organoids, with particular emphasis on how the order of APC and TP53 acquisition influences tumor phenotype. These mutations are well known to be frequent, truncal, and often co-occurring in colorectal cancer. While it is increasingly appreciated that mutational order can shape tumor behavior, studies directly comparing the phenotypic consequences of alternative APC-TP53 mutation orders remain rare. This work therefore addresses a relevant and timely question.

Strengths:

A major strength of the study is its focus on previously unexplored biology, combined with the generation of multiple isogenic murine organoid models with controlled mutational sequences. The authors employ careful and robust quality control of the CRISPR-mediated alterations, and the inclusion of both in vitro and in vivo experiments strengthens the relevance of the work.

Weaknesses:

There are, however, several limitations that should be considered when interpreting the findings. First, KRAS G12D activation is used as the initiating alteration, whereas APC loss is generally believed to be the initiating event in most human colorectal cancers. Second, the analysis is restricted to comparing only two mutation orders (KAT versus KTA), which limits the breadth of conclusions that can be drawn about mutation ordering more generally. Finally, key RNA-sequencing and in vivo experiments rely on a limited number of isogenic lines, which constrains interpretability.

The study aimed to systematically investigate how the accumulation and sequence of driver mutations influence colorectal cancer initiation. The data provide intriguing evidence that the relative timing of APC and TP53 loss may impact tumor initiation and survival in a hostile microenvironment. However, given the limited number of biological replicates, these observations should be interpreted with caution and would benefit from further validation.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this study, Li et al. used genetically engineered murine intestinal organoids to investigate how the temporal order of oncogenic mutations influences cell state and tumourigenicity of colorectal epithelial cells. By sequentially introducing Apc and Trp53 loss-of-function mutations in alternate orders within a Kras^G12D background, the authors generated isogenic organoid lines for both in vitro and in vivo characterisation. Bulk RNA-seq reveals expected transcriptional changes with relatively modest differences between the two triple-mutant configurations (KAT vs KTA). The key finding emerges from transplantation assays: while KAT and KTA organoids show equivalent tumourigenic potential in immunodeficient mice, only KAT organoids form tumours in immunocompetent hosts (5/10 vs 0/10), suggesting that mutation order shapes susceptibility to immune-mediated clearance. The experiments are well-executed, and the conclusions are generally supported by the data.

Strengths:

The experimental system is well-designed for the question. By combining a Kras^G12D transgenic background with sequential CRISPR-mediated knockout of Apc and Trp53 in alternate orders, the authors generated truly isogenic organoid lines that differ only in mutational sequence. This is technically non-trivial and provides a clean platform for dissecting order effects, a question otherwise difficult to address experimentally.

The authors performed comprehensive baseline characterisation of these organoids, including morphological and histological assessment, quantification of organoid-forming efficiency and proliferation, and bulk RNA-seq profiling. While these analyses revealed no major differences between KAT and KTA organoids, and the observed enhancement of epithelial stemness upon Apc loss and proliferative advantage conferred by Trp53 loss are largely expected, the systematic nature of this characterisation establishes a useful methodological template for future organoid-based studies.

The authors further investigated the functional impact of mutational order using subcutaneous transplantation assays. By comparing tumour formation in immunodeficient versus immunocompetent hosts, the authors uncover a genuinely unexpected finding: KAT and KTA organoids behave equivalently in the absence of adaptive immunity, but diverge dramatically when immune pressure is applied (KAT: 5/10; KTA: 0/10). This observation is arguably the most compelling aspect of the study and opens an interesting line of inquiry.

We greatly appreciate your comments on this study.

Weaknesses:

The authors acknowledge that initiating with Kras^G12D does not reflect the typical human sporadic CRC trajectory, where APC loss is usually the first event. While this design choice was pragmatic, it means the observed order effects are contextualised within an artificial starting point. It remains unclear whether the Apc/Trp53 order would matter in a Kras-wild-type background, or whether the Kras-driven cellular state is a prerequisite for these phenotypes to emerge.

We agree with the reviewer that initiating tumorigenesis with Kras^G12D does not fully recapitulate the most common trajectory of sporadic human CRC, where APC loss typically occurs first. We had noted this point in the original Discussion and further clarified it more explicitly in the Introduction part of the revised manuscript as shown in Line 97–103.

Our experimental design was intended to establish a controlled and genetically tractable system to interrogate the principle of mutation order effects. In this context, Kras^G12D activation provides a stable oncogenic baseline that facilitates sequential genome engineering and comparison of isogenic lines.

Although APC loss is frequently the initiation event, a recent study has suggested that Kras^G12D priming can reshape the selective landscape for subsequent driver events, including Apc alterations (PMID: 41339549). Consistent with this notion, our data indicate that Kras^G12D activation induces a permissive oncogenic cellular state that may influence the phenotypic consequences of later mutations. We therefore speculate that the Kras^G12D-primed context may contribute to the observed order-dependent effects.

We agree that testing Apc Trp53 order in a Kras-wild-type background would be an important future direction, and we have pointed this out explicitly in the revised Discussion as shown in Line 549–554.

Subcutaneous implantation provides a tractable readout of tumourigenicity, but the cutaneous immune microenvironment differs substantially from that of the intestinal mucosa. Given that the central claim concerns immune-mediated selection, orthotopic transplantation would more directly test whether the observed order effects hold in a physiologically relevant context.

In the present study, we employed subcutaneous transplantation as a widely used platform to assess tumorigenic potential under controlled immune conditions. This approach offers high reproducibility, straightforward tumour monitoring, and has been broadly applied in organoid-based cancer studies in both immunodeficient (PMID: 23273993, 23776211, 32209571, 33055221) and immunocompetent (PMID: 32209571, 33055221, 41672595) settings.

Importantly, our primary goal was to determine whether mutation order influences susceptibility to immune-mediated clearance, rather than to model the full complexity of the intestinal niche. The clear divergence between KAT and KTA specifically in immunocompetent hosts supports the existence of intrinsic mutation order-dependent immune vulnerability.

Nevertheless, we fully agree with the reviewer that orthotopic transplantation would provide a more physiologically relevant immune microenvironment and represents also an important direction for future investigation. We have explicitly discussed this limitation and highlight orthotopic validation as an important future direction in the revised Discussion as shown in Line 563–571.

The ssGSEA comparison involves only 14 ATK tumours, and the key comparisons (Figure 6E) yield borderline significance (p=0.052). More fundamentally, since mutation order cannot be inferred from the clinical samples, the authors are correlating organoid-derived IFN signatures with tumour immunophenotypes without direct evidence that these patients' tumours followed a KAT-like trajectory. The reasoning becomes circular: KAT organoids define the signature used to identify KAT-like clinical tumours.

We thank the reviewer for raising this important point. We would like to clarify that our intention was not to infer the actual mutation order in clinical samples, which indeed cannot be reliably reconstructed from bulk tumour RNA-seq data.

Instead, our goal was to determine whether the transcriptional programs distinguishing KAT and KTA organoids could be observed in human CRC cohorts. In this context, the organoid-derived IFN-related signature was used as a molecular reference to assess potential clinical correlation, rather than to classify tumours by evolutionary trajectory.

We agree that the statistical significance in Figure 6E is modest (p = 0.052), and we have revised the text (Line 478–480) to present this analysis more cautiously as a suggestive trend rather than definitive evidence. We also clarified this limitation explicitly in the revised manuscript (Line 537–542) to avoid overinterpretation.

Furthermore, the most striking finding of the study, that KTA organoids fail to form tumours in immunocompetent hosts while KAT organoids can, lacks a mechanistic follow-up. The transcriptomic differences between KAT and KTA are modest when cultured as monocultures, yet their in vivo fates diverge dramatically. The authors do not address why these subtle intrinsic differences translate into such divergent immune susceptibility, nor do they characterise the immune response adequately (beyond limited CD4/CD8 IHC at tumour peripheries).

We thank the reviewer for this important point. We agree that the mechanistic basis underlying the differential immune susceptibility between KAT and KTA remains incompletely resolved.

A practical limitation of the current study is that KTA grafts failed to establish tumours in immunocompetent hosts, which precluded downstream histological and immune profiling of established lesions. As a result, our in vivo immune characterization of KTA grafts is nearly impossible.

Nevertheless, our transcriptomic analyses indicate that KAT and KTA organoids differ in interferon-response and immune-related programs prior to transplantation, and those differentially expressed genes were consistently preserved in tumour cells derived from immunodeficient hosts. These results suggest the presence of intrinsic tumour-cell-autonomous differences may influence immune recognition or clearance.

We have expanded the Discussion to outline several non-mutually exclusive mechanisms that could account for this phenotype, including altered interferon responsiveness, differential antigen presentation capacity, and changes in tumour cell-intrinsic immune escape programs (Line 527–533). These hypotheses are consistent with the transcriptional differences observed prior to transplantation and provide a framework for future mechanistic investigation. We agree that deeper immune profiling (e.g., immune infiltrate composition, antigen presentation status, and functional immune assays) will be important to fully elucidate the mechanism and represents a key direction for future work.

Reviewer #2 (Public review):

Summary:

This study addresses an important and timely question in colorectal cancer biology by systematically examining the effects of the common driver mutations APC, KRAS G12D, and TP53 in murine colorectal organoids, with particular emphasis on how the order of APC and TP53 acquisition influences tumor phenotype. These mutations are well known to be frequent, truncal, and often co-occurring in colorectal cancer. While it is increasingly appreciated that mutational order can shape tumor behavior, studies directly comparing the phenotypic consequences of alternative APC-TP53 mutation orders remain rare. This work, therefore, addresses a relevant and timely question.

Strengths:

A major strength of the study is its focus on previously unexplored biology, combined with the generation of multiple isogenic murine organoid models with controlled mutational sequences. The authors employ careful and robust quality control of the CRISPR-mediated alterations, and the inclusion of both in vitro and in vivo experiments strengthens the relevance of the work.

We greatly appreciate your comments on this study.

Weaknesses:

There are, however, several limitations that should be considered when interpreting the findings. First, KRAS G12D activation is used as the initiating alteration, whereas APC loss is generally believed to be the initiating event in most human colorectal cancers.

We sincerely thank the reviewer for their insightful comments regarding the initiation of tumorigenesis with a Kras mutation rather than the more canonical Apc loss, which was also raised by the reviewer #1. We fully agree that the Apc-first represents the most prevalent sequence in human colorectal cancer (CRC), We have more clearly explained the rationale for our experimental design in the revised Introduction part as outlined in our response to reviewer #1.

Second, the analysis is restricted to comparing only two mutation orders (KAT versus KTA), which limits the breadth of conclusions that can be drawn about mutation ordering more generally.

We thank the reviewer for this critical concern, which we agree is essential for strengthening the robustness and generality of our findings. However, as a proof-of-concept study of Apc and Trp53 loss, two major oncogenic events in CRC, serves as a biologically meaningful starting point for dissecting order-dependent effects. Although it is of great significance to compare all six possible mutation orders of these three driver genes, generating and thoroughly characterizing all genotypes (with identical replicates) represents a substantial undertaking beyond the scope of this initial study.

Finally, key RNA-sequencing and in vivo experiments rely on a single isogenic line, which substantially constrains interpretability.

The aim of the study was to systematically investigate how mutation accumulation and order influence colorectal cancer initiation. While the data suggest that the relative timing of APC and TP53 loss may be particularly important for tumor initiation, the absence of biological replication makes it difficult to draw robust conclusions. Engraftment efficiency and tumor behavior can be influenced by many factors for a single clone, including additional passenger mutations acquired during culturing, as well as epigenetic differences that are independent of the engineered mutations.

We thank the reviewer for this concern. We apologize that we have not made a clear presentation of our data source. Indeed, for all major in vitro and in vivo assays of double and triple mutants, we analyzed at least two independently derived clones per genotype. These independent clones harbour distinct mutations in target genes and were treated as biological replicates throughout the study.

To improve clarity and transparency, we have revised the relevant figure legends and further provided a Table S5 to explicitly indicate the clonal origin of each data point throughout the study.

eLife Assessment

This important study shows that prenatal alcohol exposure produces lasting changes in amyloid precursor protein processing, including altered APP C-terminal fragments and Notch intracellular domain levels, that persist into adulthood and track with progressive spatial learning and memory deficits, in both wild-type and 3xTg-AD mice. The convincing study was carefully designed and combined biochemical, histological, and behavioral approaches across multiple ages. The work will interest researchers studying fetal alcohol spectrum disorders, Alzheimer's disease risk, and the developmental origins of neurodegeneration. Nonetheless, reviewers identified opportunities to strengthen the conclusions with additional samples and controls.

Reviewer #1 (Public review):

Summary:

The manuscript by Montenegro and colleagues reports a uniquely significant set of compelling results from a carefully designed study. The findings are fundamental and should substantially advance our understanding of whether prenatal exposure to high levels of alcohol produces neural changes that are precursors to the development of Alzheimer-like pathology in an animal model of Fetal Alcohol Spectrum Disorder (FASD). The quest was to test whether prenatal exposure to high-dose alcohol in the mouse would result in selective damage that would result in Alzheimer disease-like cellular disorder and mnemonic impairment. Both outcomes emerged and were exacerbated in relevant transgenic mice. The untoward effect on memory endured and even worsened with age.

Strengths:

The authors noted the importance of using a validated animal model to test their hypotheses related to AD-like outcomes because human postmortem data are unavailable and even in vivo data are limited to younger FASD cohorts. The authors further noted limitations, which also anticipate experiments that could chart the temporal course of the effect and windows of prenatal alcohol exposure that result in damage or resilience. In short, as the authors state on lines 435-7, "These data provide the first experimental evidence that developmental alcohol exposure impacts core proteolytic pathways central to AD/ADRD pathogenesis."

Weaknesses:

Addressing the following would clarify several points in an already well-written paper:

(1) It would be useful to have a timeline of the study, much like the one provided for the water maze protocol. On that timeline, please include the sample sizes examined, ages at exposure, and other pertinent procedures, indicating which animals remained alive for testing, etc.

(2) What were the attrition rates for each study group?

(3) What are the human age equivalents of the maternal mice?

(4) It would be helpful to see a graph of the BECs of each animal relative to the doses given. That would clarify how the alcohol exposure amount and timing are the same and where they are different for all exposed mice, given that the alcohol levels were somewhat different by group, as noted in the Methods. Were these BEC differences at all related to group differences in outcome measures or memory performance?

Reviewer #2 (Public review):

Summary:

In this study, the impact of prenatal alcohol (PAE) on amyloid precursor protein (APP) C-terminal fragments and notch intracellular domain (NICD) levels in adulthood is measured in 3xTg-AD mice.

Prenatal alcohol alters gamma secretase activity with development and aging. This could have implications for Alzheimer's disease risk in populations without inherited Alzheimer's risk genetics.

Strengths:

Strengths include the model, the use of orthogonal approaches, and the rigorous, high-quality data.

Weaknesses:

Some figures lack prenatal alcohol treatment in the 3xTg-AD mice.

Some overstatements should be tempered. For instance, one cannot conclude that the changes in CTFs are driving the changes in learning and memory (as suggested in the last line of the abstract) without a direct intervention testing this. For instance, though PAE caused a more robust learning deficit at 6 mo in WT, the impact on CTFs was less than it was at 3 mo. PAE did not significantly change CTFs or learning/memory in 3xTg-AD mice at 4 months, suggesting the genotype effect takes over at this point. The text should be adjusted to reflect this.

Conclusion:

In summary, this is a rigorous assessment of the long-term impacts of PAE on CTFs and learning/memory in adult WT and 3xTg-AD mice.

Reviewer #3 (Public review):

Summary:

The goal of this study was to test the hypothesis that prenatal alcohol exposure (PAE) can affect Alzheimer's disease (AD) pathogenesis using biochemical proxies, histology, and a behavioral paradigm sensitive to AD-related memory decline. Major strengths include the breadth of techniques used, consideration of different AD-related molecular markers, and the use of different ages as well as appropriate controls. The authors largely achieved their aims to show that PAE does affect amyloid precursor fragments (CTFs) and notch signaling very early on as well as long-term effects in adulthood, which may uncover a previously underappreciated mechanism that may contribute to AD-related neuropathology and behavioral outcomes during lifespan.

Strengths:

(1) Several techniques are used to address molecular, behavioral, and histological PAE-related changes.

(2) There is use of appropriate controls and an AD-relevant mouse model.

(3) Different ages are used to address age-related and long-term effects in AD and control mice.

(4) The novelty of results shows early changes in amyloid-related processes, affected by PAE.

Weaknesses:

(1) It is unclear as to whether there are sex differences, particularly in the adult cohort.

(2) More clarity is needed on sample size per cohort and whether mice that were used for anatomy and biochemical analyses were previously used for behavior. Including a table and noting any overlap would be useful.

(3) In many instances, two-way ANOVAs with treatment (PAE vs vehicle) and genotype as factors will be useful to report (e.g., Figure 1).

(4) In Figure 5 and line 253, it is stated that older mice have more severe deficits, but there are no direct statistical comparisons with younger AD mice.

(5 Lines 270-271 refer to mice as "presymptomatic", but these mice do have behavioral symptoms. Do the authors mean no neuropathology yet? Any data showing lack of robust neuropathology would be useful.

Author response:

Public Reviews:

Reviewer #1 (Public review):

(1) It would be useful to have a timeline of the study, much like the one provided for the water maze protocol. On that timeline, please include the sample sizes examined, ages at exposure, and other pertinent procedures, indicating which animals remained alive for testing, etc.

We agree with the reviewer that a timeline would be helpful for clarifying the PAE exposure paradigm and the subsequent sample collection and processing. Sample sizes vary across the different analyses; therefore, we have indicated the sample size for each experiment in the corresponding figure. To further improve clarity, we will add Author response image 1, which will include a schematic of the overall experimental timeline, including the PAE regimen, collection time points, and sample processing, as shown below.

Author response image 1.

(2) What were the attrition rates for each study group?

We thank the reviewer for raising this important point. There was no attrition of animals within the experimental groups; the number of animals included at the beginning and end of the study remained the same. However, we did observe a reduction in litter size following prenatal alcohol exposure (PAE). In our 3xTg-AD colony, litters typically consisted of approximately 8 pups under control conditions, whereas PAE litters occasionally contained 4–6 pups. Thus, the reduction in animal numbers reflects decreased litter size associated with PAE rather than attrition during the study. We would also like to clarify that the primary scope of this study was not to provide a terminal/end-point analysis of disease progression, but rather to investigate the emergence of Alzheimer’s disease (AD)-related phenotypes during early adulthood following PAE. Accordingly, our longitudinal experimental design focused on identifying the earliest molecular, synaptic, behavioral, and neuropathological alterations that emerge during this period. This approach allowed us to examine whether PAE accelerates or precipitates the onset of AD-related symptomatology in the 3xTg-AD model, rather than following the animals through advanced disease stages. We will clarify this rationale in the revised manuscript.

(3) What are the human age equivalents of the maternal mice?

We appreciate the reviewer’s question regarding the age of the maternal mice. The dams used in our study were young adult females (2 to 3 months of age) at the time of breeding. Because chronological age does not translate linearly between mice and humans, particularly during development and reproductive maturation, we have avoided assigning a precise human-age equivalent. Based on established comparative developmental frameworks and calculations, these animals represent a 20 years old young-adult in the reproductive stage, rather than an advanced maternal-age condition [1]. We will clarify this point in the revised manuscript.

(4) It would be helpful to see a graph of the BECs of each animal relative to the doses given. That would clarify how the alcohol exposure amount and timing are the same and where they are different for all exposed mice, given that the alcohol levels were somewhat different by group, as noted in the Methods. Were these BEC differences at all related to group differences in outcome measures or memory performance?

We appreciate the reviewer’s suggestion to provide a more detailed representation of the BEC data. We agree that displaying the BECs for individual animals would provide additional clarity regarding the consistency of alcohol exposure across groups. We have therefore included the individual BEC values in the new Figure 1. We observed some variability in BECs between the 3xTg-AD and B6129 groups, as noted in the Methods. Importantly, however, the average alcohol consumption was comparable between the two genotypes, indicating that the difference in BECs was not due to differences in the amount of alcohol consumed. All dams in the PAE groups reached BECs above 0.08 g/dL, the commonly used legal blood alcohol concentration limit in the United States, supporting the use of our paradigm as a binge-like alcohol exposure model. We further examined whether the variability in BECs was associated with the differences observed in outcome measures, including memory performance. We did not find evidence that the differences in BECs accounted for the group differences in behavioral or molecular outcomes. Thus, although some intergroup variability in BECs was present, the overall alcohol exposure was comparable, and the observed phenotypic differences were not attributable to differences in alcohol consumption.

Reviewer #2 (Public review):

(1) Some figures lack prenatal alcohol treatment in the 3xTg-AD mice.

We appreciate the reviewer’s observation and agree that the rationale for the different experimental groups across the figures should be clarified. The primary focus of this study is to characterize the effects of prenatal alcohol exposure (PAE) in wild-type B6129 mice, with the 3xTg-AD mice serving primarily as a disease-model reference to determine whether the effects observed following PAE in wild-type animals overlap with or resemble features of AD pathology. Accordingly, the initial figures focus on the effects of PAE in B6129 mice and include the non-exposed 3xTg-AD group as a reference for the AD phenotype. The last two figures specifically address the effects of PAE in the 3xTgAD model, with the 3xTg-AD mice becoming the experimental subject of interest rather than serving solely as a disease reference. For this reason, the PAE-3xTg-AD group is not included in the earlier figures, whereas it is included in the final two figures where the effect of PAE on the AD model is directly evaluated. We will clarify this experimental rationale in the revised manuscript and figure legends.

(2) Some overstatements should be tempered. For instance, one cannot conclude that the changes in CTFs are driving the changes in learning and memory (as suggested in the last line of the abstract) without a direct intervention testing this. For instance, though PAE caused a more robust learning deficit at 6 mo in WT, the impact on CTFs was less than it was at 3 mo. PAE did not significantly change CTFs or learning/memory in 3xTg-AD mice at 4 months, suggesting the genotype effect takes over at this point. The text should be adjusted to reflect this.

We appreciate the reviewer’s careful consideration of this point. We agree that the relationship between APP CTF accumulation and learning and memory deficits should not be interpreted as causal in the absence of a direct intervention experiment. We were careful in choosing the wording throughout the manuscript to describe these findings as associated changes rather than evidence of a causal interaction. Our data demonstrate the presence of APP CTF accumulation and learning and memory deficits following PAE, but they do not establish that CTF accumulation directly drives the behavioral phenotype. We therefore will temper the language in the Abstract and throughout the manuscript to avoid overstatement. We also acknowledge that the relationship between these phenotypes is not necessarily linear across age: although PAE produced a more pronounced learning deficit at 6 months in B6129 mice, the magnitude of APP CTF accumulation was greater at the earlier time point. Importantly, we consider the possibility that the greater APP CTF accumulation observed at earlier ages may represent an early molecular insult whose functional consequences become evident later in life. In this context, the temporal dissociation between the molecular and behavioral phenotypes could be consistent with a “two-hit” model, in which an early-life insult induced by PAE creates or primes a pathological vulnerability that subsequently manifests as cognitive dysfunction with ageing [2]. We recognize, however, that this interpretation remains a hypothesis and would require longitudinal mechanistic studies to establish. This temporal relationship may also contribute to the broader concept of early-life origins of AD/ADRD, suggesting that prenatal environmental exposures may initiate molecular alterations during neurodevelopment that remain detectable or predispose the brain to later-life dysfunction. Similarly, the absence of significant changes in APP CTFs or learning and memory in 4-month-old 3xTg-AD mice suggests that the effects of the AD genotype may become dominant at this stage. Consistent with this interpretation, we state in the Discussion that future studies are needed to identify and experimentally test the direct molecular pathways affected by PAE that ultimately contribute to learning and memory impairment. We will revise the text accordingly to make this distinction clear while highlighting the potential significance of an early molecular insult preceding the later emergence of behavioral phenotypes.

Reviewer #3 (Public review):

(1) It is unclear as to whether there are sex differences, particularly in the adult cohort.

We appreciate the reviewer’s comment regarding potential sex differences. We agree that considering sex as a biological variable is important, particularly for the adult cohorts. To address this point, we will include identifying marks for male and female animals separately in our plots where sample size permits. This will allow the reader to better evaluate potential sex-dependent effects of PAE and to determine whether the observed phenotypes are consistent across sexes. We will also clarify this approach in the revised manuscript.

(2) More clarity is needed on sample size per cohort and whether mice that were used for anatomy and biochemical analyses were previously used for behavior. Including a table and noting any overlap would be useful.

We appreciate the reviewer’s suggestion and agree that greater clarity regarding the sample sizes and use of animals across analyses is important. The sample size for each cohort and experimental group is indicated in the corresponding figures, and we will make this information more explicit in each figure legend to facilitate interpretation. Animals that underwent behavioral testing were subsequently used for biochemical analyses, allowing us to examine molecular changes in the same animals in which behavioral phenotypes were characterized. In contrast, for the neonatal cohort, we performed both anatomical and biochemical analyses, to assess the distribution and extent of APP CTF accumulation across the brain during this early developmental period. This approach was selected to provide a broader assessment of the spatial distribution of APP CTF accumulation at birth. We will clarify these experimental details in the revised Methods and figure legends.

(3) In many instances, two-way ANOVAs with treatment (PAE vs vehicle) and genotype as factors will be useful to report (e.g., Figure 1).

We appreciate the reviewer’s suggestion regarding the use of two-way ANOVA with treatment and genotype as factors. However, we respectfully disagree that this approach is appropriate for all of the analyses presented in this manuscript. Our experimental design and the specific biological questions addressed in each experiment were not uniform across cohorts. In particular, the primary objective of the study was to characterize the effects of PAE in B6129 mice, with the 3xTg-AD mice serving primarily as a disease-model reference, while the effects of PAE in the 3xTg-AD model were specifically examined in the later experiments. Therefore, combining genotype and treatment as factors across all datasets would not always reflect the experimental questions or the structure of the cohorts. In addition, some experiments did not include all four groups, making a two-way ANOVA inappropriate for those analyses. Nevertheless, we agree that a two-way ANOVA may be informative for experiments in which both genotype and treatment are fully represented and the experimental design supports this analysis. We will therefore consider and apply two-way ANOVA, where appropriate, to those datasets, including evaluation of the main effects of genotype and treatment and their interaction. We will clarify the statistical approach and its rationale in the revised Methods and figure legends.

(4) In Figure 5 and line 253, it is stated that older mice have more severe deficits, but there are no direct statistical comparisons with younger AD mice.

We appreciate the reviewer’s observation. We agree that, in the absence of a direct statistical comparison between age groups, the statement that older mice have “more severe deficits” may be too strong. Our intention was to describe the apparent progression of the phenotype across age rather than to imply that we had statistically demonstrated an age-dependent increase in severity. We have therefore revised the text in Figure 5 and at line 253 to use more cautious language, describing the greater magnitude of the observed deficits in older mice without implying a direct statistical comparison between age groups. We agree that a formal conclusion regarding age-dependent progression would require a statistical analysis, which we will include in the revised manuscript.

(5) Lines 270-271 refer to mice as "presymptomatic", but these mice do have behavioral symptoms. Do the authors mean no neuropathology yet? Any data showing lack of robust neuropathology would be useful.

We appreciate the reviewer’s careful observation. We agree that the term “presymptomatic” was not sufficiently precise, particularly because the mice already exhibit measurable behavioral alterations at this age. Our intention was not to suggest that these animals were free of phenotypic abnormalities, but rather that they were at an early stage of disease progression, before the emergence of robust neuropathological features. We have therefore revised the terminology to avoid referring to these mice as “presymptomatic.” Instead, we describe them as being in an early stage of disease development, characterized by emerging behavioral and molecular alterations but without the extensive cardinal neuropathology typically associated with later stages of the 3xTg-AD phenotype. We agree that the distinction between behavioral symptoms and neuropathological progression is important. In this study, our focus was on the emergence of early AD-related phenotypes during young adulthood, rather than on establishing the absence of neuropathology. We have therefore avoided making a definitive claim regarding the lack of neuropathology and have revised the text to more accurately reflect the scope of our data.

Additional References

(1) Dutta, S. & Sengupta, P. Men and mice: Relating their ages. Life Sci. 152, 244–248 (2016).

(2) Gunn, J. S. et al. Exploring the ‘Multiple-Hit Hypothesis’ of Neurodegenerative Disease: Bacterial Infection Comes Up to Bat’. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1, 138 (2019).

plato directly explains the symbols: the cave = world of sight; the journey upward = the soul gaining knowledge

After learning the truth, the freed people struggles to fit back into the old world

plato directly explains the symbols: the cave = world of sight: the journey upward = the soul gaining knowledge

learning the truth can be uncomfortable and confusing at first

shows how easily people can mistake appearance or copy for the real thing

they mistake the shadows for reality because that is all they know

represents appearance false knowledge and not the full truth

represents people being trapped by what they have always believed

represents ignorance and a limited view of reality

Hegemony.

Even though they know stealing is wrong, they still want the pineapples. Tortoise's gossip became a punishment that actually changed their behavior.

The story shows that embarrassment and gossip can be used to discourage people from doing wrong.

I understand why Squirrel is upset because she thought being forgiven meant the situation was over.

Is this really forgiveness if Tortoise is still telling everyone about what she did?

Tortoise shows that he is willing to forgive Squirrel.

Squirrel realizes she went too far and is asking for another chance.

Tortoise is trying to find another way to stop Squirrel since he can't fight her.

Squirrel has an advantage because Tortoise can't reach her.

Squirrel knows she is doing something wrong but continues anyway.

I think Tortoise is being reasonable because he gives Squirrel a chance to stop before taking stronger action.

Tortoise now knows exactly who is causing the problem.

Squirrel's actions are causing Tortoise to lose money and make his farm less valuable.

This is worse than just eating a pineapple because they are ruining food that could have been sold or given away.

I understand that they are hungry, but they are taking advantage of Tortoise's farm.

Tortoise's farm is the main location where the conflict happens.

The research food technologist is would be a pretty cool career. I didn't even know this was a job. I think the part of going to space would be scary but seeing all the living things up there would be so cool.

This surprised me because I thought AI was more advanced than this, and I didn’t think it could or would make up information while making it seem completely real. This further shows why it is important to fact-check information from AI, especially when the information is used for something important.

This was something that stood out to me because I did not know that AI could create information that is biased or misleading. I think that this is important to know because it shows us that we shouldn’t automatically believe everything AI tells us. We should check the information before we use it or believe it.

This is what surprised me the most in this article because I did not know that implicit bias was something that happens to everyone. I used to think that bias was something that only certain people had, but now I know that our thoughts and decisions can be influenced by things around us without us even realizing it.

This stood out to me because it shows that people can be influenced by things without even realizing that they are affecting their decisions. I think this is important to the main point because the article explains that implicit bias can happen even when someone is trying to make a fair decision.

I found this interesting because Wallace explains how people can slowly become controlled by things like money, power, or being seen as smart without realizing it. We can get so used to wanting certain things that we stop questioning why they matter so much to us. I think his point is that we should pay attention to what we are making important in our lives instead of just automatically following those desires.

This is one of the main ideas I got from the speech. We cannot always control what happens around us, but we can have some control over how we react to it. If we automatically assume everything is terrible, we are probably going to have a bad experience. Wallace is saying that being aware gives us the opportunity to look at the same situation differently.

This example stood out to me because we never really know what someone else is going through. The woman yelling at her kid might seem rude and annoying, but Wallace gives another possibility that completely changes how we see her. It shows that sometimes we judge people based on one moment without knowing the whole story

I think this part is important because Wallace shows how easy it is to automatically assume that other people are annoying or getting in our way. We usually only see the situation from our own point of view. He is saying that we can stop and think about what other people might be dealing with instead of immediately judging them.

I think this is one of the main ideas of the whole speech. Sometimes things can be right in front of us and we still don't notice them because we are so used to them. It makes me think about how people can get caught up in their normal routines without stopping.

In this quote, "what man or deity Has carried off your child" refers to how Hecta, goddess of witch craft and sorcerer, ask Demeter who would be dumb enough to kidnap her daughter. For it we'll known that Demet love her daughter so dearly she refused all who ask for Persephone's hand in marriage. So to learn that the reason for Demeter cry was because of someone had kidnap Persephone, was a great surprise.

This refers to Gaia, the Titan of earth and grandmother to the five children of Rhea and Kronos. She allows mortals to plant and walk upon her land or body with the agreement of Zeus.

"Will of Zeus" means that if things happen like a good harvest or rain has appeared during a long drought Zeus allow this including allowing men to be kings and allowing them to rule. This is examples of Will of Zeus.

PLA has huge correlation to credential completion

repair callout

sp

sp

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spaces

years

FEW

SHOWS

The Moral of this is that we should listen to others advice and be willing to accept correction instead of always thinking we are right.

The King finally realizes that his mistakes and is willing ro change.

This is the biggest lesson in the story. His behavior caused people to stop trying to help him.

I understand why he is embarrassed, but he should of realize that this happened because he refused everyone's advice.

This story is showing that refusing advice can lead to embarrassment and problems.

I think the drummer is going to be the person who finally teaches the King a lesson.

His refusal to listen has now caused other people to stop correcting him.

I think this is ironic because he could have avoided the embarrassing situation if he had listened to everyone.

This shows how stubborn he is. Multiple people warned him, but he still refused to listen.

I feel like eating the melon is going to cause a problem because everyone is warning him not to.

Why would the chiefs crown him if they knew he was not going to listen to anyone?

This started to makes me think he was becoming selfish. A good king should think about his people, not just himself.

This is a major change from his father. He refuses to listen to people who are trying to help him.

This decision caused serious consequences for his father and his future as king.

I think this was irresponsible because he knew his father needed him.

The King understands that listening to other people helps him rule well.

The King seems like a good ruler because his people respect and love him.

Interesting about Fiat

Remember this.

The "new" workforce profile?

East Asia is very diverse and has a multitude of backgrounds, advancements, and philosophies without history. To group all these different countries under one category of "East Asia" is acceptable because all fall under the geographical topic, but there are many differences between these countries

Mamara is finally being recognized for her talent instead of her appearance.

I think this shows respect for Mamara and protects her from being embarrassed.

The Duke shows kindness by helping her instead of judging her.

This proves that the people who judged her because of her sore were wrong.

I think Mamara will use the contest to prove everyone wrong.

I think this is unfair because they are judging her before seeing what she can do.

This gives Mamara a chance to show her cooking ability.

I think this sore is going to cause problems for Mamara even though it doesn't affect her cooking.

Mamara is talented, but the story is going to show that people focus on something else about her.

How commonplace was literacy for philosophical texts in this time period and geography? The anti-dogmatism is inspiring.

I found it very interesting that this foundational idea of religious freedom and rejection of supremacy was present at this point in history. In modern day, most would agree that religious tolerance has only become more prevalent, yet many more are socially persecuted for subscribing to the idea that all religions are equal in liberty and right.

I think this shows how their ideas challenge the usual story that the enlightenment was mainly European. Including thinkers like Yacob and Amo gives a more complete picture of this period.

This stood out to me because Yacob was arguing for equality before many European Enlightenment thinkers. This shows that Enlightenment style ideas were developing outside of Europe too.

I wonder if this will ever be corrected in the dictionary? How many times has this happened and we just have not found as solid proof as in this scenario?

I find it really interesting how Yacob was able to come to this conclusion and write about his views, yet we only remember and honor Western men for this idea. Makes you wonder about where those men heard/read Yacob's views in the first place.

I think it is important to look at the context of the reading, as well as decide what you want to get out of the reading. This will give you a good starting block before you start reading. It will make it easier to actively read.

I have never thought to ask these questions when reading before. I think these could be very beneficial questions to ask because knowing why we are drawn to certain types of writing can help us determine how we should write our own pieces.

This is interesting to me. I have never realized that when I read, I always read for the content and pay no attention to the writer's style. I agree that this is due to schooling. We are always taught to analyze the text by finding the main idea of the passage, even in standardized testing.

This is talking about the importance of actively reading. This means you are asking questions and staying engaged rather than just going through the motions of reading words. It is beneficial because it could reduce the need for rereading and give you a good understanding of how the different parts of a paper work together to create a big picture.

IT is talking about data and things that we see can be as I understand it understood in a lot of different ways. the Couch photo really sells this idea with its silly transformation from couch into a bunk bed

I am curious to further explore the correlation between lower income and vaccine hesitancy. I wonder if this stems from a sense of disdain or distrust in the government, or lack of education. Health literacy is a major component of engaging in proper healthcare activities, and I wonder if there is just not enough information that is getting to these lower income brackets. As nurses, we have to constantly educate our patients on an individual and micro level. I would like to engage in more community- based work to provide education on vaccines in a digestible manner and perhaps increase feelings of safety around receiving this care.

I think this changed the way I thought about the Enlightenment being a European event and now I think it may have been shaped by ideas moving between Europe and Asia.

This is interesting to me because it suggests that Enlightenment ideas may not have developed only in Europe. Hume's ideas could have been influenced by knowledge from other cultures.

1.-3. 2.-2 3.-1.

This class seems very interesting. I like that we are going to be exploring the impact of communities through writing. I am excited to dive deeper into these topics.

I like that you are covering writing in communities in many different ways. This gives us a broader understanding of the different things that influence a community, as well as the opportunity to have an impact on our community.

I am glad that this is outlined in the syllabus. Most classes use Word, but each person has their own preferences. I personally prefer Google Docs.

I appreciate that you offer an alternative to using AI in this course. Writing is interpreted differently by every person, so I like that you are not expecting perfect work, but hopefully work that we are proud of as writers.

Directly reflects anti-eugenics agenda

Substantial gap between Cobb's research and when it was recognized by other scholars in the field

Couldn't trust anyone else, therefore built his own lab

I believe, in relation to the conversations had between all the teachers and students in this article, that this method of questing and answering these writing concepts that the student doesn't understand can help them overall talk through there lack of understanding and help them clarify what they might what to look into moving forward.

Throughout the conversation, the teacher is effective in pushing the student along and helping the student work through what they think needs improving as far as their writing. Towards the end of the conversation, the teacher assists the student by asking thoughtful questions that leaves the student with a place to start editing her written work.

After reading the conversation between the student and the teacher, I agree with this sentence and how, throughout the conversation, the teacher did give the student enough time to explain things for themselves and work towards a better paper. Instead, the teacher citied mostly what they though was wrong or needed to be change and didn't help the student work through those problems.

I appreciate how the teacher is being very transparent with the student and constantly asking for their opinion with these open ended questions.

This paragraph mainly sets up the importance of an agenda during these meetings and how crucial it is, both the consultant and the student, to understand what is needing to be accomplished during this meeting.

Similar to the talks we had in training, there is not exactly a correctly rubric to follow when responding to every student. A knowledgeable consultant must be adaptable and be able to quickly understand the wants and needs of the students who are coming to them and how they can best fill those requirements.

I could also imagine one of the many issues that could come from typing rather than writing could be the inability to distinguish other's handwriting which will likely prevent in some sort of way reading as well, since typing and majority of the fonts online are the same easy-to-read text while physical hand written work is not always that way.

makes sense to me. thinking about and understanding the information with head-pictures before recording sounds a lot better than just in one ear and out your fingers to a keyboard.

Interesting idea, wonder its impact on the hippocampus.

Seems like handwriting makes different regions of the brain active compared to typing. Making memory more consolidated

This is interesting, I have heard scientific research on this topic

Craig French recommends soaking rusty typewriter springs in Diet Coke and then wiping them with aluminum foil to remove rust.

https://www.facebook.com/groups/705152958470148/posts/1354433773542060

Respecting each other is something that is very encouraged and requested. Respecting each other leads to an easier and more productive atmosphere.

synonyme: natural

https://www.facebook.com/groups/705152958470148/posts/1355607023424735

The Corona Zephyr and Smith-Corona Skyriter typewriter cases used to have two strips of leather sewn over a metal internal strip on their case handles.

Most of these vintage metal cases are missing the leather straps leaving only an uncomfortable metal strip.

This is how they think they can defeat Spain.

Enhancing your public speaking skills allows for benefits in personal, professional, and public scenarios because it equips the communicator with the skills to encode their thoughts and convey them effectively in whatever speech given. Specific examples are speeches at weddings, persuasive campaign addresses, and public safety notices.

This probably changed Europe for a while.

via Greg Hubert

via Milton Solomon at https://www.facebook.com/groups/705152958470148/posts/1354754280176676/

This might have been the start of wild pigs in America.

Multiple countries are selling spices, but people don't know who's selling all of them.

IL

Typewriter repair people colloquially called the Underwood Touchmaster 5 the "gray fives".

via Jim Tuxbury on Facebook

via Jim Tuxbury on Facebook

They were showing peace by exchanging gifts.

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

Learn more at Review Commons

Reply to the reviewers

We provide below a point-by-point response to reviewers’ comments, describing changes that were made to the manuscript. We also uploaded a "Full Revision" file that contains a general statement in addition to these responses to reviewers. We sincerely thank both reviewers for their thorough reading and suggestions that we believe contributed to a better concision and clarity of the revised manuscript.

Reviewer #1

Major Comments

1. TDH3 has been a major model in the study of the evolution of gene expression as developed by the authors. While most conclusions derived from this system relate to genetic mechanisms, this study attempted to identify the molecular/cellular mechanisms. Although I greatly appreciate the author's comprehensive efforts, I would suggest that conclusions regarding molecular/cellular mechanisms should be made with greater caution, especially avoiding over-generalized conclusions that may be specific to P_TDH3. Thus, I suggest going over the manuscript again and adjusting some statements if they are over-generalizing, as well as adding an explicit discussion of this limitation.

As suggested by the reviewer, we went over the manuscript and made sure that we specifically mentioned the TDH3 promoter in conclusions that may be specific to this promoter. In addition, we further addressed this limitation by including new results of an experiment where we measured the effects of yme2, chs1 and msh1 mutations on expression noise of three additional yeast promoters (PFBA1, PACT1, PTEF1). We found that the three mutations had similar effects on expression noise driven by PTDH3, PACT1 and PTEF1, but different or no effect on PFBA1 expression noise. Therefore, some of our conclusions may not be specific to the TDH3 promoter, such as the importance of mitochondrial state in regulating expression noise of nuclear genes. We described these findings in Results, in the new Figure 7 and in associated supplements. We also added a paragraph in the Discussion and we included new co-authors who performed these additional experiments. We believe these new results will increase the impact of the study.

1. On the surface, it may seem reasonable to ask for changed noise by unchanged mean in order to distinguish independent regulators. However, from a mechanistic perspective, this is quite demanding. In the current prevailing model (transcriptional burst) of noise origination, mutations are not assumed to be noise-specific without affecting the mean. See e.g. PMID: 31320634. If noise and mean are intrinsically coupled, looking for noise-only regulatory mechanisms would imply something very different. This may mean, for example, that we are seeking one single mutation that changes noise and mean through one mechanism, while at the same time reverting mean to its wildtype value through another mechanism. It is likely that this is one of the reasons why causal mutations are difficult to identify.

This is a very sensible comment. We agree that in the model of noise originating from transcriptional bursts, cis-acting mutations are not expected to alter noise without affecting the mean. However, trans-acting mutations may affect transcriptional bursts of a given gene via different mechanisms that compensate each others at the level of mean expression but not at the level of expression noise. Even though mutations impacting noise without altering mean expression may be less common than mutations affecting both mean and noise, they do exist as we showed here. It is possible that we did not identify mutations in transcription factors known to regulate TDH3 promoter activity because such mutations would affect both mean expression and noise. We added two sentences in the discussion to mention this hypothesis. In addition, while the transcriptional burst model can explain the coupling between intrinsic noise and mean expression, it does not apply to mutations impacting extrinsic noise.

Regarding the difficulty to identify mutations altering only expression noise, we found a causal mutations in three out of five mutants analyzed. Interestingly, the three successes corresponded to mutations that increase noise, and the two failures to mutations that decrease noise, suggesting that mutations decreasing noise may be particularly difficult to identify. Finally we note that expression noise is a complex genetic trait in natural populations. An interesting case is shown in Fehrmann et al. 2013, where three trans-acting alleles (loci on chr7, 8 & 13) had a strong individual contribution on noise, partially coupled to mean changes. When combined together, their cumulative effect was very strong on both mean and noise, although the wild strain from which these alleles originate showed an elevated noise but no remarkable change in mean as compared to a reference strain. This implies that other (unmapped) loci “buffer” expression mean in this wild strain against the action of the three noise-acting loci, a scenario comparable to the one suggested by the reviewer.

1. L209-214. The authors state that they selected 254 mutants with the largest noise changes from a library of 1,241 strains (L209-214). However, I cannot match this statement when contrasting figure1-figure supplement 1 (254 mutants) and Figure 1a (1,241 strains). Is this caused by experiments conducted in different labs ? Are there any intuitive ways for the authors to show the strains (and their parameter distribution) that produced consistent results across the two batches of data? E.g. using gray/black dots for un-/repeatable strains. Also, are mean expression levels similarly (un-)repeatable compared to noise ?

Both fluorescence screens were performed in the same laboratory, using the same instrument and following the same protocol. We clarified in the text and figures how the 254 mutants were selected for the secondary screen. In particular, we colored dots on Figure 1 and on Figure 1 – figure supplement 1 to highlight strains with reproducible or non-reproducible change in expression noise in both assays. The apparent lack of reproducibility between the first screen of 1241 strains and the second screen of 254 mutants is not surprising because the 254 strains were not picked randomly among the 1241 initial strains: they were picked because they showed the largest expression changes (either for mean or noise) in the first screen. Statistically, the most extreme effects on mean expression and expression noise observed among the 1241 strains are expected to be over-estimated on average. This phenomena is similar to the “Winner’s curse” in economy. When measuring a quantitative trait for a large number of samples with a certain degree of uncertainty, values that fall in the tails of the distribution (the most extreme values) are statistically expected to be less accurately estimated than values falling near the mean of all samples. To address the last question of the reviewer, mean expression levels were found to show better repeatability than expression noise, probably because error bars (variation among replicate samples) tended to be much smaller (one order of magnitude) for mean expression than for expression noise.

1. How were the five strains analyzed chosen? Are they the only strains fulfilling the criteria on L211-214?

The five strains were picked arbitrarily among nine strains that matched the criteria mentioned in the text. We added a sentence to mention this point. We moved to Supplementary File 5 the section describing how the five strains were chosen to make the main text shorter and easier to read.

1. L243-247. I didn't understand the logic why m2 is included, please elaborate.

We modified the text to clarify why we picked mutation m2 as a candidate. The logic is that there was no strong statistical evidence to exclude the mutation (because of lower statistical power relative to other mutations).

1. The equation for extrinsic noise (L899) seem to be slightly differently from that in Fu and Pachter 2016. The product of mean(RFP) and mean(YFP) is multiplied by 2 here, but not in Fu and Pachter 2016.

We made a typo in the text and corrected it in the revised version. We verified in our R scripts that we used the correct version from Fu and Pachter (with product of mean(RFP) and mean(YFP) not multiplied by 2), which was the case. We are particularly thankful to the reviewer for the thorough proofreading of the manuscript.

1. The experimental design to exclude noise from partitioning for yme2 is really nice. It would have been great if we had gotten to the bottom of this. (This is not a question so no response is needed)

No response requested.

1. The authors demonstrate that a nonsense mutation in CHS1 increases extrinsic noise via impaired chitin septum reparation in daughter cells. However, glucosamine treatment itself alters cell size (Figure 5-figure supplement 3a), which correlates with noise levels. This raises the question: do the observed changes in extrinsic noise stem from glucosamine-induced changes in cell size or from the impaired chitin repair caused by the CHS1 mutation itself? To disentangle these effects, an alternative approach to modulating chitin synthesis that does not alter cell size should be employed.

We do not think that glucosamine-induced changes in cell size can explain the effect of glucosamine treatment on extrinsic noise in chs1 mutant. We observed that glucosamine treatment had a stronger impact on cell size in WT cells than in chs1(G1752a) mutant cells (Figure 5 – figure supplement 3a). However, glucosamine treatment had a much stronger impact on extrinsic noise of chs1(G1752a) mutant cells than WT cells (Figure 5g). Therefore, there is no direct correspondence between the effect of glucosamine on cell size and the effect of glucosamine on extrinsic noise. Even though glucosamine drastically reduced cell size in WT cells, it had almost no impact on expression noise in these cells. We added a sentence in the revised Results to clarify this point.

Our hypothesis is that glucosamine increases chitin synthesis not only during repair of the chitin septum, but more globally at all stages of the cell cycle (as shown by Bulik et al., 2003), which may reduce cell size. The global impact of glucosamine on cell wall chitin levels could rescue defects caused by chs1 mutation on chitin septum repair. Previous studies showed that CHS1 was not involved in global chitin synthesis, but only in the repair of chitin septum in daughter cells.

1. Why did glucosamine doses not significantly impact cell growth rates during the first phase after addition (Figure5 -figure supplement 4) ? Additionally, I can seem to find the experimental details for glucosamine dosing in the Methods.

We specified the dose of glucosamine in the revised Methods. We did not observe a significant impact of glucosamine on growth rates during exponential growth either in the first growth phase or in the second growth phase after addition. However, glucosamine increased the duration of the lag phase in the second phase of growth. We do not know exactly why, but we speculate it is because the chitin cell wall becomes thicker after diauxic shift in presence of glucosamine, leading to a delay to resume cell division after cells are exposed to fresh medium with glucose.

1. Figure 2-figure supplement 2g-l are missing.

We included the missing panels in the revised figure.

1. The current manuscript is a bit lengthy (although nicely comprehensive). After deciding the journal, I suggest it would need to be more concised and logically streamlined.

We agree that the main text is lengthy, with methodological explanations sometime disrupting the main message. For this reason, we included in the revised version a new Supplementary File 5 where we moved these explanations that were important yet not essential for the reader to understand the main conclusions.

Minor points

1. P12,L345, "may not only by caused by" should be "may not only be caused by", ,and "YFP an RFP" should be "YFP and RFP" in the same sentence

We corrected these mistakes.

1. P19,L565, "sensitivite" is misspelled and should be "sensitive".P21,L630, "mitochondria dysfunction" should be "mitochondrial dysfunction."

We corrected these mistakes.

1. Typo in Figure 3's legend "** 0.001 > P {greater than or equal to} 0.001", which should read "0.01 > P {greater than or equal to} 0.001." This error appears again in Figure 4's legend.

We corrected these mistakes.

1. P9, L219 "Table 1" should be "Supplementary File 1"?

We added the number of mutations per strain in Table 1.

1. Inconsistent tetrad numbers: methods state 22 tetrads (L844) , results mention 21 tetrads ( L262 ) , and figure( figure1 -figure supplement 3) legends indicate 20 tetrads. Please clarify the correct number.

Thank to the reviewer for mentioning this inconsistency. In fact, we dissected 22 tetrads but only included 21 tetrads that showed the expected segregation of all genetic markers in the fluorescence assay. Finally, we reported fluorescence measurements for 20 tetrads due to a possible contamination for the remaining tetrad. We clarified this in the Methods.

1. The speculated retrograde response pathway is interesting. Can the authors propose some specific experiments to test that ?

To test the involvement of the retrograde pathway in PTDH3 intrinsic noise, one could mutate negative or positive regulators of the retrograde signaling in wild-type cells or in chs1 and msh1 mutant cells and quantify the effect on intrinsic noise. We proposed this experiment in the revised discussion. In previous studies, null alleles of rtg1, rtg2 or rtg3 were shown to impair the retrograde response, while specific mutations in rtg2 and deletion of mks1 were shown to activate the retrograde pathway (Garrigos et al., 2024; Jazwinski and Krete, 2012). We expect to observe an elevated intrinsic noise in wild-type cells, but not necessarily in yme2 and msh1 mutant cells, when we activate the retrograde signaling. Conversely, we expect yme2 and msh1 mutations to not alter intrinsic noise anymore when the retrograde pathway activity is impaired by mutation.

Reviewer #2

The manuscript by Martin et al. titled 'Trans-acting mutations reveal non-nuclear modulators of both intrinsic and extrinsic gene expression noise in a eukaryote.' identifies genetic mutations in yeast that can has regulate gene expression noise in trans. The manuscript is well written, and the experiments have been performed in replicates. The authors also clearly highlight the experiments where the replicates do not agree in their outcomes.

However, there are some issues that the authors need to address:

1. Introduction is too long and needs to be concise

We have shorten the introduction in the revised version. We have also moved parts of the main text in Supplementary File 5 to be more concise.

1. Lines 150-153: Do we have enough studies yet for generalizations?

We do not know other studies/examples that compared the effects of cis-acting and trans-acting mutations on mean expression and expression noise of a target gene. Therefore, we cannot generalize the results obtained for the TDH3 promoter. However, these results show that cis- and trans-acting mutations can significantly differ in their effects on expression noise (but we do not know for how many genes it is the case).

1. Line 209 - Why 254 strains? Please justify

We clarified why and how we chose these 254 strains for the secondary screen. We also added colors on Figure 1 to highlight these 254 strains.

1. Why are the authors choosing median expression and not mean expression (which is usually the norm)?

We used the median to quantify the average expression among cells as we did in previous studies with the same fluorescent reporter system because median is more robust than mean to rare outliers. However, we found the difference between median and mean fluorescence to be really small. We included a new figure (Figure 2 – figure supplement 4) showing that the effect of yme2, chs1 and msh1 mutations on expression noise were almost identical when using mean and median to calculate the noise. This is because we measured fluorescence from large number of cells for each sample (~5000) and because the distributions of fluorescence levels among cells are always unimodal with very rare outliers (as showed in Figure 3 – figure supplement 1; Figure 5 – figure supplement 1 and Figure 6 – figure supplement 1).

1. Do EMS mutants have intra-population genetic heterogeneity? This should be discussed in the text.

We sequenced the genome of the 5 EMS mutants at a coverage of ~100x, but did not find evidence of genetic heterogeneity in these strains: all mutations detected were found at a frequency near 1. In another project, we sequenced the genomes of 288 EMS mutants and detected genetic heterogeneity in 3 strains: mutations were not fixed in these strains, but found at a frequency near 0.75. We therefore expect the number of mutants with genetic heterogeneity from the collection analyzed in figure 1 to be very small. In addition, genetic heterogeneity cannot impact our conclusions because no genetic heterogeneity was detected in the 5 EMS mutants analyzed and because we constructed two independent clones to investigate the effects of each mapped mutation. We added a sentence in the main text mentioning that no genetic heterogeneity was detected in the 5 EMS mutants.

1. Line 238-240: Shouldn't the change in frequency in low, mid and high- subpopulations be tested relative to the expected distribution from the wild-type strain? This could also alter how mutations are chosen for validation. This should be mentioned in the results section and the text should be rephrased to reflect this point, although it is mentioned in the methods section

We are not completely sure to understand what statistical test the reviewer has in mind. We could not easily compare the observed frequency of mutant and wild-type alleles in low, mid and high subpopulations to expected frequencies, because these frequencies depend not only on the effect of the mutation on fluorescence among cells but also on the effect of the mutation on growth rate (which is unknown). Our strategy to compare mutation frequency in medium bulk vs low and high bulk was designed to detect mutations changing expression noise independently from their potential effect on mean expression or growth rate.

1. Line 243: The mutant name YPW2162 suddenly appears in the text - where did this strain come from?

This is the name of one of five mutants analyzed, as mentioned in Table 2 referenced in the same sentence. We modified the sentence to make it clearer: “For a fourth mutant (YPW2162), ...”

1. Line 286: 'increase' instead of 'increased' .

We corrected this error.

1. Could genomic rearrangements/copy number variation alter expression noise? For example, for m4, m5 and m6 mutants. The authors have genome data of these strains, so this can be checked.

According to the reviewer’s comment, we performed additional analyses showing that CNVs and rearrangements did not contribute to variation of expression noise in the five EMS mutants included in the mapping experiments. To detect large CNVs and aneuploidies, we computed sequencing depth in 1-kb sliding windows along the genome for each mutant. The profiles were uniform and similar to coverage profiles obtained for the reference strain. To detect rearrangements, we analyzed sequencing data using the GRIDSS module that can detect junctions between non-contiguous parts of the genome from the mapping location of paired-end reads. Using this tool, we only detected 5 rearrangements that were previously known to be present in the genome of all mutant strains relative to the reference genome (deletions at ho and ura3 loci and duplications of TDH3 promoter, CYC1 terminator as well as 41 bases from chromosome I in the PTDH3-YFP transgene inserted at the ho locus). None of these rearrangements can explain variation of expression noise among mutants. Results from GRIDSS analysis are included in Supplementary File 4 and reported in the main text.

1. Figure 2 - y-axis: What is the measure of expression noise used here? This should be mentioned in the figure captions throughout to avoid confusion.

We used the same measure of expression noise for all figures, as mentioned in the Methods. We added it in the figure captions as suggested by the reviewer to avoid confusion.

1. Line 359 - please mention the effect size here and wherever possible throughout the manuscript

In the sentence mentioned by the reviewer, we used the forward scatter signal (FSC.A) as a relative measure of cell size. A difference of FSC.A between two samples is known to reflect a difference of cell size. However, we cannot estimate the effect size on cell size because the relationship between FSC.A and cell size depends on the instrument and settings, and we have not characterized this relationship empirically. Therefore, we removed “a small effect” from the sentence and we instead only mentioned that the effect on cell size was statistically significant. Indeed, we cannot be sure that the small (and significant) reduction of FSC.A we observed corresponded to a small reduction of cell size. 12. Figure 6 - figure supplement 2 - Please mention the strains represented by grey and orange boxes

To make it more visible, we moved this information from an inset in panel b to the top of the figure.

1. One could envisage that there are many more genetic regulators of expression noise which may have not been discovered yet. This point perhaps could be discussed.

Absolutely. We added a sentence in the discussion to acknowledge that many genetic modulators of noise may still remain unknown.

1. The figure captions are too long - they should be made concise

We reduced the length of the longest figure legends. In particular, some of the text from Figure 4 legend was moved to Supplementary File 5.

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Referee #2

Evidence, reproducibility and clarity

The manuscript by Martin et al. titled 'Trans-acting mutations reveal non-nuclear modulators of both intrinsic and extrinsic gene expression noise in a eukaryote.' identifies genetic mutations in yeast that can has regulate gene expression noise in trans. The manuscript is well written, and the experiments have been performed in replicates. The authors also clearly highlight the experiments where the replicates do not agree in their outcomes.

However, there are some issues that the authors need to address:

1. Introduction is too long and needs to be concise
2. Lines 150-153: Do we have enough studies yet for generalizations?
3. Line 209 - Why 254 strains? Please justify
4. Why are the authors choosing median expression and not mean expression (which is usually the norm)?
5. Do EMS mutants have intra-population genetic heterogeneity? This should be discussed in the text.
6. Line 238-240: Shouldn't the change in frequency in low, mid and high- subpopulations be tested relative to the expected distribution from the wild-type strain? This could also alter how mutations are chosen for validation. This should be mentioned in the results section and the text should be rephrased to reflect this point, although it is mentioned in the methods section.
7. Line 243: The mutant name YPW2162 suddenly appears in the text - where did this strain come from?
8. Line 286: 'increase' instead of 'increased'
9. Could genomic rearrangements/copy number variation alter expression noise? For example, for m4, m5 and m6 mutants. The authors have genome data of these strains, so this can be checked.
10. Figure 2 - y-axis: What is the measure of expression noise used here? This should be mentioned in the figure captions throughout to avoid confusion.
11. Line 359 - please mention the effect size here and wherever possible throughout the manuscript
12. Figure 6 - figure supplement 2 - Please mention the strains represented by grey and orange boxes
13. One could envisage that there are many more genetic regulators of expression noise which may have not been discovered yet. This point perhaps could be discussed.
14. The figure captions are too long - they should be made concise

Significance

Although the effect of cis-acting mutations on expression noise has been studied, there remains a significant gap in understanding whether and how trans-acting mutations could alter protein expression noise. This is where the manuscript provides interesting and important insights into the role of trans mutations on expression noise through careful experimental dissection. The manuscript also elucidates an influence of mitochondria on expression noise and therefore, on phenotypic plasticity. The manuscript will be of interest to the researchers working on gene expression regulation and gene expression noise.

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Referee #1

Evidence, reproducibility and clarity

Summary

This manuscript investigates how rare trans-acting mutations can alter the cell-cell variability ("noise") of gene expression in yeast without affecting mean expression. Using a Saccharomyces cerevisiae strain carrying a PTDH3-YFP reporter, the authors screened 1241 EMS-mutagenized clones by flow cytometry and identified five candidate mutants with significantly altered fluorescence noise but unchanged mean expression. They crossed each mutant to a wild-type mapping strain and performed bulk segregant analysis to identify candidate causative mutations. Ultimately, three single-nucleotide substitutions were confirmed to reproducibly affect noise: a CHS1(G1752A) nonsense mutation, a YME2(G1234A) mutation, and an MSH1(G1262A) mutation. Site-directed reconstitution of each mutation (in a clean background with dual reporters) showed that CHS1(G1752A) increased extrinsic noise primarily in small daughter cells, YME2(G1234A) increased intrinsic noise, and MSH1(G1262A) increased intrinsic noise in a growth-phase-dependent manner. Notably, all three genes encode non-nuclear functions: CHS1 (chitin synthase I, cell-wall repair), YME2 (inner mitochondrial membrane protein), and MSH1 (mitochondrial DNA repair ATPase). The authors conclude that mitochondrial state and cell-wall integrity can modulate expression noise of a nuclear gene, highlighting novel trans-acting noise regulators. The experimental approach (random mutagenesis + BSA + targeted validation) is sound, and the conclusions - that these three mutations each increase noise in specific ways - are generally well supported by the data. This is an excellent work. It is thought-provoking and very comprehensive. It is also very well written (but a bit lengthy in its current form) and mostly technically sound. I would ultimately recommend publication. Yet, if I am to contribute to the strength of the paper and the robustness of the conclusion, here are some important points.

Major Comments

1. TDH3 has been a major model in the study of the evolution of gene expression as developed by the authors. While most conclusions derived from this system relate to genetic mechanisms, this study attempted to identify the molecular/cellular mechanisms. Although I greatly appreciate the author's comprehensive efforts, I would suggest that conclusions regarding molecular/cellular mechanisms should be made with greater caution, especially avoiding over-generalized conclusions that may be specific to P_TDH3. Thus, I suggest going over the manuscript again and adjusting some statements if they are over-generalizing, as well as adding an explicit discussion of this limitation.
2. On the surface, it may seem reasonable to ask for changed noise by unchanged mean in order to distinguish independent regulators. However, from a mechanistic perspective, this is quite demanding. In the current prevailing model (transcriptional burst) of noise origination, mutations are not assumed to be noise-specific without affecting the mean. See e.g. PMID: 31320634. If noise and mean are intrinsically coupled, looking for noise-only regulatory mechanisms would imply something very different. This may mean, for example, that we are seeking one single mutation that changes noise and mean through one mechanism, while at the same time reverting mean to its wildtype value through another mechanism. It is likely that this is one of the reasons why causal mutations are difficult to identify.
3. L209-214. The authors state that they selected 254 mutants with the largest noise changes from a library of 1,241 strains (L209-214). However, I cannot match this statement when contrasting figure1-figure supplement 1 (254 mutants) and Figure 1a (1,241 strains). Is this caused by experiments conducted in different labs ? Are there any intuitive ways for the authors to show the strains (and their parameter distribution) that produced consistent results across the two batches of data? E.g. using gray/black dots for un-/repeatable strains. Also, are mean expression levels similarly (un-)repeatable compared to noise ?
4. How were the five strains analyzed chosen? Are they the only strains fulfilling the criteria on L211-214?
5. L243-247. I didn't understand the logic why m2 is included, please elaborate.
6. The equation for extrinsic noise (L899) seem to be slightly differently from that in Fu and Pachter 2016. The product of mean(RFP) and mean(YFP) is multiplied by 2 here, but not in Fu and Pachter 2016.
7. The experimental design to exclude noise from partitioning for yme2 is really nice. It would have been great if we had gotten to the bottom of this. (This is not a question so no response is needed)
8. The authors demonstrate that a nonsense mutation in CHS1 increases extrinsic noise via impaired chitin septum reparation in daughter cells. However, glucosamine treatment itself alters cell size (Figure 5-figure supplement 3a), which correlates with noise levels. This raises the question: do the observed changes in extrinsic noise stem from glucosamine-induced changes in cell size or from the impaired chitin repair caused by the CHS1 mutation itself? To disentangle these effects, an alternative approach to modulating chitin synthesis that does not alter cell size should be employed.
9. Why did glucosamine doses not significantly impact cell growth rates during the first phase after addition (Figure5 -figure supplement 4) ? Additionally, I can seem to find the experimental details for glucosamine dosing in the Methods.
10. Figure 2-figure supplement 2g-l are missing.
11. The current manuscript is a bit lengthy (although nicely conprehensive). After deciding the journal, I suggest it would need to be more concised and logically streamlined.

Minor points

1. P12,L345, "may not only by caused by" should be "may not only be caused by", ,and "YFP an RFP" should be "YFP and RFP" in the same sentence
2. P19,L565, "sensitivite" is misspelled and should be "sensitive".P21,L630, "mitochondria dysfunction" should be "mitochondrial dysfunction."
3. Typo in Figure 3's legend "** 0.001 > P {greater than or equal to} 0.001", which should read "0.01 > P {greater than or equal to} 0.001." This error appears again in Figure 4's legend.
4. P9, L219 "Table 1" should be "Supplementary File 1"?
5. Inconsistent tetrad numbers: methods state 22 tetrads (L844) , results mention 21 tetrads ( L262 ) , and figure( figure1 -figure supplement 3) legends indicate 20 tetrads. Please clarify the correct number.
6. The speculated retrograde response pathway is interesting. Can the authors propose some specific experiments to test that ?

Significance

The study represents a conceptually interesting attempt to move beyond cis-acting noise modulators by identifying non-nuclear proteins that regulate nuclear gene expression randomness. A significant advance is the identification of mitochondria as a possible regulator of intrinsic noise. This study provides evidence that mitochondrial integrity can independently affect the stochastic synthesis of a nuclear gene. The results of this study will be of interest to evolutionary biologists and geneticists interested in the regulation of gene expression and the origins of phenotypic heterogeneity. Furthermore, phenotypic heterogeneity is of considerable significance when discussing realistic issues such as drug and antibiotic resistance. This manuscript provides critical mechanistic insight, and showcases a very comprehensive effort to reveal those mechanisms. I am an evolutionary geneticist more familiar with computational and theoretical considerations of phenotypic heterogeneity.

What would be the ideal sample size for an experiemnt like this?

He was holding the door shut to not let them in.

The impact of storytelling based on this quote can change the way people see everything. For example, in this quote it tells us that in real life we know certain things are dangerous, but in stories we see them as something more complex and beautiful.

Here the author provides scientific information on oxytocin levels in the brain to make a point about why we as readers get attached to characters. Oxytocin which is found in nursing mothers provides that bonded feeling people find with each other, and according to the author, with our favorite characters from the stories we interact with. This causes us to be emotionally bonded to a story in a way me may not be were this not a process that occurs in our brains. How would stories be different if we couldn't emotionally connect to our characters?

The threat of danger or harm to our children is a constant theme in story telling because it makes almost everyone afraid. People desire to protect their and others children from perceived dangers, so narratives that showcase a decided threat are often circulated very quickly. As we see often politically, if you want to make a group out to be a threat you make a story where children are being harmed, like in the trans rights movement today, or even civil rights in the 1960s.

The author highlights here how impactful culturally storytelling can be. Storytelling as a means to educate and to entertain has been around as long as humans had any means of communication with each other, and each story tells a lot about where it was from and what those people believed or felt at the time. The storys we were told as children, and ones that get read to children today also reflect our modern and changing worldviews, so understadning the science behind it is vital to understanding ourselves, and what ideas or biases we may have based on the stories we are surrounded by.

De esta sección me parece importante que los recursos listados vienen acompaños de su fecha de consulta, lo que está en coherencia con la clase y la idea de ver la trazabilidad de los eventos de un proceso.

En este segmento, me parece importante el término iterativo que hace referencia a la repetición del proceso con el objetivo de mejorarlo paulatinamente, y la idea de incorporar la retroalimentación y hacerla parte del resultado final, porque creo que estamos acostumbrados en la universidad a hacer entregas finales sobre las que no volvemos nunca y cuya única retroalimentación es una nota, sin dar espacio a una continuidad ni a un proceso de mejora que se pueda rastrear.

Did certain types of mud work better?

What food did they have to stay good for that long?

How did they travel that far?

regarding the goal of liberal arts system and goals, when people are caught up in wanting to enter a profession for the fruit of it (money, opportunities, or just being "skilled") what can make people want to be draw into the development of once self if it has more of a inner awareness that often does not reap immediate or tangible results?

They focused on things other than just what they needed to survive.

This is when they switched from being nomadic to farming and living in one place.

Was mad but still had to actually believe

Still did not believe and go to the alter

Another sensory detail

T F F F T

Learning to live by myself and having to manage my own time.

Reading for fun and watching both TV and movies.