Push

for - adjacency - science - spirituality - 0% of reality - Donald Hoffman

for - adjacency - Christian teaching - infinite intelligence - loving God - loving your neighbor - loving yourself - all the same - Donald Hoffman

for - A Answer - you know it when you let go of all concepts and you don't try. If you're trying to get there, then you don't see what you already are. - Donald Hoffman

for - Q ? - Did someone create that consciousness? - Donald Hoffman

for - A Answer - the infinite knows itself though infinite number of perspectives - Donald Hoffman

for - Q ? - What is the meaning of life - Donald Hoffman

for - example - hard problem of consciousness - Simulation Theory - Donald Hoffman

for - quote / key insight - consciousness has created the brain as an icon to describe how it's creating this headset - Donald Hoffman

for - adjacency - subjective vs objective reality - examining our most fundamental assumptions of reality, self and other Donald Hoffman - This is a difficult one for many people who reify objective reality to understand - It requires deep analysis and insight into our fundamental assumptions of how we employ anguage, learned while we were in our child development stage - Donald Hoffman is asking us to take that journey to uproot these most fundamental assumptions of self and other, long forgotten, but thoughtlessly projected into the present moment like an automaton

for - key insight / quote - the reason to love your neighbor - Donald Hoffman - The reason to love your neighbor as yourself is because - your neighbor IS YOUR (TRUE) SELF, just with a different headset. - And the only reason we have problems is - we don't realize how incredible you are. - So you are that which is creating this VR simulation with all of its beauty, all of its complexity. - All the complexity is you and you're doing it effortlessly.

adjacency - infinite intelligence - hologram metaphor - your neighbor is your (true) self - Deep Humanity motto - Join together (instead of Join us) - face behind the mask - Reflecting on this, it occurred to me that the Deep Humanity motto of "Join together, NOT join me/us" is deeply connected to what is being discussed in this annotation. - The problem with "joining me" is that it reflects we are still stuck in the ego reification paradigm while "join together" reflects awareness that the boundless intelligence is the true face behind the mask of each different species and each different individual of each species

for - adjacency - hologram metaphor - infinite intelligence - Jesus - Mathew 25 - Donald Hoffman - Interfaith - Ubuntu - I am because you are - I am you and you are me

for - adjacency - infinite intelligence - perspectival knowing - hologram - Donald Hoffman

for - key insight / quote - the reified ego is the root cause of all the problems in the world - we reify because we don't know who we REALLY are - Donald Hoffman - All the egoic stuff that we do causes all the problems in the world because - you don't know who you are. - You're creating this whole thing. - You're not a little player. - You're the inventor of this whole thing. - You have nothing to prove and - you don't need to be better than anybody else. - They're also master creators. - They're creating entire universes that they perceive as well. - And my own take on on this is that - you and I are really the same one reality - just looking at itself through two different headsets, - two different avatars and having a conversation. - And maybe that's what is required for this one infinite intelligence to sort of know itself.

• adjacency - poverty mentality - ego - problems of the world - samsara - nirvana - hologram model - Alan Watts - God playing hide and seek - Donald Hoffman
• When we don't believe we can be this, we limit ourselves
  • That is, we suffer from self-inflicted poverty mentality
• When he says we are the one same reality,
  • he is echoing the common spiritual teaching of the holographic metaphor where
    • the one nameless is distilling itself in so many separate identities to know itself,
  • Similiar to many spiritual teacher's teachings
    • Alan Watts referred to it as God playing Hide and Seek with itself

for - adjacency - perspectival knowing - rendering - learned in child development - language usage - This is an interesting use of the word "render" to demonstrate how even shared human experiences are still uniquely seen from different perspectives - We impute objective reality, for instance of the cup, even though we are each uniquely rendering it in different ways - It is a direct result of our child development in which we learned how to employ words to label such social contexts - We establish rules for word usage at an early age, but we forget the original conditions which gave rise to them - When we remind ourselves of the original motivation, it is a bit of a shock to the system how strange this reality is

for - adjacency - constructed reality - umwelt - species perspectival knowing - misunderstanding - sensory signals - map and territory - Donald Hoffman - We have to be careful how we interpret his claim here, as it is often easily misunderstood. - He means that evolution itself, reality itself has constructed this unique set of sense organs, that creates a unique human umwelt in which - the sensory signals give us a very specific map of reality, NOT reality itself - In this way, our sensory signals construct a very unique map of reality, which is different from the way all other species construct their maps

for - example - poverty mentality - adjacency - poverty mentality - ego reification - othering - competition - If I believe my own spacetime story that - I am this body - thoughts are simply epiphenomena of the brain - then I don't feel very empowered or spacious - instead, I feel small and insignificant - and it motivates me to compete with others to make myself feel better - In this way, my own poverty mentality, based on the wrong-headed belief that I am the map (not the territory) - leads to identity and ego reification and othering

for - quote - who you are beyond your headset - Donald Hoffman - If you want to understand the truth of who you are beyond just this headset description of you - then you have to - lay aside all concepts period and - just know yourself by being yourself, - not by putting a concept between you and yourself. - adjacency - headset - perspectival knowing - Donald Hoffman - unquestioned assumption of other perspectives - imputation - external observable proxy - to private, inner world - As I read Hoffman's use of the word "headset", it brought up some associations with the idea of "perspectival knowing" - There is the perspectival knowing of a species, - but also of the individual of a species - For humans, perspectival knowing must be contextualized within an imputation: - that other perspectives exist - in other words, that other private worlds exist - and ultimately, this is a widely accepted imputation of an inner private world - based upon public, external observable behavioral proxies - This imputation of the other is a fundamental imputation and assumption of the human condition which we all take for granted, - but because it is so foundational, never question

for - adjacency - spirituality - science - silence of thoughts in meditation - descriptions of reality - map and territory - Donald Hoffman - nice adjacency - if our thoughts are dependent on and built upon inputs from our senses - and our senses only provide us with a map, and not the territory, - then thinking will only ever keep us in the map world

for - quote - Almost all of us think of ourselves as an object in spacetime only here for a short amount of time and will soon die - Donald Hoffman When I say you transcend any scientific

• Almost all of us think of ourselves as
  • an object in spacetime only here for a short amount of time and will soon die.
• When I say you transcend any scientific theory,
  • that means the theory that I am just a 160lb object in spacetime is just a theory and it's not the truth.
• That's not the truth about who I am.
• That's just a theory that I have because spacetime itself is just a theory.
• Nothing inside spacetime is anything but my headset interpretation of a reality that infinitely transcends anything I can experience.

for - definition - learning by ostensive definition - adjacency - ostensible definition - parents - external proxy - children's private experiences - This is a very deep insight and important point - Parents are stewards of culture and they lead their children into a world of shared names - It is important to note that - the parent who teaches the child the name for some aspect of reality - only ever has a proxy to the child's private experience of reality - That proxy is the externally observed behaviour of the child - In fact, we fundamentally only ever have public external proxies to the private, "inner" lives of others

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Maybe to focus on

These registers are all very important for teachers to have and to know about how to swap between each of them as well as instances in which we would have to use each of them. For example, using a casual register could be expected to be used before class starts, maybe after a lesson is completed, etc. Formal is for when we are teaching a lesson or potentially when we are disciplining a student. Urgent is, clearly, for when there is a life or death situation happening or something of a high caliber. Fire, fire drill, physical fight, etc.

I think this is something important to think about because as students thinking about how we're going to handle our future students when things start to get off the rail is something we don't often think about at the front of our minds.

eLife Assessment

This study demonstrates the potential role of 17α-estradiol in modulating neuronal gene expression in the aged hypothalamus of male rats, identifying key pathways and neuron subtypes affected by the drug. While the findings are useful and provide a foundation for future research, the strength of supporting evidence is incomplete due to the lack of female comparison, a young male control group, unclear link to 17α-estradiol lifespan extension in rats, and insufficient analysis of glial cells and cellular stress in CRH neurons.

Reviewer #1 (Public review):

Summary:

Previous studies have shown that treatment with 17α-estradiol (a stereoisomer of the 17β-estradiol) extends lifespan in male mice but not in females. The current study by Li et al, aimed to identify cell-specific clusters and populations in the hypothalamus of aged male rats treated with 17α-estradiol (treated for 6 months). This study identifies genes and pathways affected by 17α-estradiol in the aged hypothalamus.

Strengths:

Using single-nucleus transcriptomic sequencing (snRNA-seq) on hypothalamus from aged male rats treated with 17α-estradiol they show that 17α-estradiol significantly attenuated age-related increases in cellular metabolism, stress, and decreased synaptic activity in neurons.

Moreover, sc-analysis identified GnRH as one of the key mediators of 17α-estradiol's effects on energy homeostasis. Furthermore, they show that CRH neurons exhibited a senescent phenotype, suggesting a potential side effect of the 17α-estradiol. These conclusions are supported by supervised clustering by neuropeptides, hormones, and their receptors.

Weaknesses:

However, the study has several limitations that reduce the strength of the key claims in the manuscript. In particular:

(1) The study focused only on males and did not include comparisons with females. However, previous studies have shown that 17α-estradiol extends lifespan in a sex-specific manner in mice, affecting males but not females. Without the comparison with the female data, it's difficult to assess its relevance to the lifespan.

(2) Its not known whether 17α-estradiol leads to lifespan extension in male rats similar to male mice. Therefore, it is not possible to conclude that the observed effects in the hypothalamus, are linked to the lifespan extension. The manuscript cited in the introduction does not include lifespan data on rats.

(3) The effect of 17α-estradiol on non-neuronal cells such as microglia and astrocytes is not well described (Fig.1). Previous studies demonstrated that 17α-estradiol reduces microgliosis and astrogliosis in the hypothalamus of aged male mice. Current data suggest that the proportion of oligo, and microglia were increased by the drug treatment, while the proportions of astrocytes were decreased. These data might suggest possible species differences, differences in the treatment regimen, or differences in drug efficiency. This has to be discussed.

A more detailed analysis of glial cell types within the hypothalamus in response to drug should be provided.

(4) The conclusion that CRH neurons are going into senescence is not clearly supported by the data. A more detailed analysis of the hypothalamus such as histological examination to assess cellular senescence markers in CRH neurons, is needed to support this claim.

Revised submission:

Some of the concerns were addressed in this revised version, and the authors responded and addressed study design limitations in both sexes/ages.

However, there are still some concerns that were not sufficiently addressed:<br /> While the term "senescent" was changed to "stressed," some histological/ cellular validation of this phenotype is still needed.

Some discussion on the sex-specific effects of 17α-estradiol in the hypothalamus is still required. Previous studies in mice demonstrated that 17α-estradiol reduced hypothalamic microgliosis and astrogliosis in male but not female UM-HET3 mice.

Additionally, the provided analysis on astrocytes and microglia is superficial.

Reviewer #2 (Public review):

Summary:

Li et al. investigated the potential anti-ageing role of 17α-Estradiol on the hypothalamus of aged rats. To achieve this, they employed a very sophisticated method for single-cell genomic analysis that allowed them to analyze effects on various groups of neurons and non-neuronal cells. They were able to sub-categorize neurons according to their capacity to produce specific neurotransmitters, receptors, or hormones. They found that 17α-Estradiol treatment led to an improvement in several factors related to metabolism and synaptic transmission by bringing the expression levels of many of the genes of these pathways closer or to the same levels to those of young rats, reversing the ageing effect. Interestingly, among all neuronal groups, the proportion of Oxytocin-expressing neurons seems to be the one most significantly changing after treatment with 17α-Estradiol, suggesting an important role of these neurons on mediating its anti-ageing effects. This was also supported by an increase in circulating levels of oxytocin. It was also found that gene expression of corticotropin-releasing hormone neurons was significantly impacted by 17α-Estradiol even though it was not different between aged and young rats, suggesting that these neurons could be responsible for side effects related to this treatment. This article revealed some potential targets that should be further investigated in future studies regarding the role of 17α-Estradiol treatment in aged males.

Strengths:

• The single nucleus mRNA sequencing is a very powerful method for gene expression analysis and clustering. The supervised clustering of neurons was very helpful in revealing otherwise invisible differences between neuronal groups and helped identify specific neuronal populations as targets.

• There is a variety of functions used that allowed the differential analysis of a very complex type of data. This led to a better comparison between the different groups in many levels.

• There were some physiological parameters measured such as circulating hormone levels that helped the interpretation of the effects of the changes in hypothalamic gene expression.

Weaknesses:

• One main control group is missing from the study, the young males treated with 17α-Estradiol.

• Even though the technical approach is a sophisticated one, analyzing the whole rat hypothalamus instead of specific nuclei or subregions makes the study weaker.

• Although the authors claim to have several findings, the data fail to support these claims.

• The study is about improving ageing but no physiological data from the study demonstrated such claim with the exception of the testes histology which was not properly analyzed and was not even significantly different between the groups.

• Overall, the study remains descriptive with no physiological data to demonstrate that any of the effects on hypothalamic gene expression is related to metabolic, synaptic or other function.

Comments on revisions:

The authors revised part of the manuscript to address some of the reviewers' comments. This improved the language and the text flow to a certain extent. They also added an additional analysis including glial cells. However, they failed to address the main weaknesses brought up by the reviewers and did not add any experimental demonstration of their claims on lifespan expansion induced by 17α-estradiol in rats (the cited study does not include lifespan in rats). In addition, they insisted i keeping parts in the discussion that are not directly linked to any of the papers' findings.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public Review):

Summary:

Previous studies have shown that treatment with 17α-estradiol (a stereoisomer of the 17β-estradiol) extends lifespan in male mice but not in females. The current study by Li et al, aimed to identify cell-specific clusters and populations in the hypothalamus of aged male rats treated with 17α-estradiol (treated for 6 months). This study identifies genes and pathways affected by 17α-estradiol in the aged hypothalamus.

Strengths:

Using single-nucleus transcriptomic sequencing (snRNA-seq) on the hypothalamus from aged male rats treated with 17α-estradiol they show that 17α-estradiol significantly attenuated age-related increases in cellular metabolism, stress, and decreased synaptic activity in neurons.

Thanks.

Moreover, sc-analysis identified GnRH as one of the key mediators of 17α-estradiol's effects on energy homeostasis. Furthermore, they show that CRH neurons exhibited a senescent phenotype, suggesting a potential side effect of the 17α-estradiol. These conclusions are supported by supervised clustering by neuropeptides, hormones, and their receptors.

Thanks.

Weaknesses:

However, the study has several limitations that reduce the strength of the key claims in the manuscript. In particular:

(1) The study focused only on males and did not include comparisons with females. However, previous studies have shown that 17α-estradiol extends lifespan in a sex-specific manner in mice, affecting males but not females. Without the comparison with the female data, it's difficult to assess its relevance to the lifespan.

This study was originally designed based on previous findings indicating that lifespan extension is only effective in males, leading to the exclusion of females from the analysis. The primary focus of our research was on the transcriptional changes and serum endocrine alterations induced by 17α-estradiol in aged males compared to untreated aged males. We believe that even in the absence of female subjects, the significant effects of 17α-estradiol on metabolism in the hypothalamus, synapses, and endocrine system remain evident, particularly regarding the expression levels of GnRH and testosterone. Notably, lower overall metabolism, increased synaptic activity, and elevated levels of GnRH and testosterone are strong indicators of health and well-being in males, supporting the validity of our primary conclusions. However, including female controls would enhance the depth of our findings. If female controls were incorporated, we propose redesigning the sample groups to include aged male control, aged female control, aged female treated, aged male treated, as well as young male control, young male treated, young female control, and young female treated. We regret that we cannot provide this data in the short term. Nevertheless, we believe this reviewer’s creative idea presents a valuable avenue for future research on this topic. In this study, we emphasize the role of 17α-estradiol in overall metabolism, synaptic function, GnRH, and testosterone in aged males and underscore the importance of supervised clustering of neuropeptide-secreting neurons in the hypothalamus.

(2) It is not known whether 17α-estradiol leads to lifespan extension in male rats similar to male mice. Therefore, it is not possible to conclude that the observed effects in the hypothalamus, are linked to the lifespan extension.

Thanks for the reminding. 17α-estradiol was reported to extend lifespan in male rats similar to male mice (PMID: 33289482). We have added the valuable reference to introduction in the new version.  

(3) The effect of 17α-estradiol on non-neuronal cells such as microglia and astrocytes is not well-described (Figure 1). Previous studies demonstrated that 17α-estradiol reduces microgliosis and astrogliosis in the hypothalamus of aged male mice. Current data suggest that the proportion of oligo, and microglia were increased by the drug treatment, while the proportions of astrocytes were decreased. These data might suggest possible species differences, differences in the treatment regimen, or differences in drug efficiency. This has to be discussed.

We have reviewed reports describing changes in cell numbers following 17α-estradiol treatment in the brain, using the keywords "17α-estradiol," "17alpha-estradiol," and "microglia" or "astrocyte." Only a limited amount of data was obtained. We found one article indicating that 17α-estradiol treatment in Tg (AβPP(swe)/PS1(ΔE9)) model mice resulted in a decreased microglial cell number compared to the placebo (AβPP(swe)/PS1(ΔE9) mice), but this change was not significant when compared to the non-transgenic control (PMID: 21157032). The transgenic AβPP(swe)/PS1(ΔE9) mouse model may differ from our wild-type aging rat model in this context.

Moreover, the calculation of cell numbers was based on visual observation under a microscope across several brain tissue slices. This traditional method often yields controversial results. For example, oligodendrocytes in the corpus callosum, fornix, and spinal cord have been reported to be 20-40% more numerous in males than in females based on microscopic observations (PMID: 16452667). In contrast, another study found no significant difference in the number of oligodendrocytes between sexes when using immunohistochemistry staining (PMID: 18709647). Such discrepancies arising from traditional observational methods are inevitable.

We believe the data presented in this article are reliable because the cell number and cell ratio data were derived from high-throughput cell counting of the entire hypothalamus using single-cell suspension and droplet wrapping (10x Genomics).

(4) A more detailed analysis of glial cell types within the hypothalamus in response to drugs should be provided.

We provided more enrichment analysis data of differentially expressed genes between Y, O, and O.T in microglia and astrocytes in Figure 2—figure supplement 3. In this supplemental data, we found unlike that in neurons, Micro displayed lower levels of synapse-related cellular processes in O.T. compared to O.

(5) The conclusion that CRH neurons are going into senescence is not clearly supported by the data. A more detailed analysis of the hypothalamus such as histological examination to assess cellular senescence markers in CRH neurons, is needed to support this claim.

We also noted the inappropriate claim and have changed "senescent phenotype" to "stressed phenotype" and "abnormal phenotype" in both the abstract and results sections. The stressed phenotype could be induced by heightened functional activity in the cells, potentially indicating higher cellular activity. The GnRH and CRH neurons discussed in this paper may represent such a case, as illustrated by the observed high serum GnRH, testosterone, and cortisol levels. This revision suggestion is highly valuable and constructive for our understanding of the unique physiological characteristics revealed by these data.

Reviewer #2 (Public Review):

Summary:

Li et al. investigated the potential anti-ageing role of 17α-Estradiol on the hypothalamus of aged rats. To achieve this, they employed a very sophisticated method for single-cell genomic analysis that allowed them to analyze effects on various groups of neurons and non-neuronal cells. They were able to sub-categorize neurons according to their capacity to produce specific neurotransmitters, receptors, or hormones. They found that 17α-Estradiol treatment led to an improvement in several factors related to metabolism and synaptic transmission by bringing the expression levels of many of the genes of these pathways closer or to the same levels as those of young rats, reversing the ageing effect. Interestingly, among all neuronal groups, the proportion of Oxytocin-expressing neurons seems to be the one most significantly changing after treatment with 17α-Estradiol, suggesting an important role of these neurons in mediating its anti-ageing effects. This was also supported by an increase in circulating levels of oxytocin. It was also found that gene expression of corticotropin-releasing hormone neurons was significantly impacted by 17α-Estradiol even though it was not different between aged and young rats, suggesting that these neurons could be responsible for side effects related to this treatment. This article revealed some potential targets that should be further investigated in future studies regarding the role of 17α-Estradiol treatment in aged males.

Strengths:

(1) Single-nucleus mRNA sequencing is a very powerful method for gene expression analysis and clustering. The supervised clustering of neurons was very helpful in revealing otherwise invisible differences between neuronal groups and helped identify specific neuronal populations as targets.

Thanks.

(2) There is a variety of functions used that allow the differential analysis of a very complex type of data. This led to a better comparison between the different groups on many levels.

Thanks.

(3) There were some physiological parameters measured such as circulating hormone levels that helped the interpretation of the effects of the changes in hypothalamic gene expression

Thanks.

Weaknesses

(1) One main control group is missing from the study, the young males treated with 17α-Estradiol.

Given that the treatment period lasts six months, which extends beyond the young male rats' age range, we aimed to investigate the perturbation of 17α-Estradiol on the normal aging process. Including data from young males could potentially obscure the treatment's effects in aged males due to age effects, though similar effects between young and aged animals may exist. Long-term treatment of hormone may exert more developmental effects on the young than the old. Consequently, we decided to exclude this group from our initial sample design. We apologize for this omission.

(2) Even though the technical approach is a sophisticated one, analyzing the whole rat hypothalamus instead of specific nuclei or subregions makes the study weaker.

The precise targets of 17α-Estradiol within the hypothalamus remain unresolved. Selecting a specific nucleus for study is challenging. The supervised clustering method described in this manuscript allows us to identify the more sensitive neuron subtypes influenced by 17α-Estradiol and aging across the entire hypothalamus, without the need to isolate specific nuclei in a disturbed hypothalamic environment.

(3) Although the authors claim to have several findings, the data fail to support these claims. You may mean the claim as the senescent phenotype in Crh neuron induced by 17a-estradiol.

Thanks. We have changed the "senescent phenotype" to "stressed phenotype" in the abstract and results to avoid such claim. The stressed phenotype may be induced by heightened functional activity in the cells, potentially indicating higher cellular activity.

(4) The study is about improving ageing but no physiological data from the study demonstrated such a claim with the exception of the testes histology which was not properly analyzed and was not even significantly different between the groups.

The primary objective of this study is to elucidate the effects of 17α-Estradiol on the endocrine system in the aging hypothalamus; exploring anti-aging effects is not the main focus. From the characteristics of the aging hypothalamus, we know that down-regulated GnRH and testosterone levels, along with elevated mTOR signaling, are indicators of aging in these organs from previous publications (PMID: 37886966, PMID: 37048056, PMID: 22884327). The contrasting signaling networks related to metabolism and synaptic processes significantly differentiate young and aging hypothalami, and 17α-Estradiol helps rebalance these networks, suggesting its potential anti-aging effects.

(5) Overall, the study remains descriptive with no physiological data to demonstrate that any of the effects on hypothalamic gene expression are related to metabolic, synaptic, or other functions.

The study focuses on investigating cellular responses and endocrine changes in the aging hypothalamus induced by 17α-estradiol, utilizing single-nucleus RNA sequencing (snRNA-seq) and a novel data mining methodology to analyze various neuron subtypes. It is important to note that this study does not mainly aim to explore the anti-aging effects. Consequently, we have revised the claim in the abstract from “the effects of 17α-estradiol in anti-aging in neurons” to “the effects of 17α-estradiol on aging neurons.” We observed that the lower overall metabolism and increased expression levels of cellular processes in the synapses align with findings previously reported regarding 17α-estradiol. To address the lack of physiological data and the challenges in measuring multiple endocrine factors due to their volatile nature, we employed several bidirectional Mendelian analyses of various genome-wide association study (GWAS) data related to these serum endocrine factors to identify their mutual causal effects.

Reviewing Editor Comment:

Based on the Public Reviews and Recommendations for Authors, the Reviewers strongly recommend that revisions include an experimental demonstration of the physiological effects of the treatment on ageing in rats as well as the CRH-senescence link. Additional analysis of the glia would greatly strengthen the study, as would inclusion of females and young male controls. The important point was also raised that the work linking 17a-estradiol was performed in mice, and the link with lifespan in rats is not known. Discussion of this point is recommended.

We thank the reviewers for their constructive feedback. Regarding the recommendations in the Public Reviews and Recommendations for Authors:

a)  Physiological effects & CRH-senescence link:

We acknowledge that 17α-estradiol has been reported to extend lifespan in male rats, consistent with findings in male mice (PMID: 33289482). This point has now been noted in the Introduction. We regret that further experimental validation of the treatment's physiological effects on aging in rats was beyond the scope of this study.

b) Phenotype terminology:

In response to concerns about the "senescent" characterization of CRH neurons, we have revised this terminology to "stressed phenotype" throughout the abstract and results. While we were unable to conduct additional experiments to confirm senescence markers, this revised description better reflects the heightened cellular activity observed (as evidenced by elevated serum GnRH and testosterone levels), without implying confirmed senescence.

c) Glial cell analysis:

To address questions about glial cell function during treatment, we have added new enrichment analysis data of differentially expressed genes in microglia and astrocytes from young (Y), old (O), and old treated (O.T) groups in Figure 2—figure supplement 3. This analysis reveals that microglia exhibit contrasting synaptic-related cellular processes compared to total neurons.

d) Female and young controls:

We sincerely apologize for the absence of female subjects and young male controls in the current study. The reviewers' suggestion to examine the male-specific effects of 17α-estradiol using female controls represents an excellent direction for future research, which we plan to pursue in upcoming studies.

Reviewer #2 (Recommendations For The Authors):

General comments:

(1) The manuscript is very hard to read. Proofreading and editing by software or a professional seems necessary. The words "enhanced", "extensive" etc. are not always used in the right way.

Thanks for the suggestion. We have revised the proofreading and editing. The words "enhanced" and "extensive" were also revised in most sentences.

(2) The numbers of animals and samples are not well explained. Is it 9 rats overall or per group? If there are 8 testes samples per group, should we assume that there were 4 rats per group? The pooling of the hypothalamic how was it done? Were all the hypothalamic from each group pooled together? A small table with the animals per group and the samples would help.

We appreciate your reminder regarding the initial mistake in our manuscript preparation. In the preliminary submission, we reported 9 rats based solely on sequencing data and data mining. The revised version (v1) now includes additional experimental data, with an effective total of 12 animals (4 per group). Unfortunately, we overlooked updating this information in the v1 submission. We have since added detailed information in the Materials and Methods sections: Animals, Treatment and Tissues, and snRNA-seq Data Processing, Batch Effect Correction, and Cell Subset Annotation.

(3) The Clustering is wrong. There are genes in there that do not fall into any of the 3 categories: Neurotransmitters, Receptors, Hormones.

We acknowledge the error in gene clustering and have implemented the following corrections:

(a) The description has been updated to state: 'Vast majority of these subtypes were clustered by neuropeptides, hormones, and their receptors among all neurons.'

(b) Genes not belonging to these three categories have been substantially removed.

(c) The neuropeptide category (now including several growth hormones) has been expanded to 104 genes, while their corresponding receptors (including several sex hormone receptors) now comprise 105 genes.

(4) The coloring of groups in the graphs is inconsistent. It must be more homogeneous to make it easier to identify.

We have changed the colors of groups in Fig. 1D to make the color of cell clusters consistent in Fig. 1A-D.

(5) The groups c1-c4 are not well explained. How did the authors come up with these?

We have added more descriptions of c1-c4 in materials and methods in the new version.

(6) In most cases it's not clear if the authors are talking about cell numbers that express a certain mRNA, the level of expression of a certain mRNA, or both. They need to do a better job using more precise descriptions instead of using general terms such as "signatures", "expression profiles", "affected neurons" etc. It is very hard to understand if the number of neurons is compared between the groups or the gene expression.

We have changed the "signatures" to "gene signatures" to make it more accurate in meaning. The "affected neurons" were also changed to "sensitive neurons". But sorry that we were not able to find better alternatives to the "expression profiles".

(7) Sometimes there are claims made without justification or a reference. For example, the claim about the senescence of CRH neurons due to the upregulation of mitochondrial genes and downregulation of adherence junction genes (lines 326-328) should be supported by a reference or own findings.

The "senescence" here is not appropriate. We have changed it to "stressed phenotype" or "aberrant changes" in abstract and results.

(8) Young males treated with Estradiol as a control group is necessary and it is missing.

Your suggestion is appreciated; however, the treatment duration for aged mice (O.T) was set at 6 months, while the young mice were only 4 months old. This disparity makes it challenging to align treatment timelines for the young animals. The primary aim of this study is to investigate the perturbation of 17α-estradiol on the aging process, and any distinct effects due to age effect observed in young males might complicate our understanding of its role in aged males, though similar endocrine effects may exist in the young animals. Long-term treatment of hormone may exert more developmental effects on the young than the old. Therefore, we made the decision to exclude the young samples in our initial study design. We apologize for any confusion this may have caused.

Specific Comments:

Line 28: "elevated stresses and decreased synaptic activity": Please make this clearer. Can't claim changes in synaptic activity by gene expression.

We have changed it to "the expression level of pathways involved in synapse"

Line 32: "increased Oxytocin": serum Oxytocin.

We have added the “serum”.

Line 52 - 54: Any studies from rats?

Thanks. In rats there is also reported that 17α-estradiol has similar metabolic roles as that in mice (PMID: 33289482) and we have added it to the refences. It’s very useful for this manuscript.

Line 62 - 65: It wasn't investigated thoroughly in this paper so why was it suggested in the introduction?

We have deleted this sentence as being suggested.

Line 70: "synaptic activity" Same as line 28.

We have changed it to "pathways involved in synaptic activity".

Line 79: Why were aged rats caged alone and young by two? Could that introduce hypothalamic gene expression effects?

The young males were bred together in peace. But the aged males will fight and should be kept alone.

Lines 78, 99, 109-110: It is not clear how many animals per group were used and how many samples per group were used separately and/or grouped. Please be more specific.

We have added these information to Materials and methods/Animals, treatment and tissues and Materials and methods/snRNA-seq data processing, batch effect correction, and cell subset annotation.

Line 205: "in O" please add "versus young.".

We have changed accordingly.

Line 207: replace "were" with "was"

We have alternatively changed the "proportion" to "proportions".

Line 208: replace "that" with "compared to" and after "in O.T." add "compared to?"

We have changed accordingly.

Line 223: "O.T." compared to what? Figure?

We have changed it accordingly.

Line 227: Figure?

We have added (Figure 1E) accordingly.

Line 229: "synaptic activity" Same as line 28.

We have revised it.

Line 235: "synaptic activity" and "neuropeptide secretion" Same as line 28.

We have revised it.

Line 256:" interfered" please revise.

We changed to "exerted".

Line 263: "on the contrary" please revise.

We have changed "on the contrary" to "opposite".

Line 270: "conversed" did you mean "conserved"?

We have changed "conversed" to "inversed".

Line 296-298: Please explain. Why would these be side effects?

It’s hard to explain, therefore, we deleted the words "side effects".

Line 308: "synaptic activity" Same as line 28.

We have changed it to "expression levels of synapse-related cellular processes".

Line 314: "and sex hormone secretion and signaling"Isn't this expected?

Yes, it is expected. We have added it to the sentence "and, as expected, sex hormone secretion and signaling".

Line 325-328: Why is this senescence? Reference?

We have added “potent” to it.

Line 360-361: This doesn't show elevated synaptic activity.

"elevated synaptic activity" was changed to "The elevated expression of synapse-related pathways"

Line 363-364: "Unfortunately" is not a scientific expression and show bias.

We have changed it to "Notably".

Line 376: Similar as above.

Yes, we have change it to "in contrast".

Lines 382-385: This is speculation. Please move to discussion.

Sorry for that. We think the causal effects derived from MR result is evidence. As such, we have not changed it.

Line 389: Please revise "hormone expressing".

We have changed it accordingly.

Line 401: Isn't this effect expected due to feedback inhibition of the biochemical pathway? Please comment.

The binding capability of 17alpha-estradiol to estrogen receptors and its role in transcriptional activation remain core questions surrounded by controversy. Earlier studies suggest that 17alpha-estradiol exhibits at least 200 times less activity than 17beta-estradiol (PMID: 2249627, PMID: 16024755). However, recent data indicate that 17alpha-estradiol shows comparable genomic binding and transcriptional activation through estrogen receptor α (Esr1) to that of 17beta-estradiol (PMID: 33289482). Additionally, there is evidence that 17alpha-estradiol has anti-estrogenic effects in rats (PMID: 16042770). These findings imply possible feedback inhibition via estrogen receptors. Furthermore, 17alpha-estradiol likely differs from 17beta-estradiol due to its unique metabolic consequences and its potential to slow aging in males, an effect not attributed to 17beta-estradiol. For instance, neurons are also targets of 17alpha-estradiol, with Esr1 not being the sole target (PMID: 38776045). Intriguingly, neurons expressing Ar and Esr1 ranked among the top 20 most perturbed receptor subtypes during aging (O vs Y), but were no longer ranked in this group following treatment (O.T vs Y and O.T vs O comparisons). This indicates that 17α-estradiol administration attenuated age-associated perturbation in these neuronal subtypes, which may be a consequence of potential feedback (Figure 3D). Nevertheless, the precise effective targets of 17alpha-estradiol are still unresolved.

Line 409: This conclusion cannot be made because the effect is not statistically significant. Can say "trend" etc.

Thanks for the recommendation. We have added "potential" in front of the conclusion.

Line 426: "suggesting" please revise.

sorry, it’s a verb.

Lines 426-428: This is speculation. Please move to discussion.

The elevated GnRH levels in O.T., observed through EIA analysis, suggest a deduction regarding the direct causal effects of 17alpha-estradiol on various endocrine factors related to feeding, energy homeostasis, reproduction, osmotic regulation, stress response, and neuronal plasticity through MR analysis. Thus, we have not amended our position. We apologize for any confusion.

Lines 431-432: improved compared to what?

The statement have been revised as " The most striking role of 17α-estradiol treatment revealed in this study showed that HPG axis was substantially improved in the levels of serum Gnrh and testosterone".

Line 435: " Estrogen Receptor Antagonists". Please revise.

Thanks for the recommendation. We have changed it to "estrogen receptor antagonists".

Line 438" "Secrete". Please revise

Sorry, it is "secret".

Lines 439-449: None of this has been demonstrated. Please remove these conclusions.

We appreciate the reviewer's scrutiny regarding lines 439-449. While these statements should not be interpreted as definitive conclusions from our current data, we propose they serve as clinically relevant discussion points worthy of exploration. Our findings demonstrate 17α-estradiol's role in modulating testosterone levels in aged males. This mechanistic insight warrants consideration of its therapeutic potential for age-related hypogonadism - a hypothesis we believe merits discussion given the compound's specific endocrine effects.

Lines 450-457: No females were included in this study. Why? Also, why is this discussed? It is relevant but doesn't belong in this manuscript since it was not studied here.

Testosterone levels are crucial for male health, while estradiol levels are essential for the health and fertility of females. Previous studies have demonstrated that 17α-estradiol does not contribute to lifespan extension in females. Given the effects of 17α-estradiol on males—specifically, its role in promoting testosterone and reducing estradiol levels—we believe it is important to discuss the potential sex-biased effects of 17α-estradiol, as this could inform future investigations. We have refined this section to clarify that these points represent mechanistic hypotheses derived from our male data and existing literature, not conclusions about unstudied female physiology. This framing maintains the discussion's scientific value while respecting the study's scope.

Lines 458-459: This was not demonstrated in this article. Please remove.

We have restricted the claim to "expression level of energy metabolism in hypothalamic neurons".

Line 464: "Promoted lifespan extension" Not demonstrated. Please remove.

At the end of the sentence it was revised as "which may be a contributing factor in promoting lifespan extension".

Line 466: "Showed" No.

The whole sentence was deleted in the new version.

Line 483: "the sex-based effects". Not studied here.

Since the changes in testosterone levels are significant in this dataset and this hormone has a sex-biased nature, we find it worthwhile to suggest this as a topic for future investigation. We have added "which needs further verification in the future" at the end of this sentence.

listening to this song could be deadly and others who survive it forget about it like brainwashing.

It still brings sailors to jump overboard and the want to hear the secret ends

talking more on how important the song is to them and shows how it is less doubtful

shows how the song is irresistible

This section was a description of the natives that lived there. It seems like this guy came off patronizing in some way, as if they needed saving from their "primitive" way of living.

Discussion: Servants = Taxpayers to the king. Christians = People who are civilized compared to their "primitive ways"

Discussion Question: If the eukaryotic nucleus originated from a giant virus, that infected an archael ancestor of the eukaryotes (Bell, 2020), wouldn't that mean the original cell was an archaeal cell? And how "giant" does the virus have to be to be able to replicate this now?

https://www.sciencedirect.com/science/article/pii/S0168170220310753

"How did the discovery of the new Asgard Archaea change the way researchers understood eukaryotic evolution and the three branched tree of life?" Once scientists realized the newly discovered Asgard arhcaea genomes had genes that produced eukaryotic signature proteins, everything they knew about the origin of a eukaryotic cell was questioned. According to our understanding of the three domains of the tree of life, these proteins should not have been found in the Asgard archaea.

Advancement in technology happens frequently. We started with only one Asgard genome and within short amout of time, up to 2022, (the year of this publication), there were many more. According to an article in 2024, (Leao, et al, 2024), there were 869 Asgardarchaeota genomes. With the number of these genomes continually expanding, there is bound to be more research opportunities available, and each year ahead.

https://www.nature.com/articles/s41467-024-50195-2

"Why does research seek to understand the evolutionary history of the eukaryotic cell?" With new breakthroughs with the discovery of the Asgard Archaea, there are still so many studies that need to be performed to gain more knowledge about the origin of the eukaryotic cell. Scientists continue to develop more research and continue to search for answers to questions that seem impossible to answer, because the idea of understanding where life came from has endless possibilities for future advancement. It might hold the key to understanding diseases and how to cure them. Or it might offer answers about climate change and how our ecosystems function. It could possibly help farmers and ranchers, by eliminating animal and plant diseases. The potential is endless.

"Why do you think it is so hard for scientists to agree on a theory that supports the rise of the eukaryotic cell?" There is still so much debate on the origin of the nucleus, whether it has an archaeal origin or possibly came from a giant virus. I think once more research has been done, if possible, on the nucleus origin, we will be able to determine whether or not an update needs to happen to the current tree of life.

When Lady Penelope tickles my tummy when I was a baby

Chicken Little on Disney Junior

Ponies looking out your bedroom window

Bluey Cookalongs

Making a list of things to ask a support worker who was once a former psychologist

Tin Tin hanging upside down from the trees with her red panda friend

Feeling the gentleness of Lady Penelope's body near my skin during spa treatment

Playing the the pool with Alan and Tin Tin, and squirting Virgil with a water gun

Tin Tin playing with her peacock friend

Tin Tin and her animal friends falling asleep together on the peaceful beach

Staying up during a blackout

Alan hugging and cuddling me to the point where he indulges in tickles

Sharing a shower with Lady Penelope

Bacon frosting!

I Love Sugar (shop)

* Risk = arriesgar. * Downstairs = del piso de abajo. * Drinking buddies = compañeros de bebida.

* before = delante de. * right = justo.

* thug = matón , rufián. * listen up = escucha (imperativo).

* prove yourself = demuestra tu valía.

* Take on = asumir. * Hosting = anfitrión. * El demonio asume la personalidad del demonio anfitrion.

• whatever = lo que/lo que sea.
• have = tomar/ tener lo que se te ofrezca

* Plum = ciruela. * Butter = mantequilla. * cinnamon = canela.

* Asleep = dormir como adjetivo. * crapper = escusado.

* waste = despediciar.

* east = "este" de dirección cardinal.

* Holed up = Encerrado. * The rest is up to you = el resto depende de ustedes. * to be up to somewhere (modismo) es depender de x.

* get = recibir.

* Give up on (modismo) = rendirme con. * El with en el ingles es mas limitado que en el español. Solo se usa para indicar compañía como un adicional a la oración que complementa sin cambiar el significado de esta. Ejemplo: I wrote a thesis with my best friend.

• Cuando quieras usar los otros usos de con de las otras formas ,como en el español, hara referencia a una phrasal verb o preposicional-phrasal verb que lleve on.

* owe = debo a.

so why is this guy even important. he was a rapist to my people. FUCK Christopher Columbus

thats it. this can't be 100% accurate

They're going into middle-class areas and moving out the middle-class residents.

gentrification is REAL

The last part is NOT true. They are, in fact, making it harder to live there so that the people who are making the neighborhood bad move out along with their low income.

gentrification is what needs to happen if neighborhoods want to see improvement in the poor neighborhoods.

this can't be a good thing for either countries

I heard of this. Canada is definitely a friendlier country for this. this should be global. Like, how are charging millions of dollars for birth and and expensive medical treatment

!!!

key detail

good.

bad. Americans should be employed before a refugee.

I understand the US because what they're doing is putting the rightful people first. WHY DOESN"T THE SYRIAN GOVERNMENT TAKE CARE OF THEIR OWN. WHY DOES THE US ALWYAS GO THROUGH THIS

in search for a better life... so let me go to the united states where I don't belong to create a more worse life for those americans who are already struggling

absolutely awful for the true american citizens

largest river in the United states of America

the midwest aswell

interesting fact about the Boreal forest

US humans should be able to all help and benefit from our neighbor. It is stated in the bible that everyone should love their neighbor

why is Canada not apart of the US?

makes me question how something that doesn't move or eat lives for over 100 years and grows to its maximum size is even possible. How come Humans are not able to live healthy long lives

I have never heard of this tool before! Looking forward to exploring it this semester.

eLife Assessment

This is a well-done study that provides compelling data from a diverse set of approaches from single cell transcriptome data and network analysis from genetically diverse mouse cells to identify novel driver genes underlying human GWAS associations. The authors present solid evidence that network analysis of scRNA-seq data from genetically diverse mouse bone-marrow derived stromal cells can be informative for identifying human BMD GWAS driver genes. Their approach should be broadly useful and applicable to other GWAS studies.

Reviewer #1 (Public review):

In this manuscript, Dillard and colleagues integrate cross-species genomic data with a systems approach to identify potential driver genes underlying human GWAS loci and establish the cell type(s) within which these genes act and potentially drive disease.

Specifically, they utilize a large single cell RNA-seq (scRNA-seq) dataset from an osteogenic cell culture model - bone marrow-derived stromal cells cultured under osteogenic conditions (BMSC-OBs) - from a genetically diverse outbred mouse population called the Diversity Outbred (DO) stock to discover network driver genes that likely underlie human bone mineral density (BMD) GWAS loci. The DO mice segregate over 40M single nucleotide variants, many of which affect gene expression levels, therefore making this an ideal population for systems genetic and co-expression analyses.

The current study builds on previous published work from the same group that used co-expression analysis to identify co-expressed "modules" of genes that were enriched for BMD GWAS associations. In this study, the authors utilized a much larger scRNA-seq dataset from 80 DO BMSC-OBs, inferred co-expression based on Bayesian networks for each identified mesenchymal cell type, focused on networks with dynamic expression trajectories that are most likely driving differentiation of BMSC-OBs, and then prioritized genes ("differentiation driver genes" or DDGs) in these osteogenic differentation networks that had known expression or splicing QTLs (eQTL/sQTLs) in any GTEx tissue that co-localized with human BMD GWAS loci. The systems analysis is impressive, the experimental methods are described in detail, and the experiments appear to be carefully done. The computational analysis of the single cell data is comprehensive and thorough, and the evidence presented in support of the identified DDGs, including Tpx2 and Fgfrl1, is for the most part convincing. Some limitations in the data resources and methods hamper enthusiasm somewhat and are discussed below.

Overall, while this study will no doubt be valuable to the BMD community, the cross-species data integration and analytical framework may be more valuable and generally applicable to the study of other diseases, especially for diseases with robust human GWAS data but for which robust human genomic data in relevant cell types is lacking.

Specific strengths of the study include the large scRNA-seq dataset on BMSC-OBs from 80 DO mice, the clustering analysis to identify specific cell types and sub-types, the comparison of cell type frequencies across the DO mice, and the CELLECT analysis to prioritize cell clusters that are enriched for BMD heritability (Figure 1). The network analysis pipeline outlined in Figure 2 is also a strength, as is the pseudotime trajectory analysis (results in Figure 3).

Potential drawbacks of the authors' approach include their focus on genes that were previously identified as having an eQTL or sQTL in any GTEx tissue. The authors rightly point out that the GTEx database does not contain data for bone tissue, but reason that eQTLs can be shared across many tissues - this assumption is valid for many cis-eQTLs, but it could also exclude many genes as potential DDGs with effects that are specific to bone/osteoblasts. Indeed, the authors show that important BMD driver genes have cell-type specific eQTLs. Another issue concerns potential model overfitting in the iterativeWGCNA analysis of mesenchymal cell type-specific co-expression, which identified an average of 76 co-expression modules per cell cluster (range 26-153). Based on the limited number of genes that are detected as expressed in a given cell due to sparse per cell read depth (400-6200 reads/cell) and drop outs, it's surprising that as many as 153 co-expression modules could be distinguished within any cell cluster. I would suspect some degree of model overfitting is responsible for these results.

Overall, though, these concerns are minor relative to the many strengths of the study design and results. Indeed, I expect the analytical framework employed by the authors here will be valuable to -- and replicated by -- researchers in other disease areas.

Comments on revisions:

Thank you for addressing my concerns. This is an impressive study and manuscript that you should be proud of.

Reviewer #2 (Public review):

Summary:

In this manuscript, Farber and colleagues have performed single cell RNAseq analysis on bone marrow derived stem cells from DO Mice. By performing network analysis, they look for driver genes that are associated with bone mineral density GWAS associations. They identify two genes as potential candidates to showcase the utility of this approach.

Strengths:

The study is very thorough and the approach is innovative and exciting. The manuscript contains some interesting data relating to how cell differentiation is occurring and the effects of genetics on this process. The section looking for genes with eQTLs that differ across the differentiation trajectory (Figure 4) was particularly exciting.

Weaknesses:

The manuscript is, in parts, hard to read due to the use of acronyms and there are some questions about data analysis that still need to be addressed.

Comments on revisions:

Dillard et al have made several improvements to their manuscript.

(1) We previously asked the authors to determine whether any cell types were enriched for BMD-related traits since the premise of the paper is that 'many genes impacting BMD do so by influencing osteogenic differentiation or ... adipogenic differentiation'. Given the potential for the cell culture method to skew the cell type distribution non-physiologically, it is important to establish which cell types in their assay are most closely associated with BMD traits. The new CELLECT analysis and Figure 1E address this point nicely. However, it would still be nice to see the correlations between these cell types and BMD traits in the mice as this would provide independent evidence to support their physiological importance more broadly.

(2) Shortening the introduction.

(3) Addressing limitations that arise from not accounting for founder genome SNPs when aligning scRNA-seq data.

(4) The main take-away of this paper is, to us, the development of a single cell approach to studying BMD-related traits. It is encouraging that the cells post-culture appear to be representative of those pre-culture (supplemental figure 3).

However, the authors seem to have neglected several comments made by both reviewers. While we share the authors' enthusiasm for the single cell analytical approach, we do not understand their reluctance to perform further statistical tests. We feel that the following comments have still not been addressed:

(1) The manuscript still contains the following:

"To provide further support that tradeSeq-identified genes are involved in differentiation, we performed a cell type-specific expression quantitative trait locus (eQTL) analysis for each mesenchymal cell type from the 80 DO mice. We identified 563 genes (eGenes) regulated by a significant cis-eQTL in specific cell types of the BMSC-OB scRNA-seq data (Supplementary Table S14). In total, 73 eGenes were also tradeSeq-identified genes in one or more cell type boundaries along their respective trajectories (Supplementary Table S9)."

The purpose of this paragraph is to convince readers that the eGenes approach aligns with the tradeSeq approach (and that their approach can therefore be trusted). It is essential that such claims are supported by statistical reasoning. Given that it would be very simple to perform permutation/enrichment analyses to address this point, and both reviewers requested similar analyses, we do not understand the author's reluctance here. Otherwise, this section should be rewritten so that it does not imply that the identification of these genes provides support for their approach.

(2) Given that a central purpose of this manuscript is to establish a systematic workflow for identifying candidate genes, the manuscript could still benefit from more explanation as to why the authors chose to highlight Tpx2 and Fgfrl1. Tpx2 does already have a role in bone physiology through the IMPC. The authors should comment on why they did not explore Kremen1, for instance, as this gene seems important for the transition to both OB1 and 2.

A final minor comment is that it would be very helpful if the authors could indicate if the DDGs in Table 1 are also eGenes for the relevant cell type. This is much more meaningful than looking through GTEx.

Author response:

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

Reviewer #1 (Public review): 

In this manuscript, Dillard and colleagues integrate cross-species genomic data with a systems approach to identify potential driver genes underlying human GWAS loci and establish the cell type(s) within which these genes act and potentially drive disease. Specifically, they utilize a large single-cell RNA-seq (scRNA-seq) dataset from an osteogenic cell culture model - bone marrow-derived stromal cells cultured under osteogenic conditions (BMSC-OBs) - from a genetically diverse outbred mouse population called the Diversity Outbred (DO) stock to discover network driver genes that likely underlie human bone mineral density (BMD) GWAS loci. The DO mice segregate over 40M single nucleotide variants, many of which affect gene expression levels, therefore making this an ideal population for systems genetic and co-expression analyses. The current study builds on previously published work from the same group that used co-expression analysis to identify co-expressed "modules" of genes that were enriched for BMD GWAS associations. In this study, the authors utilize a much larger scRNA-seq dataset from 80 DO BMSC-OBs, infer co-expression-based and Bayesian networks for each identified mesenchymal cell type, focused on networks with dynamic expression trajectories that are most likely driving differentiation of BMSC-OBs, and then prioritized genes ("differentiation driver genes" or DDGs) in these osteogenic differentiation networks that had known expression or splicing QTLs (eQTL/sQTLs) in any GTEx tissue that colocalized with human BMD GWAS loci. The systems analysis is impressive, the experimental methods are described in detail, and the experiments appear to be carefully done. The computational analysis of the single-cell data is comprehensive and thorough, and the evidence presented in support of the identified DDGs, including Tpx2 and Fgfrl1, is for the most part convincing. Some limitations in the data resources and methods hamper enthusiasm somewhat and are discussed below. Overall, while this study will no doubt be valuable to the BMD community, the cross-species data integration and analytical framework may be more valuable and generally applicable to the study of other diseases, especially for diseases with robust human GWAS data but for which robust human genomic data in relevant cell types is lacking. 

Specific strengths of the study include the large scRNA-seq dataset on BMSC-OBs from 80 DO mice, the clustering analysis to identify specific cell types and sub-types, the comparison of cell type frequencies across the DO mice, and the CELLECT analysis to prioritize cell clusters that are enriched for BMD heritability (Figure 1). The network analysis pipeline outlined in Figure 2 is also a strength, as is the pseudotime trajectory analysis (results in Figure 3). One weakness involves the focus on genes that were previously identified as having an eQTL or sQTL in any GTEx tissue. The authors rightly point out that the GTEx database does not contain data for bone tissue, but the reason that eQTLs can be shared across many tissues - this assumption is valid for many cis-eQTLs, but it could also exclude many genes as potential DDGs with effects that are specific to bone/osteoblasts. Indeed, the authors show that important BMD driver genes have cell-type-specific eQTLs. Furthermore, the mesenchymal cell type-specific co-expression analysis by iterative WGCNA identified an average of 76 co-expression modules per cell cluster (range 26-153). Based on the limited number of genes that are detected as expressed in a given cell due to sparse per-cell read depth (400-6200 reads/cell) and dropouts, it's hard to believe that as many as 153 co-expression modules could be distinguished within any cell cluster. I would suspect some degree of model overfitting here and would expect that many/most of these identified modules have very few gene members, but the methods list a minimum module size of 20 genes. How do the numbers of modules identified in this study compare to other published scRNA-seq studies that use iterative WGCNA? 

In the section "Identification of differentiation driver genes (DDGs)", the authors identified 408 significant DDGs and found that 49 (12%) were reported by the International Mouse Knockout [sic] Consortium (IMPC) as having a significant effect on whole-body BMD when knocked out in mice. Is this enrichment significant? E.g., what is the background percentage of IMPC gene knockouts that show an effect on whole-body BMD? Similarly, they found that 21 of the 408 DDGs were genes that have BMD GWAS associations that colocalize with GTEx eQTLs/sQTLs. Given that there are > 1,000 BMD GWAS associations, is this enrichment (21/408) significant? Recommend performing a hypergeometric test to provide statistical context to the reported overlaps here. 

We thank the reviewer for their constructive feedback and thoughtful questions. In regards to the iterativeWGCNA, a larger number of modules is sometimes an outcome of the analysis, as reported in the iterativeWGCNA preprint (Greenfest-Allen et al., 2017). While we did not make a comparison to other works leveraging this tool for scRNA-seq, it has been used broadly across other published studies, such as PMID: 39640571, 40075303, 33677398, 33653874. While model overfitting, as you mention, may be a cause for more modules, our Bayesian network analysis we perform after iterativeWGCNA highlights smaller aspects of coexpression modules, as opposed to focusing on the entirety of any given module.

We did not perform enrichment or statistical tests as our goal was to simply highlight attributes or unique features of these genes for additional context.

Reviewer #2 (Public review): 

Summary: 

In this manuscript, Farber and colleagues have performed single-cell RNAseq analysis on bone marrow-derived stem cells from DO Mice. By performing network analysis, they look for driver genes that are associated with bone mineral density GWAS associations. They identify two genes as potential candidates to showcase the utility of this approach. 

Strengths: 

The study is very thorough and the approach is innovative and exciting. The manuscript contains some interesting data relating to how cell differentiation is occurring and the effects of genetics on this process. The section looking for genes with eQTLs that differ across the differentiation trajectory (Figure 4) was particularly exciting. 

Weaknesses: 

The manuscript is in parts hard to read due to the use of acronyms and there are some questions about data analysis that need to be addressed. 

We thank the reviewer for their feedback and shared enthusiasm for our work. We tried to minimize the use of technical acronyms as much as we could without compromising readability. Additionally, we addressed questions regarding aspects of data analysis. 

Reviewer #1 (Recommendations for the authors):

(1) For increased transparency and to allow reproducibility, it would be necessary for the scripts used in the analysis to be shared along with the publication of the preprint. Also, where feasible, sharing the processed data in addition to the raw data would allow the community greater access to the results and be highly beneficial. 

Thank you for this suggestion. The raw data will be available via GEO accession codes listed in the data availability statement. We will make available scripts for some analyses on our Github (https://github.com/Farber-Lab/DO80_project) and processed scRNA-seq data in a Seurat object (.rds) on Zenodo (https://zenodo.org/records/15299631)

(2) Lines 55-76: I think the summary of previous work here is too long. I understand that they would like to cover what has been done previously, but this seems like overkill. 

Good suggestion. We have streamlined some of the summary of our previous work.

(3) Did the authors try to map QTL for cell-type proportion differences in their BMSC-OBs? While 80 samples certainly limit mapping power, the data shown in Figs 4C/D suggest that you might identify a large-effect modifier of LMP/OB1 proportions. 

We did try to map QTL for cell type proportion differences, but no significant associations were identified. 

(4) Methods question: Does the read alignment method used in your analysis account for SNPs/indels that segregate among the DO/CC founder strains? If not, the authors may wish to include this in their discussion of study limitations and speculate on how unmapped reads could affect expression results. 

The read alignment method we used does not account for SNPs/indels from the DO founder strains that fall in RNA transcripts captured in the scRNA-seq data. We have included this as a limitation in our discussion (line 422-424). 

(5) Much of the discussion reads as an overview of the methods, while a discussion of the results and their context to the existing BMD literature is relatively lacking in comparison.

We have added additional explanation of the results and context to the discussion (line 381-382, 396-407). 

(6) Figure 1E and lines 146-149: Adjusted p values should be reported in the figure and accompanying text instead of switching between unadjusted and adjusted p values. 

We updated Figure 1e to portray adjusted p-values, listed the adjusted p-values in legend of Figure 1e, and listed them in the main text (line 153-154).

(7) Why do the authors bring the IMPC KO gene list into the analysis so late? This seems like a highly relevant data resource (moreso than the GTEx eQTLs/sQTLs) that could have been used much earlier to help identify DDGs. 

Given that our scRNA-seq data is also from mice, we did choose to integrate information from the IMPC to highlight supplemental features of genes in networks (i.e., genes that have an experimentally-tested and significant effect on BMD in mice). However, our primary goal was to inform human GWAS and leverage our previous work in which we identified colocalizations between human BMD GWAS and eQTL/sQTL in a human GTEx tissue, which is why this information was used to guide our network analysis.

(8) Does Fgfrl1 and/or Tpx2 have a cis-eQTL in your BMSC-OB scRNA-seq dataset? 

We did not identify cis-eQTL effects for Fgfrl1 and Tpx2.

(9) Figure 4B-C: These eQTLs may be real, but based on the diplotype patterns in Figure 4C, I suspect they are artifacts of low mapping power that are driven by rare genotype classes with one or two samples having outlier expression results. For example, if you look at the results in Fig 4C for S100a1 expression, the genotype classes with the highest/lowest expression have lower sample numbers. In the case of Pkm eQTL showing a PWK-low effect, the PWK genome has many SNPs that differ from the reference genome in the 3' UTR of this gene, and I wonder if reads overlapping these SNPs are not aligning correctly (see point 4 above) and resulting (falsely) in lower expression values for samples with a PWK haplotype. 

As mentioned above, our alignment method did not consider DO founder genetic variation that is specifically located in the 3’ end of RNA transcripts in the scRNA-seq data. We have included this as a limitation in our discussion (line 422-424).

In future studies, we intend to include larger populations of mice to potentially overcome, as you mention, any artifacts that may be attributable to low statistical power, rare genotype classes, or outlier expression.

Reviewer #2 (Recommendations for the authors):

Major Points 

(1) The authors hypothesize "that many genes impacting BMD do so by influencing osteogenic differentiation or possibly bone marrow adipogenic differentiation". However, cell type itself does not correlate with any bone trait. Does this indicate that the hypothesis is not entirely correct, as genes that drive these phenotypes would not be enriched in one particular cell type? The authors have previously identified "high-priority target genes". So, are there any cell types that are enriched for these target genes? If not, this would indicate that all these genes are more ubiquitously expressed and this is probably why they would have a greater effect on the overall bone traits. Furthermore, are the 73 eGenes (so genes with eQTLs in a particular cell type that change around cell type boundaries) or the DDGs (Table 1) enriched for these high-priority target genes? 

The bone traits measured in the DO mice are complex and impacted by many factors, including the differentiation propensity and abundance of certain cell types, both within and outside of bone. Though we did not identify correlations between cell type abundance and the bone traits we measured, we tailored our investigations to focus on cellular differentiation using the scRNA-seq data. However, future studies would need to be performed to investigate any connections between cellular differentiation, cell type abundance, and bone traits.

We did not perform enrichment analyses of either the target genes identified from our other work or eGenes identified here, but instead used the target gene list to center our network analysis and the eGenes to showcase the utility of the DO mouse population.

(2) The readability of the paper could be improved by minimising the use of acronyms and there are several instances of confusing wording throughout the paper. In many cases, this can be solved by re-organising sentences and adding a bit more detail. For example, it was unclear how you arrived at Fgfrl1 or Tpx2.

One of the goals of our study was to identify genes that have (to our knowledge) little to no known connection to BMD. We chose to highlight Fgfrl1 and Tpx2 because there is minimal literature characterizing these genes in the context of bone, which we speak to in the results (line 296-297). Additionally, we prioritized these genes in our previous work and they were identified in this study by using our network analyses using the scRNA-seq data, which we mention in the results (line 276-279).

(3) Technical aspects of the assay. In Figure 1d you show that the cell populations vary considerably between different DO mice. It would be useful to give some sense of the technical variance of this assay given that the assay involves culturing the cells in an exogenous environment. This could take the form of tests between mice within the same inbred strain, or even between different legs of the same DO mice to show that results are technically very consistent. It might also be prudent to identify that this is a potential limitation of the approach as in vitro culturing has the potential to substantially change the cell populations that are present. 

We agree that in vitro culturing, in addition to the preparation of single cells for scRNA-seq, are unavoidable sources of technical variation in this study. However, the total number of cells contributed by each of the 80 DO mice after data processing does not appear to be skewed and the distribution appears normal (see added figures, now included as Supplemental Figure 3). Therefore, technical variation is at least consistent across all samples. Nevertheless, we have mentioned the potential for technical variation artifacts in our study in the discussion (line 414-416).

(4) Need for permutation testing. "We identified 563 genes regulated by a significant eQTL in specific cell types. In total, 73 genes with eQTLs were also tradeSeq-identified genes in one or more cell type boundaries". These types of statements are fine but they need to be backed up with permutation testing to show that this level of enrichment is greater than one would expect by chance. 

We did not perform enrichment tests as our only goal was to 1. determine if eQTL could be resolved in the DO mouse population using our scRNA-seq data and 2. predict in what cell type the associated eQTL and associated eGene may have an effect.

(5) The main novelty of the paper seems to be that you have used single-cell RNA seq (given that you appear to have already detailed the candidates at the end). I don't think this makes the paper less interesting, but I think you need to reframe the paper more about the approach, and not the specific results. How you landed on these candidates is also not clear. So the paper might be improved by more robustly establishing the workflow and providing guidelines for how studies like this should be conducted in the future. 

We sought to not only devise a rigorous approach to analyze our single cell data, but also showcase the utility of the approach in practice by highlighting targets for future research (i.e., Fgfrl1 and Tpx2).

Our goal was to identify novel genes and we landed on these candidate genes (Fgfrl1 and Tpx2) because they had substantial data supporting their causality and they have yet to be fully characterized in the context of bone and BMD (line 295-297).

In regards to establishing the workflow, we have included rationale for specific aspects of our approach throughout the paper. For example, Figure 2 itemizes each step of our network analysis and we explain why each step is utilized throughout various parts results (e.g., lines 168-170, 179-181, 191-193, 202-203, 257-260, 276-277).

We have added a statement advocating for large-scale scRNA-seq from genetically diverse samples and network analyses for future studies (line 436-438).

Minor Points 

(1) In the summary you use the word "trajectory". Trajectories for what? I assume the transition between cell types, but this is not clear. 

We added text to clarify the use of trajectory in the summary (line 34).

(2) This sentence: "By 60 identifying networks enriched for genes implicated in GWAS we predicted putatively causal genes 61 for hundreds of BMD associations based on their membership in enriched modules." is also not clear. Do you mean: we predicted putatively causal genes by identifying clusters of co-expressed genes that were enriched for GWAS genes?" It is not clear how you identify the causal gene in the network. Is this just based on the hub gene? 

The aforementioned sentence has since been removed to streamline the introduction, as suggested by Reviewer 1.

In regards to causal gene identification, it is not based on whether it is hub gene. We prioritized a DDG (and their associated networks) if it was a causal gene that we identified in our previous work as having eQTL/sQTL in a GTEx tissue that colocalizes with human BMD GWAS.

(3) Figure 3C. This is good but the labels are quite small. Would be good to make all the font sizes larger. 

We have enlarged Figure 3C.

(4) Line 341 in the Discussion should be "pseudotemporal". 

We have edited “temporal” to “pseduotemporal”.

Humidifiers near the detector (person) can decrease the exposure to airborne pathogens.

Evidence that increased humidity decreases exposure.

Does more humidity in the room mean less change of exposure to aerosols if you are located far from the source. Additionally, time spent in the room from start of aerosolization has to be taken into consideration.

Examples of how aerosolization can occur that then leads to the pathogen particles becoming airborne.

So by increasing the humidity in a classroom/lab there could be more control over the dispersal of airborne pathogens?

Yes but instead of us being given a few options of what we can spend our time watching or looking at, we now get to decide what content we consume. Whether it be binging a show, or doom scrolling, participating in online forums we are not relegated to what's given to us, we find what we are looking for to consume.

I feel like this statement can be attributed to Soap Operas. They really did cater to the housewife who was home everyday. My grandmother used to stop all activities when her "stories" came on and if she wasn't home to catch them she learned to use her VHS recorder to tape them for when she got home.

This process is rather artificial, it's possible to allow shadowing variable of the context by changing the weakening rule.

Hi!

Not so much anymore, now we have news channels that only talk about the same news all day long. Circling back to the same topics that they had touched on earlier in the day.

I feel like with the change in streaming availability that this is not so much the case anymore for younger watchers. We pick and chose when we want to watch our new/favorite show. We can binge in little bits when we have time. We are not bound by the timing of television. And more so we have many more options of things besides tv, Podcasts, audiobooks. Television is no longer the go to for entertainment.

When I still had cable television years ago, I would switch between shows during commercial breaks so that I could catch multiple shows or pieces of shows at the same time. I have a serious hate for advertisements so I try to not watch them at all.

This seems like a more natural and sensible way to deal with advertisements. Creating breaks so that can add in more adds is a terrible setup and the audience recognizes that.

I have discovered some of my favorite musicians and styles of music just by getting to shows early and actually listening to the opening acts or the intermission act of a show.

This quote stuck out to me as well because it shows how language change is totally natural and unstoppable. Prof. Garley mentions the comparison between modern-day English and Shakespearean English, and that really made me think deeply about how vastly different the two are. The way we speak now is so far removed from how people communicated back then, and it proves the point that living languages are always shifting with how people actually use them. It also pushes back on prescriptive rules that try to hold onto outdated forms (like whom) even though most speakers don’t use them anymore. To me, it shows that change in language isn’t corruption, it’s proof that the language is very much alive and always adapting.

This quote resonated with me because it highlights the distinction between prescriptive and descriptive grammar. Prescriptive rules aren’t really about how language works, but more about social habits that people turned into “rules” over time, enforced by teachers, editors, and grammar books. The table manners comparison makes sense; just like elbows on the table doesn’t stop you from eating, breaking these grammar “rules” doesn’t necessarily stop language from working. It also made me think about how these rules are made up by people, which raises the bigger question of who really gets to decide what’s considered “proper” English. Is it people in higher social classes? And historically, has this “standard English” been tied to white, upper-class norms through what linguists call "standard language ideology"?

This underlines how other people's judgements try to limit our potential. Even though her boss told her that her writing was her weakness and that she should move on and try something else, that criticism did not define her abilities or her future success. The author's story is the perfect example that even when outside opinions are discouraging and limiting, our personal conviction can carry us through and allow us to grow beyond the limits that others try to place on us.

This shows the unfair reality that people are judged by the way that they speak rather than by who they are. When the doctor only stepped in when the daughter who spoke "perfect English" appeared, the bias against those who communicate differently was made readily apparent. Even though she voiced her concerns over her family history with brain tumors, she wasn't taken seriously because her language was imperfect. This reveals how language can create barriers to respect and care, even when the person's needs are serious and just as real and valid as the needs of those who speak "perfect English."

This segment suggests that because the author's mother expressed her thoughts imperfectly, others assumed her thoughts were imperfect as well. From my own experiences in foreign language classes, I know that even when my grammar and/or vocabulary was limited, my ideas were still there, and they were complex and meaningful. Language fluency does not determine the depth of thought, it changes the way that those thoughts are expressed.

This shows how language is deeply linked to perspective. Even though many people cannot understand the author's mother, the author understands her on a basis that goes farther than grammar, she understands the love, culture, and perspective that her language carries. It shows that even though strangers cannot understand, her words are reflective of her own lived experience and that language is not solely defined by "perfection" in sound, but rather by the truth and worldview that form the basis for the words.

A concessão de gratuidade de justiça não afasta a responsabilidade do beneficiário pelas despesas processuais e honorários.

O benefício somente suspende a exigibilidade pelo prazo de até 5 anos, podendo o credor realizar a cobrança de tais débitos uma vez comprovado a alteração da situação econômica do devedor.

Gerry Anderson characters attacking the owner of the boring charity shop in Invergordon

Toy Story Land's Alien Pizza Planet

Shark Tale (2004)

Thunderbirds returning to BBC 2 in September 2000

Bluey Fancy Restaurant frozen food range

Going around town in a white bunny costume

Alan taming the 'itchy monster' on my toddler self's head

Toy Story Animated Storybook

A satisfying Look At Life short

Lounging on the sofa with crisps and jellybeans by your side

The softness of nature

Fact is observing reality and having evidence so you can’t deny the real fact and opinion is saying what you believe yet having no evidence

We should be informed consumers of the information made available to us because decisions based on this information have significant consequences . And i truly agree the way you taking in and process the information is critical to how you may act based on little information

Use of research helps to illustrate a point and study

To my understanding meaning its important for research so we are relying on a hypothesis more than a theory . To have actual evidence proof rather then just a guessed prediction based on limited knowledge or a non sufficient evidence just rely on someone’s belief

How are children being influenced by the media that are exposed to. I feel not just children are being easily influenced a lot of adults as well . The children are just a easier target since there still growing and learning the sense of reality what’s real or not or how things function being more vulnerable or gullible

Hello, l'm looking forward to learning more about how I can deepen my understanding of grammar and syntax, as well as how it may apply to me, and help me improve as a journalist.

eLife Assessment

This manuscript introduces a potentially valuable large-scale fMRI dataset pairing vision and language, and employs rigorous decoding analyses to investigate how the brain represents visual, linguistic, and imagined content. The current manuscript blurs the line between a resource paper and a theoretical contribution, and the evidence for truly modality-agnostic representations remains incomplete at this stage. Clarifying the conceptual aims and strengthening both the dataset technicality and the quantitative analyses would improve the manuscript's significance for the fields of cognitive neuroscience and multimodal AI.

Reviewer #1 (Public review):

Summary:

The authors introduce a densely-sampled dataset where 6 participants viewed images and sentence descriptions derived from the MS Coco database over the course of 10 scanning sessions. The authors further showcase how image and sentence decoders can be used to predict which images or descriptions were seen, using pairwise decoding across a set of 120 test images. The authors find decodable information widely distributed across the brain, with a left-lateralized focus. The results further showed that modality-agnostic models generally outperformed modality-specific models, and that data based on captions was not explained better by caption-based models but by modality-agnostic models. Finally, the authors decoded imagined scenes.

Strengths:

(1) The dataset presents a potentially very valuable resource for investigating visual and semantic representations and their interplay.

(2) The introduction and discussion are very well written in the context of trying to understand the nature of multimodal representations and present a comprehensive and very useful review of the current literature on the topic.

Weaknesses:

(1) The paper is framed as presenting a dataset, yet most of it revolves around the presentation of findings in relation to what the authors call modality-agnostic representations, and in part around mental imagery. This makes it very difficult to assess the manuscript, whether the authors have achieved their aims, and whether the results support the conclusions.

(2) While the authors have presented a potential use case for such a dataset, there is currently far too little detail regarding data quality metrics expected from the introduction of similar datasets, including the absence of head-motion estimates, quality of intersession alignment, or noise ceilings of all individuals.

(3) The exact methods and statistical analyses used are still opaque, making it hard for a reader to understand how the authors achieved their results. More detail in the manuscript would be helpful, specifically regarding the exact statistical procedures, what tests were performed across, or how data were pooled across participants.

(4) Many findings (e.g., Figure 6) are still qualitative but could be supported by quantitative measures.

(5) Results are significant in regions that typically lack responses to visual stimuli, indicating potential bias in the classifier. This is relevant for the interpretation of the findings. A classification approach less sensitive to outliers (e.g., 70-way classification) could avoid this issue. Given the extreme collinearity of the experimental design, regressors in close temporal proximity will be highly similar, which could lead to leakage effects.

(6) The manuscript currently lacks a limitations section, specifically regarding the design of the experiment. This involves the use of the overly homogenous dataset Coco, which invites overfitting, the mixing of sentence descriptions and visual images, which invites imagery of previously seen content, and the use of a 1-back task, which can lead to carry-over effects to the subsequent trial.

(7) I would urge the authors to clarify whether the primary aim is the introduction of a dataset and showing the use of it, or whether it is the set of results presented. This includes the title of this manuscript. While the decoding approach is very interesting and potentially very valuable, I believe that the results in the current form are rather descriptive, and I'm wondering what specifically they add beyond what is known from other related work. This includes imagery-related results. This is completely fine! It just highlights that a stronger framing as a dataset is probably advantageous for improving the significance of this work.

Reviewer #2 (Public review):

Summary:

This study introduces SemReps-8K, a large multimodal fMRI dataset collected while subjects viewed natural images and matched captions, and performed mental imagery based on textual cues. The authors aim to train modality-agnostic decoders--models that can predict neural representations independently of the input modality - and use these models to identify brain regions containing modality-agnostic information. They find that such decoders perform comparably or better than modality-specific decoders and generalize to imagery trials.

Strengths:

(1) The dataset is a substantial and well-controlled contribution, with >8,000 image-caption trials per subject and careful matching of stimuli across modalities - an essential resource for testing theories of abstract and amodal representation.

(2) The authors systematically compare unimodal, multimodal, and cross-modal decoders using a wide range of deep learning models, demonstrating thoughtful experimental design and thorough benchmarking.

(3) Their decoding pipeline is rigorous, with informative performance metrics and whole-brain searchlight analyses, offering valuable insights into the cortical distribution of shared representations.

(4) Extension to mental imagery decoding is a strong addition, aligning with theoretical predictions about the overlap between perception and imagery.

Weaknesses:

While the decoding results are robust, several critical limitations prevent the current findings from conclusively demonstrating truly modality-agnostic representations:

(1) Shared decoding ≠ abstraction: Successful decoding across modalities does not necessarily imply abstraction or modality-agnostic coding. Participants may engage in modality-specific processes (e.g., visual imagery when reading, inner speech when viewing images) that produce overlapping neural patterns. The analyses do not clearly disambiguate shared representational structure from genuinely modality-independent representations. Furthermore, in Figure 5, the modality-agnostic encoder did not perform better than the modality-specific decoder trained on images (in decoding images), but outperformed the modality-specific decoder trained on captions (in decoding captions). This asymmetry contradicts the premise of a truly "modality-agnostic" encoder. Additionally, given the similar performance between modality-agnostic decoders based on multimodal versus unimodal features, it remains unclear why neural representations did not preferentially align with multimodal features if they were truly modality-independent.

(2) The current analysis cannot definitively conclude that the decoder itself is modality-agnostic, making "Qualitative Decoding Results" difficult to interpret in this context. This section currently provides illustrative examples, but lacks systematic quantitative analyses.

(3) The use of mental imagery as evidence for modality-agnostic decoding is problematic. Imagery involves subjective, variable experiences and likely draws on semantic and perceptual networks in flexible ways. Strong decoding in imagery trials could reflect semantic overlap or task strategies rather than evidence of abstraction.

The manuscript presents a methodologically sophisticated and timely investigation into shared neural representations across modalities. However, the current evidence does not clearly distinguish between shared semantics, overlapping unimodal processes, and true modality-independent representations. A more cautious interpretation is warranted. Nonetheless, the dataset and methodological framework represent a valuable resource for the field.

Reviewer #3 (Public review):

Summary:

The authors recorded brain responses while participants viewed images and captions. The images and captions were taken from the COCO dataset, so each image has a corresponding caption, and each caption has a corresponding image. This enabled the authors to extract features from either the presented stimulus or the corresponding stimulus in the other modality. The authors trained linear decoders to take brain responses and predict stimulus features. "Modality-specific" decoders were trained on brain responses to either images or captions, while "modality-agnostic" decoders were trained on brain responses to both stimulus modalities. The decoders were evaluated on brain responses while the participants viewed and imagined new stimuli, and prediction performance was quantified using pairwise accuracy. The authors reported the following results:

(1) Decoders trained on brain responses to both images and captions can predict new brain responses to either modality.

(2) Decoders trained on brain responses to both images and captions outperform decoders trained on brain responses to a single modality.

(3) Many cortical regions represent the same concepts in vision and language.

(4) Decoders trained on brain responses to both images and captions can decode brain responses to imagined scenes.

Strengths:

This is an interesting study that addresses important questions about modality-agnostic representations. Previous work has shown that decoders trained on brain responses to one modality can be used to decode brain responses to another modality. The authors build on these findings by collecting a new multimodal dataset and training decoders on brain responses to both modalities.

To my knowledge, SemReps-8K is the first dataset of brain responses to vision and language where each stimulus item has a corresponding stimulus item in the other modality. This means that brain responses to a stimulus item can be modeled using visual features of the image, linguistic features of the caption, or multimodal features derived from both the image and the caption. The authors also employed a multimodal one-back matching task, which forces the participants to activate modality-agnostic representations. Overall, SemReps-8K is a valuable resource that will help researchers answer more questions about modality-agnostic representations.

The analyses are also very comprehensive. The authors trained decoders on brain responses to images, captions, and both modalities, and they tested the decoders on brain responses to images, captions, and imagined scenes. They extracted stimulus features using a range of visual, linguistic, and multimodal models. The modeling framework appears rigorous, and the results offer new insights into the relationship between vision, language, and imagery. In particular, the authors found that decoders trained on brain responses to both images and captions were more effective at decoding brain responses to imagined scenes than decoders trained on brain responses to either modality in isolation. The authors also found that imagined scenes can be decoded from a broad network of cortical regions.

Weaknesses:

The characterization of "modality-agnostic" and "modality-specific" decoders seems a bit contradictory. There are three major choices when fitting a decoder: the modality of the training stimuli, the modality of the testing stimuli, and the model used to extract stimulus features. However, the authors characterize their decoders based on only the first choice-"modality-specific" decoders were trained on brain responses to either images or captions, while "modality-agnostic" decoders were trained on brain responses to both stimulus modalities. I think that this leads to some instances where the conclusions are inconsistent with the methods and results.

First, the authors suggest that "modality-specific decoders are not explicitly encouraged to pick up on modality-agnostic features during training" (line 137) while "modality-agnostic decoders may be more likely to leverage representations that are modality-agnostic" (line 140). However, whether a decoder is required to learn modality-agnostic representations depends on both the training responses and the stimulus features. Consider the case where the stimuli are represented using linguistic features of the captions. When you train a "modality-specific" decoder on image responses, the decoder is forced to rely on modality-agnostic information that is shared between the image responses and the caption features. On the other hand, when you train a "modality-agnostic" decoder on both image responses and caption responses, the decoder has access to the modality-specific information that is shared by the caption responses and the caption features, so it is not explicitly required to learn modality-agnostic features. As a result, while the authors show that "modality-agnostic" decoders outperform "modality-specific" decoders in most conditions, I am not convinced that this is because they are forced to learn more modality-agnostic features.

Second, the authors claim that "modality-specific decoders can be applied only in the modality that they were trained on, while "modality-agnostic decoders can be applied to decode stimuli from multiple modalities, even without knowing a priori the modality the stimulus was presented in" (line 47). While "modality-agnostic" decoders do outperform "modality-specific" decoders in the cross-modality conditions, it is important to note that "modality-specific" decoders still perform better than expected by chance (figure 5). It is also important to note that knowing about the input modality still improves decoding performance even for "modality-agnostic" decoders, since it determines the optimal feature space-it is better to decode brain responses to images using decoders trained on image features, and it is better to decode brain responses to captions using decoders trained on caption features.

"In fact, discontinuing invasive ventilation in favor of noninvasive respiratory support has been considered the single best approach that neonatologists can implement to reduce BPD."

Does reduction of invasive respiratory support decrease incidence of BPD? As discussed in respiratory class readings volutrauma, barotrauma, and breath stacking can still occur when utilizing non-invasive support.

Could you be more precise here? E.g. granularity? Mabye, this is a bad idea. Just an input.

Before, you state, that k has the least direct relation to fertilisation. So why is it particularly interesting? A clarifying sentence might help here.

Its not so nice to use : after :

I suggest to rephrase the sentences, e.g. to The desorbable P parameter ( P desorb ) behaved very ...

suggestion: including those of the standard STP

I always assumed the 'Black Plague' originated in Europe so I was surprised to see evidence that the plague began in Kyrgyzstan.

I always liked science so I researched that bit further and found that traces of the plague bacteria were discovered in the enamel of the deceased buried in Kyrgyzstan, the deceased who were said to have died of pestilence.

I feel this is a wise bit of advice to consider. Some people tend to be takers without offering anything themselves. Confucius tells us to reciprocate and to not impose on others what we would not do for ourselves. This reminded me of a saying a store manager I knew once said in that nobody should expect from others what they would not do themselves. As a society we would not get very far if we all expected things from others without contributions ourselves.

Confucius had been a Chinese philosopher.

This compares to our society in how our laws and secular teachings were influenced by Socretes, Plato, and Aristotle.

I found this bit interesting as positions of leadership here are based on scholarly and intellectual merit, not democracy or hereditary. This section made me consider my town's local city council. I reside in a small rural town of less than 3,000 people. When there are elections for school board representatives, city council, or other positions where one would run for...most people (locals born and raised here) in town vote for their relatives or family friend over the most qualified competent candidate. Right now the town itself is not faring well due to irresponsible spending, and that nobody on the outside wants to move to this town or start businesses here.

eLife Assessment

This study provides new important insights concerning pathogen variant-specific reproduction parameters from molecular sequencing and case finding. The methods for inferring which variants will likely emerge in subsequent epidemic cycles are solid. This article is of broad interest to infectious disease epidemiology researchers and mathematical modellers of the COVID-19 pandemic.

Reviewer #1 (Public review):

In this manuscript, the authors describe a new method to more accurately estimate the fitness advantage of new SARS-CoV-2 variants when they emerge. This was a key public health question during the pandemic and drove a number of important policy choices during the latter half of the acute phase of the pandemic. They attempt to link fitness to expected wave size. The analyses are tested on data from 33 different US states for which the data were considered sufficient. The main novelty of the method is that it links the frequency of variants to the number of cases and thus estimates fitness in terms of the reproduction number.

The results with the new method appear to be more consistent estimates of fitness advantage over time, suggesting that the methods suggested are more accurate than the comparator methods.

Given that the paper presents a methodological advancement, the absence of a simulation study is a weakness. I am satisfied that the trends estimated via the different approaches suggest a useful advancement for a difficult problem. However, the work would have been considerably stronger if synthetic data had been used to illustrate without doubt how the revised method better captures underlying, pre-specified differences in fitness.

Reviewer #2 (Public review):

Summary:

This study develops a joint epidemiological and population genetic model to infer variant-specific effective reproduction numbers Rt and growth advantages of SARS-CoV-2 variants using US case counts and sequence data (Jan 2021-Mar 2022). For this, they use the commonly used renewal equation framework, observation models (negative binomial with zero inflation and Dirichlet-multinomial likelihoods, both to account for overdispersion). For the parameterization of Rt, again, they used a classic cubic spline basis expansion. Additionally, they use Bayesian Inference, specifically SVI. I was reassured to see the sensitivity analysis on the generation time to check effects on Rt.

This is an incredibly robust study design. Integrating case and sequence data enables estimation of both absolute and relative variant fitness, overcoming limitations of frequency-only or case-only models. This reminds me of https://www.medrxiv.org/content/10.1101/2023.01.02.23284123v4.full

I also really appreciated the flexible and interpretable parameterization of the renewal equations with splines. But I may be biased since I really like splines!

The approach is justified, however, it has some big limitations. Specifically, there are some notable weaknesses, that I detail below.

(1) The model does not account for demographic stochasticity or transmission overdispersion (superspreading), which are known to affect SARS-CoV-2 dynamics and can bias Rt, especially in low incidence or early introduction phases.

(2) While the authors explore the sensitivity of generation time, the reliance on fixed generation time parameters (with some adjustments for Delta/Omicron) may still bias results

(3) There is no explicit adjustment for population immunity, which limits the ability to disentangle intrinsic variant fitness (even though the model allows for inclusion of covariates - this to me is one of two major flaws in the study.

(4) The second major flaw in my opinion is that there is no hierarchical pooling across states - each state is modeled independently. A hierarchical Bayesian model could borrow strength across states, improving estimates for states with sparse data and enabling more robust inference of shared variant effects.

I would strongly recommend the following things in order of priority, where the first two points I consider critical.

(1) Implement a hierarchical model for variant growth advantages and Rt across states.

(2) Include time-varying covariates for vaccination rates, prior infection, and non-pharmaceutical interventions directly. This would help disentangle intrinsic variant transmissibility from changes in population susceptibility and behavior.

(3) Extend the renewal model to a stochastic or branching process framework that explicitly models overdispersed transmission.

(4) It would be good to allow for multiple seeding events per variant and per state. This can be informed by phylogeography in a minimum effort way and would improve the accuracy of Rt.

(5) By now, I don't think it will be a surprise that addressing sampling bias is standard, reweighting sequence data or comparing results with independent surveillance data to assess the impact of non-representative sequencing.

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The equation CaCl₂(aq) + Na₂CO₃(aq) → CaCO₃(s) + 2NaCl(aq) shows that an aqueous solution of calcium chloride reacts with an aqueous solution of sodium carbonate to produce a solid precipitate of calcium carbonate and an aqueous solution of sodium chloride. This is a double displacement reaction that can be further classified as a precipitation reaction because it forms an insoluble solid.

a popular ancient Roman winter festival honoring the god Saturn.

What are these "savage societies"? What are these "tribes" he is referring to?

Perhaps this is why being a part of a team is such a unique experience. You may not otherwise have anything in common with a teammate, and outside of the sport you may never have crossed paths or become friends, but because you were on the same team, there is a unique bond formed that never really disappears.

The spoil sport rejects the rules and thus rejects the game itself. The cheater rejects the rules but still continues to take part in the game-world.

The idea that the tension element confers a kind of ethical aspect to play is one that I agree with; as the author says, when a game is full of tension, the players character is revealed

I didn't understand what this meant so I looked it up; apparently "warp and woof" refers to the fundamental components of weaving, and as an idiom it refers to the "essential foundation or base" of a structure. So, basically what he's saying is that repetition and alternation are essential components on which play is built.

I am curious to learn more about this link between play and the "sacred sphere." I do understand how the author tied the origins of ritual to play, but how exactly do they connect now? Would praying or going to ones place of worship for service be considered a form of play?

I like that the author is taking time to emphasize that play and non-seriousness cannot be conflated.

Interesting. Sometimes I am tired and don't feel like going to basketball practice or training. But I do go, because I feel like I have to, and in general I do like the sport, just not all the time. According to this argument, on the when I don't feel like going, I am not actually playing?

This thought makes me think of the way watching a dog or another animal run around highlights its muscles and athleticism. I've never thought about it this way, but physical play does, in a way, display the body in it's most active and beautiful form.

from the beginning, we've used fiction to work through our sorrow

double meaning: shepherd, tyrant

wow just like lotr

Rhetoric:Art of persuasion

Look at the context, summarization, before analyzing

Is the author consistent and supportive of thesis or claim? Rhetorical moves? Tone?Objective? Does the author know the audience? Do you believe the author? Does the flow of text make sense? Logical reasoning? Emotional appeal?

This passage truly reveals the danger of waiting until we don't have fear to speak because silence will only grow heavier the longer it lingers. Even though fear will often never completely disappear, if we wait for it to go away completely, it is more likely that our silence will completely overtake us, and our thoughts and beliefs will never be shared.

This also serves as a powerful reminder because if we truly embody the truths that we hold deeply in our lives, we will share them every opportunity we get. By living and speaking these truths, we teach others through our actions and words, which gives them an extra layer of power and credibility.

This is such a powerful segment of this piece. In this definition, the author combines all of her personal experiences, and you can feel her conviction and determination radiating from the words. In so doing, she once again underscores the importance of speaking up because without standing up for what you believe in, the voices of others will overtake you, and you will lose a piece of yourself in the process.

This particular passage highlights how silence often stems from fear or hesitation, leading us to betray ourselves in a way by holding back our true voices. Although silence may feel safer, it limits our growth and impact. Because of this, choosing to speak is powerful, in that it confronts our fear, sparks change, and affirms our identity and the influence our voices can have both on those around us and on our situation.

Rhetorical situation: context or set of circumstances out of which a text arises

How rhetorical situation(context) shapes the rhetorical act(text)

Concepts; author, audience, setting, purpose, text

Author authority and identity, values/pperspective

Audience engagement, demographic, assumptions of author, where article is published, context audience receives

Setting, did something specific happen that provided motivation to speak out?

Purpose, what’s being achieved

Text format, image, wiritten essay, protest?

A trace