DOI: 10.1038/s44318-024-00233-y

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What is this?

DOI: 10.1038/s44318-024-00233-y

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What is this?

DOI: 10.1038/s44318-024-00233-y

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What is this?

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

With socioeconomic development, more and more people are obese which is an important reason for sub-fertility and infertility. Maternal obesity reduces oocyte quality which may be a reason for the high risk of metabolic diseases for offspring in adulthood. Yet the underlying mechanisms are not well elucidated. Here the authors examined the effects of maternal obesity on oocyte methylation. Hyper-methylation in oocytes was reported by the authors, and the altered methylation in oocytes may be partially transmitted to F2. The authors further explored the association between the metabolome of serum and the altered methylation in oocytes. The authors identified decreased melatonin. Melatonin is involved in regulating the hyper-methylation of high-fat diet (HFD) oocytes, via increasing the expression of DNMTs which is mediated by the cAMP/PKA/CREB pathway.

Strengths:

This study is interesting and should have significant implications for the understanding of the transgenerational inheritance of GDM in humans.

Thank you for your positive comments to our manuscript.

Weaknesses:

The link between altered DNA methylation and offspring metabolic disorders is not well elucidated; how the altered DNA methylation in oocytes escapes reprogramming in transgenerational inheritance is also unclear.

Thanks. These are very good questions. There is a long way to completely elucidate the relationship between methylation and offspring metabolic disorders, and the underlying mechanisms of obtained methylation escaping the reprogramming during development. We would like to explore these in the future.

Reviewer #2 (Public Review):

This manuscript offers significant insights into the impact of maternal obesity on oocyte methylation and its transgenerational effects. The study employs comprehensive methodologies, including transgenerational breeding experiments, whole genome bisulfite sequencing, and metabolomics analysis, to explore how high-fat diet (HFD)-induced obesity alters genomic methylation in oocytes and how these changes are inherited by subsequent generations. The findings suggest that maternal obesity induces hyper-methylation in oocytes, which is partly transmitted to F1 and F2 oocytes and livers, potentially contributing to metabolic disorders in offspring. Notably, the study identifies melatonin as a key regulator of this hyper-methylation process, mediated through the cAMP/PKA/CREB pathway.

Strengths:

The study employs comprehensive methodologies, including transgenerational breeding experiments, whole genome bisulfite sequencing, and metabolomics analysis, and provides convincing data.

Thank you for your positive comments to our manuscript.

Weaknesses:

The description in the results section is somewhat verbose. This section (lines 126~227) utilized transgenerational breeding experiments and methylation analysis to demonstrate that maternal obesity-induced alterations in oocyte methylation (including hyper-DMRs and hypo-DMRs) can be partially transmitted to F1 and F2 oocytes and livers. The authors should consider condensing and revising this section for clarity and brevity.

Thanks for your suggestions. We have re-written this parts in the revised manuscript.

There is a contradiction with Reference 3, but the discrepancy is not discussed. In this study, the authors observed an increase in global methylation in oocytes from HFD mice, whereas Reference 3 indicates Stella insufficiency in oocytes from HFD mice. This Stella insufficiency should lead to decreased methylation (Reference 33). There should be a discussion of how this discrepancy can be reconciled with the authors' findings.

Thanks for your suggestions. As reported by Reference 33, STELLA prevents hypermethylation in oocytes by sequestering UHRF1 from the nuclei which recruits DNMT1 into nuclei. Han et al. reported that obesity induced by high-fat diet reduces STELLA level in oocytes. These indicate that STELLA insufficiency might induce hypermethylation in oocytes, although significant hypermethylation in obese oocytes is not reported by Han et al. using immunofluorescence. This contradiction may be caused by the limited sample sizes (n=14) used by Han et al. We have added a brief discussion in the revised manuscript.

Reviewer #3 (Public Review):

Summary:

Maternal obesity is a health problem for both pregnant women and their offspring. Previous works including work from this group have shown significant DNA methylation changes for offspring of obese pregnancies in mice. In this manuscript, Chao et al digested the potential mechanisms behind the DNA methylation changes. The major observations of the work include transgenerational DNA methylation changes in offspring of maternal obesity, and metabolites such as methionine and melatonin correlated with the above epigenetic changes. Exogenous melatonin treatment could reverse the effects of obesity. The authors further hypothesized that the linkage may be mediated by the cAMP/PKA/CREB pathway to regulate the expression of DNMTs.

Strengths:

The transgenerational change of DNA methylation following HFD is of great interest for future research to follow. The metabolic treatment that could change the DNA methylation in oocytes is also interesting and has potential relevance to future clinical practice.

Thank you for your positive comments to our manuscript.

Weaknesses:

The HFD oocytes have more 5mC signal based on staining and sequencing (Fig 1A-1F). However, the authors also identified almost equal numbers of hyper- and hypo-DMRs, which raises questions regarding where these hypo-DMRs were located and how to interpret their behaviors and functions. These questions are also critical to address in the following mechanistic dissections as the metabolic treatments may also induce bi-directional changes of DNA methylation. The authors should carefully assess these conflicts to make the conclusions solid.

Thanks for the helpful comments and suggestions. As presented in Fig. 1F, there is an increase of methylation level in promoter and exon regions and there is a decrease in intron, utr3 and repeat regions. According to the suggestions, we further analyzed the distribution of DMRs, and found that hypo-DMRs were mainly distributed at utr3, intron, repeat, and tes regions compared with hyper-DMRs (Fig. S3). These suggest that the distribution of DMRs in genome is not random.

The transgenerational epigenetic modifications are controversial. Even for F0 offspring under maternal obesity, there were different observations compared to this work (Hou, YJ., et al. Sci Rep, 2016). The authors should discuss the inconsistencies with previous works.

Thanks for the suggestions. There are contradictions on the whole genome DNA methylation of oocytes in obese mice. Hou YJ et al. in 2016 reported that obesity reduces the whole genome DNA methylation of NSN GV oocytes using immunofluorescence. In 2018, Han LS et al. reported that the whole genome 5mC of oocytes is not significantly influenced by obesity using immunofluorescence, but they find the Stella level is reduced in oocytes by obesity. Stella locates in the cytoplasm and nuclei of oocytes and sequesters Uhrf1 from the nuclei. Stella knockout in oocytes results in about twofold increase of global methylation in MII oocytes via recruiting more DNMT1 into nuclei. These suggest that the global methylation of oocytes in obese mice should be increased, but the similar methylation in oocytes between obese and non-obese mice is reported by Han LS et al. Thus, the contradiction may be induced by the different sample size in our manuscript and previous studies, and Hou YJ and colleagues just examined the methylation of NSN GV oocytes. As present in Stella+/- oocytes, the global methylation of oocytes is normal, which suggest that the insufficiency of Stella may be not the main reason for the increased methylation of oocytes in obese mice. We have added a brief discussion in the revised manuscript.

In addition to the above inconsistencies, the DNA methylation analysis in this work was not carefully evaluated. Several previous works were evaluating the DNA methylation in mice oocytes, which showed global methylation levels of around 50% (Shirane K, et al. PLoS Genet, 2013; Wang L., et al, Cell, 2014). In Figure 1E, the overall methylation level is about 23% in control, which is significantly different from previous works. The authors should provide more details regarding the WGBS procedure, including but not limited to sequencing coverage, bisulfite conversion rate, etc.

Thanks for the good questions. Smallwood et al. reported the the CG methylation of MII oocyte is about 33.1% (Smallwood et al. Nature Methods, 2014) using single-cell genome-wide bisulfite sequencing. Shirane K et al. reported that the average methylation level of GV oocytes is 37.9%. Kobayashi H et al. Reported that the CG methylation in GV oocytes is about 40% (Kobayashi H et al. Plos Genet. 2012). CG methylation in fully grown oocytes is about 38.7% (Maenohara S et al. Plos Genet. 2017). The variation of methylation in oocytes is associated with sequencing methods, sequencing depth, and mapping rates. In the present study, whole genome bisulfite sequencing (WGBS) for small sample and methylation analysis were performed by NovoGene. The reads are 31613641 to 37359643, unique mapping rate is ≥32.88%,  conversation rate is > 99.44%, and sequencing depth is 2.45 to 2.75. Relative information is presented in Table S1. The sequencing depth might be a reason for the inconsistence. But we further confirmed our sequencing results using bisulfite sequencing (BS), and the result is similar between BS and WGBS results. These findings suggest that our results are reliable.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) Since the results show that melatonin may play a role in hyper-methylation, the authors need to give some basic information in the Introduction section.

Thanks. We added more information in the section of Introduction.

(2) There are many differential metabolites identified. Besides melatonin, other differential metabolites are involved in the altered methylation in oocytes

These is a good question. We firstly filtered the differential metabolites which may be involved in methylation, and then further filtered these metabolites according to the relative DNA methylation pathways and published papers. After that, we confirmed the concentrations of relative metabolites in the serum using ELISA. Certainly, we can not completely exclude all the metabolites which might involved in regulating DNA methylation.

(3) The altered methylation would be found in the F1 tissues. Did the authors examine the other parts besides the liver?

Thank you. In the present study, we didn’t examined the DNA methylation in the other tissues besides the liver. We agree that the altered methylation should be observed in the other tissues.

(4) Did the authors try or guess how many generations the maternal obesity-induced genomic methylation alterations can be transmitted?

Thanks. This is a good question. Takahashi Y and colleagues reported that obtained DNA methylation at CpG island can be transmitted across multiple generations using DNA methylation-edited mouse (Takahashi Y et al. 2023, cell). Similar inheritance is also reported by other studies using different models.

(5) The F2 is indirectly affected by maternal obesity, so the evidence is not enough to prove the transgenerational inheritance of the altered methylation.

Thanks. We find the altered DNA methylation in F2 tissue and oocytes is similar to that in F1 oocytes. These suggest the altered DNA methylation in F2 oocytes should be at least partly transmitted to F3. Previous paper (Takahashi Y et al. 2023, cell) confirms that obtain DNA methylation in CpG island can be transmitted across several generations through paternal and maternal germ lines. Certainly, it’s better if it is examined in F3 tissues.

Reviewer #2 (Recommendations For The Authors):

(1) Figure Font Size: The font sizes in the figures are quite inconsistent. Please try to uniform the font size of similar types of text.

Thanks for your suggestions. We re-edited the relative figures in the revised manuscript.

(2) Figure Clarity: Ensure that all critical information in the figures is clearly visible, such as in Figure 3C.

Thank you. We revised this figure.

(3) Figure 1B, C: The position of the asterisks ("**") is not centered in the corresponding columns, and the font size is too small. Please correct this and address similar issues in other figures.

Thank you for your suggestions. We re-edited these in the revised figures.

(4) Line 126: The current expression is confusing. It may be revised to: "Both the oocyte quality and the uterine environment can contribute to adult diseases, which may be mediated by epigenetic modifications."

Thanks. We revised this sentence in the revised manuscript.

(5) Missing Panel in Figure 3: Figure 3 is missing panel 3N.

Thank you so much. We corrected it in the revised manuscript.

(6) Figure Panel Order: Please adjust the order of the panels in the figures to follow a logical reading sequence.

Thank you. We changed the orders in the revised manuscript.

(7) Line 493: Correct "inthe" to "in the".

Thank you. We revised it.

(8) Lines 102-106: Polish the wording and expression, an example as follows: "We analyzed the differentially methylated regions (DMRs) in oocytes from both HFD and CD groups and identified 4,340 DMRs. These DMRs were defined by the criteria: number of CG sites {greater than or equal to} 4 and absolute methylation difference {greater than or equal to} 0.2. Among these, 2,013 were hyper-DMRs (46.38%) and 2,327 were hypo-DMRs (53.62%) (Fig. 1G). These DMRs were distributed across all chromosomes (Fig. 1H). "

Thank you! We re-wrote these parts in the revised manuscript.

Reviewer #3 (Recommendations For The Authors):

The sample numbers should be annotated in the figure legend for all the bar plots using Image J. The lines in Figures 2B and 2C were without error bars. How many mice were used for these plots?

Thanks for your suggestions. We added the sample size in the revised manuscript. We made a mistake when we prepared the pictures for figure 2B and figure 2C, which resulted in missing the error bars. We have corrected these pictures. Thanks again!

The authors should revise the panel arrangement of the figures (Figure 2, Figure 5, etc) to make them more clear and readable.

Thank you! We have revised these in the revised manuscript.

The writing should be improved since there were multiple typos and unclear expressions. AI tools like Grammarly or ChatGPT may help.

Thank you! We have re-edited the language in the revised manuscript using AI tools.

Please recheck the immunofluorescence images for clear interpretability. For example, in Figure 5F (H89 treated), the GV is all the way at the edge of the oocyte, and the oocyte in the DIC image appears like it is partially lysed. The DIC images and the DAPI images are not clear enough.

Thanks for your suggestions. We have re-edited these pictures in the revised manuscript.

Another concern is that the Methods describes the immunofluorescence preparation for 5mC and 5hmC staining as a simple fixation in 4% paraformaldehyde followed by permeabilization with .5% TritonX-100, but there is no antigen exposure step described, a step that is normally required for visualizing these DNA modifications (e.g., 4N HCl).

Thanks. Sorry for that we didn’t describe the methods clearly. We have added more information about the methods in the revised manuscript.

The metabolomic analysis revealed a highly significant increase in dibutylphthalate, genistein, and daidzein in the control mice. The presence of these exogenous metabolites suggests that the diets differed in many aspects, not just fat content, so it would be very difficult to interpret the results as related to a high-fat diet alone. Both daidzein and genistein are phytoestrogens and dibutylphthalate is a plasticizer, suggesting differences in the diet and/or in the materials used to collect the samples for analysis from the mice. The Methods define the high-fat diet adequately, as the formulation can be found online using the catalog number. However, the control diet is just listed as "normal diet", so one has no idea what is in it

Thank you for your good questions. The daidzein and genistein may be from the diets and the dibutylthalate may be from the materials used to collect samples. If so, these should be similar between groups. Thus, we added the formulation of normal diet in the revised manuscript. The raw materials of normal diet include corn, bean pulp, fish meal, flour, yeast powder, plant oil, salt, vitamins, and mineral elements. According to the suggestions, we re-checked the data about these metabolites, and found that the abundance of these metabolites was low. And the result of these metabolites was at a low confidence level because the iron of these metabolites was only mapped to ChemSpider(HMDB,KEGG,LIPID MAPS). To further confirm these results, we examined these metabolites in serum using ELISA, and results revealed that the concentrations of genistein and dibutylthalate were similar between groups. These results suggest that these metabolites may be not involved in the altered methylation of oocytes induced by obesity.

Cette question est-elle vraiment utile ? (i) Je n'ai pas l'impression que le texte y réponde. (ii) Par définition, une variation du taux modifie l’orientation par rapport à un contrefactuel ou le taux n'est pas modifié.

Reviewer #2 (Public review):

Summary:

This paper introduces a new methodology for probing time-varying causal interactions in complex dynamical systems using a novel machine-learning architecture of Temporal Autoencoders for Causal Inference (TACI) combined with a novel metric (CSGI) for assessing causal interactions using surrogate data. This is a timely contribution in the field of causal inference from temporal data which has been largely restricted to stationary time series so far. However, the benchmarking of the proposed methods could be improved.

Strength:

The method's capacity to uncover piecewise time-varying non-linear dynamic systems is demonstrated on synthetic datasets as well as on two real-world applications on climate and brain activity data. A particular advantage of the approach is to train a single model capturing the dynamics of the whole time series, thereby allowing for time-varying interactions to be found without retraining over different time periods.

Weaknesses:

(1) It is not clear why the new metric Comparative Surrogate Granger Index CSGI (Eq.6) should be better than the Extended Granger Causality Index EGCI (Eq.5), which can also be used to compare the information about y(t) contained in the actual data x(t) versus in a randomized surrogate x^s(t), as implemented in the proposed metric (Eq.6).

(2) The benchmarking of the new approach TACI against earlier metrics (ie Surrogate Linear Granger, Convergent Cross Mapping, and Transfer Entropy) should be revised:

(a) The details of the computation should be provided to clarify how the different metrics are estimated notably between multidimensional variables [for instance to estimate Ty->x for x=(x_1,x_2,x_3) and y=(y_1,y_2,y_3)].

(b) Reliable implementations of the different metrics should be used, as some of the reported results do not seem right. In particular, the unidirectional examples, Eq.9 (Figure 2) and Eq.12 (Figure 5), are expected to lead to vanishing transfer entropies from Y to X, ie Ty->x =0, for all values of the coupling parameter below the synchronization threshold. This can be verified by computing transfer entropies as conditional mutual information using MIIC R package, i.e. Ty->x = I(x(t);y(t-1)|x(t-1)).

(c) Besides, some reported benchmarks focus on peculiar non-linear systems displaying somewhat "pathological" behaviors. For instance, the two Hénon maps with unidirectional coupling Eq.12 (Figure 5) lead to an equality between the two variables, i.e. y(t)=x(t) for all t, above the synchronization threshold C>0.7. This leads mathematically to zero transfer entropy upon synchronization, as I(x(t);y(<br /> d) By contrast, Eq.9 (Fig.2) leads to strongly coupled, yet non-identical variables above the synchronization threshold. This strong coupling can be shown to yield non-vanishing transfer entropies in both directions, as observed in Figure 2c, and does not correspond to "incorrect prediction of non-existent interactions", as stated in the "Summary of Results on Artificial Test Systems". Clearly synchronized variables do interact and their bidirectional transfer entropies are actually consistent with a non-causal (or bidirectional) relationship. Only a vanishing transfer entropy in one direction implies a causal relation (in the opposite direction). Likewise, vanishing transfer entropies in both directions imply either independent variables or a spurious dependency between them due to an unobserved common cause L, i.e. X<--(L)-->Y. This is usually represented with a bidirected edge (X<-->Y), which is different from a bidirectional relation corresponding to two opposite unidirectional edges (ie X-->Y and X<--Y). It is therefore surprising that TACI metric vanishes in both directions upon synchronization in this case (Eq.9, Figure 2), as one would expect to learn variable y(t) more reliably using the actual data x(<br /> e) In order to assess TACI performance on non-stationary time series, it might be more informative to benchmark it on datasets displaying intermittency rather than synchrony. In particular, the change of causal directions over time, presented as one of the motivations for the new approach, should be more thoroughly benchmarked in the paper. For instance, it would be nice to demonstrate the tracking of the spontaneous reversal of causal relation in a simple 'toggle switch' regulatory network between two mutually repressing genes + expression noise. This is something that causal inference methods assuming stationarity cannot do.

(3) Concerning the real-world applications, the analysis of the electrocorticography (ECoG) data does not seem to be in strong disagreement with the general trends of the original more detailed study by Tajima et al 2015. Could the authors better delineate what are the common versus conflicting findings between the two approaches? The main difference appears to be the near loss of interaction in the anesthetized state, which might be linked to TACI's tendency to report no interaction between synchronized variables as discussed in d) above. Does the anesthetized state correspond to a global synchrony of the brain regions? This could be easily validated by a more direct analysis of synchrony.

Reviewer #1 (Public review):

Summary:

Abdelmageed et al. investigate age-related changes in the subcellular localization of DNA polymerase kappa (POLK) in the brains of mice. POLK has been actively investigated for its role in translesion DNA synthesis and involvement in other DNA repair pathways in proliferating cells, very little is known about POLK in a tissue-specific context, let alone in post-mitotic cells. The authors investigated POLK subcellular distribution in the brains of young, middle-aged, and old mice via immunoblotting of fractioned tissue extracts and immunofluorescence (IF). Immunoblotting revealed a progressive decrease in the abundance of nuclear POLK, while cytoplasmic POLK levels concomitantly increased. Similar findings were present when IF was performed on brain sections. Further, IF studies of the cingulate cortex (Cg1), the motor cortex (M1, M2), and the somatosensory (S1) cortical regions all showed an age-related decline in nuclear POLK. Nuclear speckles of POLK decrease in each region, meanwhile, the number of cytoplasmic POLK granules decreases in all four regions, but granule size is increasing. The authors report similar findings for REV1, another Y-family DNA polymerase.

The authors then investigate the colocalization of POLK with other DNA damage response (DDR) proteins in either pyramidal neurons or inhibitory interneurons. At 18 months of age, DNA damage marker gH2AX demonstrated colocalization with nuclear POLK, while strong colocalization of POLK and 8-oxo-dG was present in geriatric mice. The authors find that cytoplasmic POLK granules colocalize with stress granule marker G3BP1, suggesting that the accumulated POLK ends up in the lysosome.

Brain regions were further stained to identify POLK patterns in NeuN+ neurons, GABAergic neurons, and other non-neuronal cell types present in the cortex. Microglia associated with pyramidal neurons or inhibitory interneurons were found to have a higher abundance of cytoplasmic POLK. The authors also report that POLK localization can be regulated by neuronal activity induced by Kainic acid treatment. Lastly, the authors suggest that POLK could serve as an aging clock for brain tissue, but POLK deserves further characterization and correlation to functional changes before being considered as a biomarker.

Strengths:

Investigation of TLS polymerases in specific tissues and in post-mitotic cells is largely understudied. The potential changes in sub-cellular localization of POLK and potentially other TLS polymerases open up many questions about DNA repair and damage tolerance in the brain and how it can change with age.

Weaknesses:

The work is quite novel and interesting, and the authors do suggest some potentially interesting roles for POLK in the brain, but these are in and of themselves a bit speculative. The majority of the findings of this paper draw upon findings from POLK antibody and its presumed specificity for POLK. However, this antibody has not been fully validated and needs further work. Further validation experiments using Polk-deficient or knocked-down cells to investigate antibody specificity for both immunoblotting and immunofluorescence should be performed. More mechanistic investigation is needed before POLK could be considered as a brain aging clock.

DOI: 10.7150/thno.97165

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La entrada de una célula a fase M depende entonces, del factor promotor de maduración, el cual tiene 2 subunidades: 1. cinasa: transfiere grupos P del ATP a residuos de serina y treonina 2. ciclina: su concentración activa o inhibe a la cinasa. A mayor concentración de esta, mayor actividad de la cinasa. Las bajas concentraciones de ciclina provocan la inhibición de las cinasas, provocando que la célula NO entre a fase M

Author response:

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

Reviewer #1 (Public Review):

Summary:

This manuscript from Mukherjee et al examines potential connections between telomere length and tumor immune responses. This examination is based on the premise that telomeres and tumor immunity have each been shown to play separate, but important, roles in cancer progression and prognosis as well as prior correlative findings between telomere length and immunity. In keeping with a potential connection between telomere length and tumor immunity, the authors find that long telomere length is associated with reduced expression of the cytokine receptor IL1R1. Long telomere length is also associated with reduced TRF2 occupancy at the putative IL1R1 promoter. These observations lead the authors towards a model in which reduced telomere occupancy of TRF2 - due to telomere shortening - promotes IL1R1 transcription via recruitment of the p300 histone acetyltransferase. This model is based on earlier studies from this group (i.e. Mukherjee et al., 2019) which first proposed that telomere length can influence gene expression by enabling TRF2 binding and gene transactivation at telomere-distal sites. Further mechanistic work suggests that G-quadruplexes are important for TRF2 binding to IL1R1 promoter and that TRF2 acetylation is necessary for p300 recruitment. Complementary studies in human triple-negative breast cancer cells add potential clinical relevance but do not possess a direct connection to the proposed model. Overall, the article presents several interesting observations, but disconnection across central elements of the model and the marginal degree of the data leave open significant uncertainty regarding the conclusions.

Strengths:

Many of the key results are examined across multiple cell models.

The authors propose a highly innovative model to explain their results.

Weaknesses:

Although the authors attempt to replicate most key results across multiple models, the results are often marginal or appear to lack statistical significance. For example, the reduction in IL1R1 protein levels observed in HT1080 cells that possess long telomeres relative to HT1080 short telomere cells appears to be modest (Supplementary Figure 1I). Associated changes in IL1R1 mRNA levels are similarly modest.

Related to the point above, a lack of strong functional studies leaves an open question as to whether observed changes in IL1R1 expression across telomere short/long cancer cells are biologically meaningful.

Statistical significance is described sporadically throughout the paper. Most major trends hold, but the statistical significance of the results is often unclear. For example, Figure 1A uses a statistical test to show statistically significant increases in TRF2 occupancy at the IL1R1 promoter in short telomere HT1080 relative to long telomere HT1080. However, similar experiments (i.e. Figure 2B, Figure 4A - D) lack statistical tests.

TRF2 overexpression resulted in ~ 5-fold or more change in IL1R1 expression. Compared to this, telomere length-dependent alterations in IL1R1 expression, although about 2-fold, appear modest (~ 50% reduction in cells with long telomeres across different model systems used). Notably, this was consistent and significant across cell-based model systems and xenograft tumors (see Figure 1). Unlike TRF2 induction, telomere elongation or shortening vary within the permissible physiological limits of cells. This is likely to result in the observed variation in IL1R1 levels.

For biological relevance, we have shown this using multiple models where telomere length was either different (patient tissue, organoids) or were altered (cell lines, xenograft models) . Where IL1 signalling in TNBC tissue and tumor organoids, and cells/xenografts were shown to impact M2 macrophage infiltration in a telomere length sensitive fashion. We made use of the tumor organoids to test M2 macrophage infiltration using IL1RA and small molecule based IL1R1 inhibition.

We have now included statistical tests in all the relevant figures and incorporated the necessary details about the tests performed in the figure legend for clarity of readers. Additionally, all data points, p values and details of statistical tests have been included in Figure wise excel sheets for both main and supplementary figures.

Reviewer #1 (Recommendations For The Authors):

There are typos throughout the manuscript. The word 'expression' is incorrectly spelled on y-axis labels throughout the manuscript (for example see Figure 1B). The word 'telomere' is incorrectly spelled in Supplementary Figure 1 legend panel A. Most errors, such as these, do not interfere with my comprehension of the manuscript. However, others made the manuscript difficult to follow. For example, I think that MDAMB231, MDAMD231, and MDAM231 are frequently used interchangeably to refer to the same cell line. This makes it very difficult to understand certain experiments.

I often found it difficult to understand which statistical test was used for a specific experiment. I suggest changing the style in the legends to more clearly connect statistical tests with specific data points.

We thank the reviewer for pointing out the typological errors. We have now made relevant corrections to both figures and text.

As stated above, we have now provided details of statistical tests performed in the figure legend for clarity of readers. Additionally, all data points, p values and details of statistical tests have been included in Figure wise excel sheets for both main and supplementary figures.

Reviewer #2 (Public Review):

This study highlights the role of telomeres in modulating IL-1 signaling and tumor immunity. The authors demonstrate a strong correlation between telomere length and IL-1 signaling by analyzing TNBC patient samples and tumor-derived organoids. Mechanistic insights revealed non-telomeric TRF2 binding at the IL-1R1. The observed effects on NF-kB signaling and subsequent alterations in cytokine expression contribute significantly to our understanding of the complex interplay between telomeres and the tumor microenvironment. Furthermore, the study reports that the length of telomeres and IL-1R1 expression is associated with TAM enrichment. However, the manuscript lacks in-depth mechanistic insights into how telomere length affects IL-1R1 expression. Overall, this work broadens our understanding of telomere biology.

The mechanism of how telomere length affects IL1R1 expression involves sequestration and reallocation of TRF2 between telomeres and gene promoters (in this case, the IL1R1 promoter). We have previously shown this across multiple genomic sites (Mukherjee et al, 2018; reviewed in J. Biol. Chem. 2020, Trends in Genetics 2023). We have described this in the manuscript along with references citing the previous works. A scheme explaining the model was provided as Additional Supplementary Figure 1, along with a description of the mechanistic model.

Figure 1-4 in main figures describe the molecular mechanism of telomere-dependent IL1R1 activation. This includes ChIP data for TRF2 on the IL1R1 promoter in long/short telomeres, as well as TRF2-mediated histone/p300 recruitment and IL1R1 gene expression. We further show how specific acetylation on TRF2 is crucial for TRF2-mediated IL1R1 regulation (Figure 5).

Reviewer #2 (Recommendations For The Authors):

The study primarily provides a snapshot of cytokine expression and telomere length at a single time point. Longitudinal studies or dynamic analyses could provide a more comprehensive understanding of the temporal relationship between telomere length and cytokine expression.

Tumor heterogeneity is a significant problem for the various therapies. The study notes significant heterogeneity in telomere length but does not investigate the implications of this heterogeneity. Understanding the role of telomere length variation in different tumor cell populations is essential for a comprehensive interpretation of the results.

The study only mentions a correlation between IL1R1 and relative telomere length but does not provide any potential clinical correlations with patient outcomes or survival. Addressing the clinical relevance of these molecular changes would improve the translational impact.

The importance of IL1R1 in prognostic and clinical outcomes of TNBC has been studied by multiple groups. The overall consensus is that higher IL1R1 leads to poor prognosis – aiding both cancer progression and metastasis. Using publicly available TCGA data, we found that IL1R1 high samples had significantly lower survival in breast cancer (BRCA) datasets. The results have now been included in the manuscript as Supplemnetray Figure 7G.

Addition in text:

“We, next, used publicly available TCGA gene expression data of breast cancer samples (BRCA) (Supplementary file 4) to assess the effect of IL1R1 expression on cancer prognosis. We categorized samples based on IL1R1 expression: IL1R1 high (N=254) and IL1R1 low samples (N= 709). It was seen that overall patient survival was significantly lower in IL1R1 high samples (Log-rank p value -0.0149) (Supplementary Figure 7G). We also checked the frequency of occurrence of various breast cancer sub-types in IL1R1 high and low samples (Supplementary Figure 7H). While invasive mixed mucinous carcinoma (the most abundant sub-type) was predominantly seen in IL1R1 low samples, metaplastic breast cancer was only found within the IL1R1 high samples. Interestingly, metaplastic breast cancer has been frequently found to be ‘triple negative’-i.e., ER-,PR- and HER2-. (Reddy et al., 2020).”

However, we could not access a TNBC (or any breast cancer dataset) that has been characterized for telomere length. Unfortunately, the clinical TNBC samples that we had access to did not have any paired short-term/long-term survival datasets. We could, in principle, use TERT/TERC expression as a proxy for telomere length; however, in our experiments, we found that telomerase activity did not positively correlate with telomere length as expected (Supplementary Figure 7C, Supplementary Figure 8D). Therefore, transcriptional signature (of telomere-associated genes) may not be a reliable indicator of telomere length.

The study lacks in-depth mechanistic insights into how telomere length affects IL1R1 expression and subsequently influences TAM infiltration. Further molecular studies or pathway analyses are necessary to elucidate the underlying mechanisms.

The mechanism involves sequestration and reallocation of TRF2 between telomeres and gene promoters (in this case, IL1R1 promoter). We have previously shown this across multiple genomic sites (Mukherjee et al, 2018). We have appropriately discussed this in the manuscript.

A schematic explaining the model has been provided as Additional Supplementary Figure 1.

We have provided ChIP data for TRF2 on IL1R1 promoter in long/short telomeres in the manuscript as well as histone/p300 ChIP and gene expression (Figure 1-4 in main figures exclusively deal with molecular mechanism of telomere dependent IL1R1 activation).  We further go on to show how specific acetylation on TRF2 might be crucial for TRF2-mediated IL1R1 regulation (Figure 5). One of the key findings herein is the fact that TRF2 can directly regulate IL1R1 expression through promoter occupancy- tested in telomere altered cell lines (HT1080, MDAMB231) and tumor xenografts (Figure 1 A, F, I- for TRF2 promoter occupancy).

Pathway analysis of HT1080 (short vs long telomere) transcriptome, shows that cytokine-cytokine receptor interaction is one of the key pathways in upregulated genes.

While we have focused on TRF2 mediated IL1R1 regulation, it is quite possible that there are other telomere sensitive pathways/mechanisms by which IL1R1 is regulated. This has been duly acknowledged in the discussion.

The manuscript title suggests modulation of immune signaling in the tumor microenvironment, yet the authors exclusively focus on CD206+ TAMs, limiting the scope. It is recommended to investigate other immune cell types for a more comprehensive understanding of changes in the immune tumor microenvironment.

As stated above, we approached the manuscript from the purview of TRF2-mediated IL1R1 regulation. In our assessment of TCGA data for breast cancer, we found that CD206 (MRC1) had the highest enrichment in IL1R1 high samples among key TAM and TIL markers- now added as Figure 8A (Details in Supplementary file 5). It also had the highest correlation with IL1R1 among the tested markers. Therefore, we proceeded to check CD206+ve TAMs.

Now the following section has been added to text:

“We further found that the total proportion of immune cells (% of CD45 +ve cells) did not vary significantly between short and long telomere TNBC samples (Supplementary Figure 8C). However, TNBC-ST samples had a higher percentage of myeloid cells (CD11B +ve) within the CD 45 +ve immune cell population. We checked in three TNBC-ST and TNBC-LT samples each and found that the percentage of M1 macrophages (CD86 high CD 206 low) in the myeloid population was lower than that of the M2 macrophages (CD 206 high CD 86 low) and unlike the latter, did not vary significantly between the TNBC-ST and TNBC-LT samples (Supplementary Figure 8C).”

Unfortunately, due to sample limitations we are unable to test this on a larger cohort of samples.

A single cell transcriptome experiment may have been a good way to have a more comprehensive immune profiling. However, with our TNBC samples, isolated nuclei for downstream processing had low viability as per 10X genomics specifications.

Does IL1R1 influence TAM recruitment or polarization within the tumor microenvironment? To assess the impact, the authors should use a marker indicative of M1-like macrophages, such as CD80 or CD86.

To address the issue of TAM recruitment vs polarization meaningfully we need to characterize tissue resident macrophages as well as macrophages in circulation. We did not have access to patient blood.  A murine breast cancer in-vivo model might be a more appropriate model to test this, which would take considerable time for us to develop. It is something that we hope to address in a follow up study.

Did the authors analyze other breast cancer subtypes for telomere length?

Unfortunately, other breast cancer sub-types besides TNBC were not available to us for experimentation.

Figure legends are very briefly written and need to be elaborated. Scale bars are also missing in images.

Add a gating strategy for flow cytometry results in Figure 8A.

Figure legend have been expanded for clarity. More prominent scale bars have been added for better visibility and reference.  A relevant gating strategy has been added as Supplementary figure 8B.

Reviewer #3 (Public Review):

Summary:

In this manuscript, entitled "Telomere length sensitive regulation of Interleukin Receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment", Dr. Mukherjee and colleagues pointed out clarifying the extra-telomeric role of TRF2 in regulating IL1R1 expression with consequent impact on TAMs tumor-infiltration.

Strengths:

Upon careful manuscript evaluation, I feel that the presented story is undoubtedly well conceived. At the technical level, experiments have been properly performed and the obtained results support the authors' conclusions.

Weaknesses:

Unfortunately, the covered topic is not particularly novel. In detail, the TRF2 capability of binding extratelomeric foci in cells with short telomeres has been well demonstrated in a previous work published by the same research group. The capability of TRF2 to regulate gene expression is well-known, the capability of TRF2 to interact with p300 has been already demonstrated and, finally, the capability of TRF2 to regulate TAMs infiltration (that is the effective novelty of the manuscript) appears as an obvious consequence of IL1R1 modulation (this is probably due to the current manuscript organization).

Here we studied the TRF2-IL1R1 regulatory axis (not reported earlier by us or others) as a case of the telomere sequestration model that we described earlier (Mukherjee et al., 2018; reviewed in J. Biol. Chem. 2020, Trends in Genetics 2023). This manuscript demonstrates the effect of the TRF2-IL1R1 regulation on telomere-sensitive tumor macrophage recruitment. To the best of our knowledge, no previous study connects telomeres of tumor cells mechanistically to the tumor immune microenvironment. Here we focused on the IL1R1 promoter and provided mechanistic evidence for acetylated-TRF2 engaging the HAT p300 for epigenetically altering the promoter. This mechanism of TRF2 mediated activation has not been previously reported. Further, the function of a specific post translational modification (acetylation of the lysine residue 293K) of TRF2 in IL1R1 regulation is described for the first time. Additional experiments showed that TRF2-acetylation mutants, when targeted to the IL1R1 promoter, significantly alter the transcriptional state of the IL1R1 promoter. To our knowledge, the function of any TRF2 residue in transcriptional activation had not been previously described. Taken together, these demonstrate novel insights into the mechanism of TRF2-mediated gene regulation, that is telomere-sensitive, and affects the tumor-immune microenvironment.

We considered the reviewer’s suggestion to reorganize the result section. Reorganizing the manuscript to describe the TAM-related results first would, in our opinion, limit focus of the new findings and discovery [and novelty of the mechanisms (as described in above response, and in response to other comments by reviewers)] of the non-telomeric TRF2-mediated IL1R1 regulation. We have tried to bring out the novelty, implications and importance of the TAM-related observations in the discussion.

Reviewer #3 (Recommendations For The Authors):

Based on the comments reported above, I would encourage the author to modify the manuscript by reorganizing the text. I would suggest starting from the capability of TRF2 to modulate macrophages infiltration. Data relative to IL1R1 expression may be used to explain the mechanism through which TRF2 exerts its immune-modulatory role. This, in my view, would dramatically strengthen the presented story.

Concerning the text, "results" should be dramatically streamlined and background information should be just limited to the "introduction" section.

The manuscript should be carefully revisited at grammar level. A number of incomplete sentences and some typos are present within the text.

We thank the reviewer for the appreciation of our work for its technical strengths.

At the onset, we agree that we have explored the TRF2-IL1R1 regulatory axis. This underscores the significance of the telomere sequestration model that we had proposed earlier (Mukherjee et al., 2018). Herein, however, we significantly extend our previous work (which was more general and intended for putting forward the idea of telomere-dependent distal gene expression) by studying TRF2-mediated regulation of IL1 signalling (which was previously unreported). In addition, mechanistic details of how telomeres are connected to IL1 signaling through non-telomeric TRF2 are entirely new, not reported before by us or others.

We have removed some text descriptions from the result section to streamline the section.

¿Como sería la solución tecnológica o innovación tecnológica que para cuantificar y valorar las relaciones de la biodiversidad con el negocio o la empresa?

AI models collapse when trained on recursively generated data by Ilia Shumailov et al.

ᔥ[[Mathew Lowry]] in AI4Communities post - MyHub Experiments Wiki (accessed:: 2024-11-06 09:43:23)

En caso de pueblos conquistados por otros de lengua distinta, el conjunto de influencias léxicas, fonéticas y gramaticales que ejerce la lengua originalmente hablada en el territorio sobre la lengua que la sustituye... How a language shows characteristics of the language/s that came before it.

Author response:

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

We thank the reviewers for their detailed comments. Several comments revolved around potential improvements in the 3D reconstructions that are obtained in later steps of the image processing pipelines for single-particle cryoEM and cryo-electron tomography. We have not investigated how our improvements in CTFFIND5 affect these downstream results and can therefore not make specific and quantitative statements in this regard. However, CTFFIND5 provided additional information about the sample that users will find useful (thickness, tilt) for selecting the data they would like to include in later processing, and how to process them. Furthermore, when the sample tilt of a thin specimen is known, local defocus estimates (e.g., per-particle defocus estimates) will be more accurate compared to estimates that ignore tilt information. In the following, we provide point-by-point responses to the reviewers’ comments.

Reviewer #1 (Public Review):

This work presents CTFFIND5, a new version of the software for determination of the Contrast Transfer Function (CTF) that models the distortions introduced by the microscope in cryoEM images. CTFFIND5 can take acquisition geometry and sample thickness into consideration to improve CTF estimation.

To estimate tilt (tilt angle and tilt axis), the input image is split into tiles and correlation coefficients are computed between their power spectra and a local CTF model that includes the defocus variation according to a tilted plane. As a final step, by applying a rescaling factor to the power spectra of the tiles, an average tilt-corrected power spectrum is obtained and used for diagnostic purposes and to estimate the goodness of fit. This global procedure and the rescaling factor resemble those used in Bsoft, Warp, etc, with determination of the tilt parameters being a feature specific of CTFFIND5 (and formerly CTFTILT). The performance of the algorithm is evaluated with tilted 2D crystals and tiltseries, demonstrating accurate tilt estimation in some cases and some limitations in others. Further analysis of CTF determination with tilt-series, particularly showing whether there is accurate or stable estimation at high tilts, might be helpful to show the robustness of CTFFIND5 in cryoET.

CTFFIND5 represents the first CTF determination tool that considers the thickness-related modulation envelope of the CTF firstly described by McMullan et al. (2015) and experimentally confirmed by Tichelaar et al. (2020). To this end, CTFFIND5 uses a new CTF model that takes the sample thickness into account. CTFFIND5 thus provides more accurate CTF estimation and, furthermore, gives an estimation of the sample thickness, which may be a valuable resource to judge the potential for high resolution. To evaluate the accuracy of thickness estimation in CTFFIND5, the authors use the Lambert-Beer law on energy-filtered data and also tomographic data, thus demonstrating that the estimates are reasonable for images with exposure around 30 e/A2. While consideration of sample thickness in CTF determination sounds ideally suited for cryoET, practical application under the standard acquisition protocols in cryoET (exposure of 3-5 e/A2 per image) is still limited. In this regard, the authors are honest in the conclusions and clearly identify the areas where thickness-aware CTF determination will be valuable at present: e.g. in situ single particle analysis and in vitro single particle cryoEM of purified samples at low voltages.

In conclusion, the manuscript introduces novel methods inside CTFFIND5 that improve CTF estimation, namely acquisition geometry and sample thickness. The evaluation demonstrates the performance of the new tool, with fairly accurate estimates of tilt axis, tilt angle and sample thickness and improved CTF estimation. The manuscript critically defines the current range of application of the new methods in cryoEM.

Reviewer #2 (Public Review):

Summary:

This paper describes the latest version of the most popular program for CTF estimation for cryo-EM images: CTFFIND5. New features in CTFFIND5 are the estimation of tilt geometry, including for samples, like FIB-milled lamellae, that are pre-tilted along a different axis than the tilt axis of the tomographic experiment, plus the estimation of sample thickness from the expanded CTF model described by McMullan et al (2015). The results convincingly show the added value of the program for thicker and tilted images, such as are common in modern cryo-ET experiments. The program will therefore have a considerable impact on the field.

I have only minor suggestions for improvement below:

Abstract: "[CTF estimation] has been one of the key aspects of the resolution revolution"-> This is a bit over the top. Not much changed in the actual algorithms for CTF estimation during the resolution revolution.

We have removed this statement in the abstract.

L34: "These parameters" -> Cs is typically given, only defocus (and if relevant phase shift) are estimated.

We have modified the introduction to reflect this. Page 3, L30-35

L110-116: The text is ambiguous: are rotations defined clockwise or counter-clockwise? It would be good to explicitly state what subsequent rotations, in which directions and around which axes this transformation matrix (and the input/output angles in CTFFIND5) correspond to.

Thank you for pointing this out. We have revised the Methods section, Page 4 L57-61,  to explicitly define the convention for the tilt axis and tilt angle. We have also modified Fig. 1b to illustrate our convention for the tilt axis.

L129-130: As a suggestion: it would be relatively easy, and possibly beneficial to the user, to implement a high-resolution limit that varies with the accumulated dose on the sample. One example of this exists in the tomography pipeline of RELION-5.

We appreciate the suggestion. However, since CTFFIND5 currently has no concept of a tilt-series and treats every micrograph independently, this would not be trivial to implement. As detailed below, CTFFIND5 in its current form is not targeted toward tomography processing, but its features might be useful for its use in pipelines for tomography processing, such as RELION-5. We made this more explicit in the conclusion section. Page 16 L390-399

Substituting Eq (7) into Eq (6) yields ksi=pi, which cannot be true. If t is the sample thickness, then how can this be a function of the frequency g of the first node of the CTF function? The former is a feature of the sample, the latter is a parameter of the optical system. This needs correction.

We have rewritten the text describing equations 7 and 6 to avoid this confusion (Page 7, L146-153). The reviewer is right that inserting Eq. 7 into Eq. 6 yields ksi=psi, as in fact Eq. 7 is derived from Eq. 6, by substituting ksi=psi, since this describes the condition for the first node. Also, in this context, nodes in the CTF function refer to the places where the term sinc(ksi) becomes zero and therefore the CTF is apparently "flat". The frequency at which this occurs is sample-thickness dependent. As explained below, the previous version of our manuscript did not point out the difference between the first zero and first node in the power spectrum. We have amended Fig. 3a to make this difference clearer.

Reviewer #3 (Public Review):

In this manuscript, the authors detail improvements in the core CTFFIND (CTFFIND5 as implemented in cisTEM) algorithm that better estimates CTF parameters from titled micrographs and those that exhibit signal attenuation due to ice thickness. These improvements typically yield more accurate CTF values that better represent the data. Although some of the improvements result in slower calculations per micrograph, these can be easily overcome through parallelization.

There are some concerns outlined below that would benefit from further evaluation by the authors.

For the examples shown in Figure 3b, given the small differences in estimated defocus1 and 2, what type of improvements would be expected in the reconstructed tomograms? Do such improvements in estimates manifest in better tilt-series reconstruction?

As explained in our preface, we do not believe that these difference would manifest in any improvements during tilt-series reconstruction and would not create any meaningful differences, even when tomograms are reconstructed with CTF correction. They might become meaningful during subtomogram averaging, but subtomograms are usually corrected using per-particle CTF estimation, similar to single-particle processing. We have included a new paragraph in the discussion to describe potential benefits of CTFFIND5 for cryo-tomography, Page 16 L390-399.

Similarly, the data shown in Figure 3C shows minimal improvements in the CTF resolution estimate (e.g., 4.3 versus 4.2 Å), but exhibited several hundred Å difference in defocus values. How do such differences impact downstream processing? Is such a difference overcame by per-particle (local) CTF refinements (like the authors mention in the discussion, see below)?

The difference in the defocus estimate (~600A) is substantially smaller than the thickness of the sample (2000A). Hence both estimates may be valid, depending on which particles inside the sample are considered. Particles with larger defocus errors could certainly be corrected by per-particle CTF refinement as long as the search range is chosen to be large enough. The main benefit of using CTFFIND5 is information for the user regarding the sample thickness to set the defocus search range appropriately.

At which point does the thickness of the specimen preclude the ice thickness modulation to be included for "accurate" estimate? 500Å? 1000Å? 2000Å? Based on the data shown in Figure 3B, as high as 969 Å thick specimens benefit moderately (4.6 versus 3.4 Å fit estimate), but perhaps not significantly, from the ice thickness estimation. Considering the increased computational time for ice thickness estimation, such an estimate of when to incorporate for single-particle workflows would be beneficial.

As explained in our preface, the main benefit for single-particle workflows will be sample tilt estimation. This will provide more accurate per-particle defocus estimates, compared to estimates that do not take the tilt into account. For single-particle samples, the ice thickness in holes is probably more efficiently monitored using the Beer-Lambert law.

It would seem that this statement could be evaluated herein: "the analysis of images of purified samples recorded at lower acceleration voltages, e.g., 100 keV (McMullan et al., 2023), may also benefit since thickness-dependent CTF modulations will appear at lower resolution with longer electron wavelengths". There are numerous examples of 300kV, 200kV, and 100kV EMPIAR datasets to be compared and recommendations would be welcomed.

Publicly available datasets recorded at 100kV and 200kV were collected in very thin ice, making it difficult to demonstrate the stated benefits. We have removed this statement.

Although logical, this statement is not supported by the data presented in this manuscript: "The improvements of CTFFIND5 will provide better starting values for this refinement, yielding better overall CTF estimation and recovery of high-resolution information during 3D reconstruction."

We have revised this statement and now explain that the sample tilt information will provide more accurate per-particle defocus estimates, compared to estimates that do not take the tilt into account, Page 17, L400-409. We did not investigate how this will affect downstream processing results.

Moreso, the lack of single-particle data evaluation does present a concern. Naively, these improvements would benefit all cryoEM data, regardless of modality.

We agree with the reviewer that all cryoEM modalities should benefit from more accurate defocus value estimates and have amended our concluding statement. However, how improved defocus values will benefit downstream processing results will depend on the processing pipeline, which includes various points of user input and data-dependent choices. We have therefore limited our analysis to the outputs of CTFFIND5.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) CTFFIND5 in cryo-ET

(1.1) CTFFIND4 is prone to unreliable CTF estimates at high tilts in cryoET, a situation that can be identified by high variability or 'unstable' estimates as a function of the tilt angle. Prof. Mastronarde recently illustrated this situation in his article JSB 216:108057, 2024 (Fig. 7). Therefore, the authors could add results to show whether the improvements to tilt estimation introduced in CTFFIND5 overcome this problem. So, in addition to the estimation of tilt angle and tilt axis in Figure 2, the estimated defocus could also be shown.

We have worked with Prof. Mastronarde to help him use CTFFIND as a tool in his cryoET processing pipeline. Mastronarde chose CTFFIND because it contains algorithms and architecture that he could optimize for his purposes. CTFFIND5 is currently lacking the concept of a tilt series and can therefore not take advantage of the additional information that comes with tilt series. Our own applications for CTFFIND5 currently do not include tomography, and our results presented in Fig. 2 were obtained for validation of the tilt estimation feature. We did not attempt to duplicate Mastronarde’s optimization for reliable tilt series processing.

Figure 2b of this manuscript already suggests that CTFFIND5 may exhibit some variability of defocus estimates at high tilts (in view of the variability of tilt axis angle). A strategy used in IMOD and TOMOCTF is to consider the tiles of a group of consecutive images (typically 35; especially at high tilts) to add more signal to the average spectrum, thus providing more reliable estimates (illustrated in Mastronarde's article JSB 216:108057, 2024, Fig. 8). Will the authors think that CTFFIND5 might include a strategy like this for cryoET tilt-series?

We currently do not have plans to develop CTFFIND5 as a tool for tomography as there are already other excellent tools available, some of them based on CTFFIND’s basic algorithms (see previous comment).

(1.2) In cryoET, the CTF is often determined on the aligned tilt-series, with the tilt axis typically running along the Y axis. Has CTFFIND5 got the option to exclude estimation of the tilt geometry (tilt angle and/or axis) and, instead, take tilt geometry directly from the alignment and/or from the microscope??. This would significantly speed up determination of the CTF (in 1-2 seconds per image, according to Table 2) while still taking advantage of all power spectra in tilted images (as described in their tilt estimation algorithm) for improved CTF estimation. This strategy would be similar to what it is done in Bsoft and IMOD.

This is an excellent idea and we may implement this in an updated version. The current version is primarily meant for lamellae and single-particle samples where we usually have a single tilt in an unknown direction. For these cases, the suggested feature will have less benefit. 

Thus, I suggest that the authors should also include results comparing CTF estimation in aligned tilt-series with CTFFIND4 and with CTFFIND5 (with no tilt estimation but indeed taking the tilt information from the alignment or the microscope into account). The results would show that CTFFIND5 is more robust than CTFFIND4, especially at high tilts.

Thank you for this suggestion. We are now showing a comparison of defocus estimates from CTFFIND4 and CTFFIND5 in Fig. 2. Indeed, in one case CTFFIND5 seems to report more robust defocus values at high tilt.

(1.3) The newer improvements in CTFFIND5 seem to be especially tailored to cryoET. The cryoET community will be highly attracted by these improvements. However, the current standard acquisition protocols (exposure of 3-5 e/A2 per image, tilts up to 60 degrees, etc) limit their full exploitation, particularly the thickness-aware CTF determination. I believe that adding a paragraph exclusively focused on cryoET and describing the potential benefits from CTFFIND5 and their limitations could enrich the Conclusion section. In this paragraph, the authors could highlight the great benefits from the tilt-aware CTF estimation. They could also discuss the current standard acquisition protocols (e.g. exposure 3-5 e/A2 per image, nominal defocus 3-5 microns, cellular thickness from 150 nm up to 200-300 nm that, at a tilt of 60 degrees, become 300 nm up to 400-600 nm) and their implications for the potential benefit from the improvements available in CTFFIND5.

This reviewer is clearly excited about the potential application of CTFFIND5 in cryoET. We are sorry that we are currently not developing CTFFIND5 in this direction.

(1.4) Apologies for insisting on cryoET in the previous points. I am just trying to suggest ideas to make CTFFIND5 even more helpful in cryoET. You can consider them now, or for a future version of the software, or just ignore them.

Thanks for your suggestions. Since there is clearly demand for tools to process tomographic tilt series, we will keep these suggestions in mind for the future development of CTFFIND.

(2) Tilt estimation

(2.1) Page 4. Tiles for the initial steps in tilt estimation are of size 128x128.  At which point tiles of larger size (e.g. 512x512) are used?. Please, define.

Thank you for pointing out this lack of clarity. For the tilt estimation, we used a tile size 128 x 128, which has been hard-coded in our program, as mentioned in line 68 on page4. For generating the final power spectrum, we usually use size 512 x 512. This tile size can be defined by the user when running the program. We have now clarified this on Page 4, L74-76.

(2.2) Page 6 and/or page 11: evaluation of tilt estimation with tilt-series.

Please indicate the acquisition details of the tilt-series used for the evaluation, especially the exposure per image. This information is neither available in this manuscript nor in Elferich et al., 2022.

Please, add these acquisition details similarly to page 9 in this manuscript (evaluation of sample thickness estimation using tomography): pixel size, exposure per image and total exposure, number of images, tilt range and interval

The same tilt-series were used to verify tilt-estimation and sample thickness. We have revised the Methods section to make this clear on Page5, L98-105 and Page 10, L202.

(2.3) Page 10. Section Results. Subsection Tilt estimation.

The authors use "defocus correction" to refer to their method for scaling the power spectra. "Defocus correction" might perhaps be a misleading term. In contrast, in page 4 the authors use the term "tilt correction". Please, revise and make it consistent throughout the manuscript.

We agree and now use “tilt correction” throughout the manuscript.

(2.4) Legend of Figure 2.

Please add what the red dashed curve represents. Also, please note there might be an error in the estimated stage tilt axis angle: the legend states "171.8" where in the main text it is "178.2" (apparently, the latter is the correct one).

Thank you for pointing this out. We have modified the legend and changed the number in the legend to 178.2°.

(3) Thickness estimation

(3.1) Line 141, page 7. The sentence reads: "The modulation of the CTF due to sample thickness t is described by the function E (current Equation 6), "  I believe that the modulation envelope of the CTF due to sample thickness is not really E (current Equation 6), but the function sinc(E). Please, revise.

We have revised the manuscript as advised, Page 7, L148.

(3.2) Line 148, page 7. The sentence reads "an estimate of the frequency g of the first node of the CTF_t function "

The concept of 'node' was introduced by Tichelaar et al. (2020). The authors should not assume that this concept is familiar to the readership. So, it is suggested that the authors should introduce this concept in this section. For instance, just after Equation 6 they could add a sentence like this: "This sinc modulation envelope increasingly attenuates the amplitude of the Thon rings with increasing spatial frequencies in an oscillatory fashion, with locations where the amplitude is zero known as nodes (Tichelaar et al., 2020)."

Thank you for this suggestion. We have revised the manuscript accordingly (Page 7, L151-156) and also marked the position of the first node in Fig. 3a.

(3.3) Line 154, page 8: A citation is lacking: "(corrected for astigmatism, as described in )". Perhaps the authors refer to the EPA (EquiPhase Averaging) method introduced by Zhang, JSB 193:1-12, 2016, 10.1016/j.jsb.2015.11.003.

Thanks for spotting this omission. We have added the appropriate reference.

(3.4) Figure 3.

(3.4.1) Perhaps, the EPA (EquiPhase Averaging) method is used to reduce the 2D CTF to 1D curves, as represented in Figure 3b and 3c. Please, mention this in the legend of the figure or in the main text referring to Figure 3. The same might apply to Figure 1c.

Thanks for spotting this omission. We have clarified that this is indeed an EPA in the figure legends.

(3.4.2) Please indicate what the colored curves represent in 3b and 3c: The fitted CTF model (dashed red) and the EPA or astimatism-corrected radial average of power spectrum (solid black) ?

Thanks for spotting this omission. We have added descriptions of the colored lines in these plots (red = modeled CTF, blue = goodness of fit).

(3.4.3) Please note that the power spectrum (solid black curves in Figure 3b and 3c) does not look the same in the top and bottom panels: Without thickness estimation (top panels), the power spectrum is in the range [0,1] in Y, as expected. However, with thickness estimation (bottom panels), the power spectrum seems to have undergone a frequencydependent transformation (a rescaling or something that makes the power spectrum oscillates around 0.5 in Y). This transformation of the power spectrum resembles the thickness-induced sinc modulation of the CTF and seems to be appropriate to better fit the new thickness-aware CTF_t model in CTFFIND5 to the (transformed) power spectrum. However, this transformation of the power spectrum is not mentioned in the manuscript at all. Instead, according to the main text (page 8), the fitting method is based on the crosscorrelation between the new CTF model and the power spectrum, so I was expecting to see the same power spectrum black curve in the top and bottom panels. Please, clarify.

Indeed, CTFFIND5 displays the power spectrum differently after thickness estimation. We have revised the methods to explain this (page8, L178-181). The reviewer is also correct that the 1D lines plots of the Thon ring patterns in Fig. 3b and 3c are not identical. These 1D plots are generated from the 2D plots according to the fitted CTF, which is needed to follow the astigmatic rings and avoid blurring of the oscillations in the radial average. This means that different CTF fits will also result in somewhat different 1D plots. However, these differences only affect the 1D EPA plots shown to the user. The actual fitting is performed against the same 2D spectra.

(3.4.4) Line 319, Page 14. "A linear fit revealed .." It would be good to add a line with the linear fit in Figure 5.

Agreed. The revised Fig. 5 now shows a line for the linear fit.

(3.5) New CTF Model

It is not clear from the text if the new CTF_t model is used at all times in CTFFIND5 or only when the user requests thickness estimation. Related to this, if the user requests both tilt estimation and thickness estimation, how is the CTF estimation process carried out in CTFFIND5?: Tilt and thickness are estimated at the same time? or one after the other (i.e. first the tilt is estimated, then followed by thickness estimation)?. Please, clarify.

The new CTF_t model is only used when the user requests thickness estimation. When both tilt-estimation and thickness estimation are requested, the tilt is estimated first and the corrected power spectrum is then fitted using the CTF_t model. We have revised the Methods section to explain this better, Page 8, L158-159.

(4) Pages 14-15. Section "CTF estimation and correction assists "

This section just shows that correction of a highly underfocused image for the CTF with phase flipping or a Wiener filter reduces the CTF-induced fringes. I do not really understand the inclusion of this section to the manuscript. There is no contribution related to CTFFIND5.  

The ability to apply a CTF correction to the input image according to Tegunov & Cramer is a new feature of apply_ctf, a program included with cisTEM. We think that this section fits into the theme of CTFFIND5 because the correction adds valuable information about the samples, such as FIB-milled lamellae.

If the authors prefer to keep this section, then please take the following points into account:

(4.1) Figure 6b: This is the only time that the term "EPA" (EquiPhase Averaging, I guess) is used in the manuscript. Please, spell it out somewhere in the manuscript, define what it means and add a proper citation, if convenient. This point is related to point 3.3 above.

We have added the appropriate reference and defined EPA in the methods section as indicated in the reply to point 3.3.

(4.2) Figure 6d. The contrast of this image is poor. Please, increase the contrast (to be similar to Figure 6c) so that the details can be better discerned. The image also shows a grainy texture, likely artefacts from the Wiener filter due to excessive amplification. Maybe the 'strength parameter' S of the deconvolution Wiener filter (Tegunov & Cramer, 2019) should be tuned down or the 'fall-off parameter' F tuned up to try to attenuate these artefacts.

Agreed. The revised figure shows panel d with increased contrast with the custom fall-off parameter set to 1.3 and the custom strength parameter set to 0.7.

(5) CTFFIND5 runtimes

Table 2 shows that estimation of tilt increases the runtime up to 39 s in an image of 4070x2892 and to 208 s in one of 2880x2046. There is a significant difference between these two cases (39 s vs. 208 s) and the first image is much larger than the second. Why does CTFFIND5 on the smaller image take so long compared to the larger image?

During tilt estimation, the images are binned to a pixel size of 5 Å. This causes micrograph 1 to be substantially smaller (in pixels) than micrographs 2 and 3, resulting in the faster runtime.

(6) Conclusions

(6.1) In the Conclusion section, the authors could elaborate a bit the insights about the sample quality provided by CTFFIND5. This is stated in the title of the manuscript, but it was hardly mentioned in the manuscript.

We have revised the conclusion to make this clearer (Page 16, L389-396). CTFFIND5 helps in estimating sample quality since (1) the sample thickness is an important determinant in the amount of high-resolution signal in a micrograph and (2) the estimated fit-resolution reflects more accurately the amount of signal present in a micrograph after tilt and sample thickness have been taken into account.

(6.2) The authors nicely identify and describe the applications where thickness-aware CTF determination will be valuable: in situ single particle analysis and in vitro single particle cryoEM of purified samples at low voltages. Perhaps, CTFFIND5 will also be of great interest for single particle cryoEM of thick specimens (e.g. capsid of large viruses with diameter in the range 120-200 nm such as PBCV-1 or HSV-1).

Agreed. We have added this case to our Conclusions. (Fig. 3d)

(7) Typographical errors:

line 161, page 8. "1.5 time" should be "1.5 times"

lines 185-191. All exposures are given in 'electrons/Angstrom', not in 'electrons/square Angstrom'

line 206, page 10. With "slides" the authors seem to mean "slices"

line 338, page 14: "describeD by Tegunov"

line 349, page 15. "power spectra"

lines 366 and 368, page 15: Note that Square Angstrom is written as "A2". Put "2" with superscript.

Thank you for pointing out these errors. They have been corrected.

(8) References:

Reference: Lucas et al., eLife 10 e68946. Year is lacking. Add year: 2021.

Reference: Yan et al. 2015 cited in line 169, page 8, does not appear in Bibliography. The authors may mean: Yan et al. 2015 JSB 192:287-296, 2015  

It would be good to cite Bsoft, as it has a procedure similar to tilt-corrected CTF estimation: Heymann, Protein Science, 2021,  

Thank you for carefully checking the cited references. We have revised the manuscript as suggested.

Reviewer #2 (Recommendations For The Authors):

I have only minor suggestions for improvement below:

L218: "these option"

Corrected

L243: "chevron-shape" -> V-shape would be more accessible language for non-native speakers.

Changed

L281: "Based on these results we conclude that CTFFIND5 will provide more accurate CTF parameters" -> Given that the maximum resolutions of the fits by the old model and the new model are nearly the same, how big would the actual advantage of the new model be for subsequent sub-tomogram averaging?

Please see our response above, Reviewer #3 (Public Review), 

L376: The correct reference for RELION per-particle CTF estimation is Zivanov et al, (2018) [https://elifesciences.org/articles/42166]. Also, the cryoSPARC paper referenced does not describe per-particle CTF estimation and should thus be removed from this context.

Thanks for pointing out these mistakes, which we have now corrected. We have chosen to keep the citation for CryoSPARC to reference the general software, but have added Ziavanov et.al. 2020 as suggested by the CryoSPARC website.

Reviewer #3 (Recommendations For The Authors):

Minor:

Figure 1A legend - authors mention boxes but only 1 box is shown.

Thank you for pointing this out. For visual clarity we decided to only show one box. We have corrected the legend.

Figure 1B - it would be nice if the boxes that contributed to the power spectra were mapped on Figure 1A

The shown power spectra are not actual data. Instead, we show power spectra with exaggerated defocus differences for visual clarity. We have revised the figure legends to make this clear. 

The Y-axis legends in Figure 2 are not aligned vertically

Corrected

Figure 3A - CTFFIND4 is missing an "I"

Corrected

Figure 3 - Y-axis legends are not aligned vertically

Corrected

Page 16, line 376, Relion should be RELION

We have revised the manuscript as advised.

Typo in equation 5, sinc versus sin?

“sinc” is correct here, since this is a thickness-dependent modulation of the CTF.

Lambert-Beer's, Lambert-Beer are used variably but curious if Beer-Lambert should be used.

We have revised the manuscript as advised.

dfasdkjfhas

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This work sets out to elucidate mechanistic intricacies in inflammatory responses in pneumonia in the context of the aging process (Terc deficiency - telomerase functionality).

Strengths:

Very interesting, conceptually speaking, approach that is by all means worth pursuing. An overall proper approach to the posited aim.

We want to thank the reviewer for taking the time to review our manuscript and for providing positive feedback regarding our research question.  

Weaknesses:

The work is heavily underpowered and may have statistical deficits. This precludes it in its current state from drawing unequivocal conclusions.

Thank you for this essential and valuable comment. We fully accept that the small sample size of the Tercko/ko mice is a major limitation of our study and transparently discuss this in our manuscript.  However, due to Animal Welfare regulations, only a reduced number of mice were approved because of the strong burden of disease. Consequently, only three non-infected and five infected mice were available to us. This reduced number of mice presents a clear limitation to our study. However, due to ethical considerations related to animal welfare and sustainability, as well as compliance with German animal welfare regulations, it is not possible to obtain additional Tercko/ko mice to increase the dataset.

The animal studies are an important aspect of our study; however, our hypothesis was also investigated at multiple levels, including in an in vitro co-culture model (Figure 5), to ensure comprehensive analysis. Thus, we clearly demonstrated that S. aureus pneumonia in Tercko/ko mice leads to a more severe phenotype, orchestrated by the dysregulation of both innate and adaptive immune response.

Reviewer #2 (Public Review):

Summary:

The authors demonstrate heightened susceptibility of Terc-KO mice to S. aureus-induced pneumonia, perform gene expression analysis from the infected lungs, find an elevated inflammatory (NLRP3) signature in some Terc-KO but not control mice, and some reduction in T cell signatures. Based on that, They conclude that disregulated inflammation and T-cell dysfunction play a major role in these phenomena.

Strengths:

The strengths of the work include a problem not previously addressed (the role of the Terc component of the telomerase complex) in certain aspects of resistance to bacterial infection and innate (and maybe adaptive) immune function.

We would like to thank the reviewer for the positive feedback regarding our aim to investigate the impact of Terc deletion on the pulmonary immune response to S. aureus.  

Weaknesses:

The weaknesses outweigh the strengths, dominantly because conclusions are plagued by flaws in experimental design, by lack of rigorous controls, and by incomplete and inadequate approaches to testing immune function. These weaknesses are as follows

(1) Terc-KO mice are a genomic knockout model, and therefore the authors need to carefully consider the impact of this KO on a wide range of tissues. This, however, is not the case. There are no attempts to perform cell transfers or use irradiation chimera or crosses that would be informative.

We thank the reviewer for bringing up this important point. The aim of our study, however; was to investigate the impact of Terc deletion in the lung and on the response to bacterial pneumonia, rather than to provide a comprehensive characterization of the Tercko/ko model itself. This characterization of different tissues and cell types has already been conducted by previous studies. For instance, studies that characterize the general phenotype of the model (Herrera et al., 1999; Lee et al., 1998; Rudolph et al., 1999) but also investigations that shed light on the impact of Terc deletion on specific cell types such as microglia (Khan et al., 2015) or T cells (Matthe et al., 2022). The impact of Terc deletion on T cells is also discussed in our manuscript in lines 89 to 105. Furthermore, a section about the general phenotype of the Terc deletion model is included in the introduction in lines 126 to 138. Thus we discussed the relevant literature regarding Tercko/ko mice in our manuscript and attempted to provide a more in-depth characterization of the lung by investigating the inflammatory response to infection as well as changes in the gene expression (Figure 2-4).  

(2) Throughout the manuscript the authors invoke the role of telomere shortening in aging, and according to them, their Terc-KO mice should be one potential model for aging. Yet the authors consistently describe major differences between young Terc-KO and naturally aging old mice, with no discussion of the implications. This further confuses the biological significance of this work as presented.

Thank you for mentioning this relevant point. We want to apologize for the confusion regarding this matter. While Tercko/ko mice are a well-established model for premature aging, these effects become more apparent with increasing generations (G) and thus, G5 and 6 mice are the most affected by Terc deletion (Lee et al., 1998; Wong et al., 2008).

Thus, while Tercko/ko mice are a common model for premature aging, this accelerated aging phenotype is predominantly apparent in later-generation Tercko/ko (G5 and 6) or aged Tercko/ko mice (Lee et al., 1998; Wong et al., 2008). Since the aim of this study was to analyze the impact of Terc deletion on the lung and its immune response to bacterial infections instead of the impact of telomere shortening and telomerase dysfunction, young G3 Tercko/ko mice (8 weeks) were used in this study. This is also mentioned in the lines 131-134. In this study, Tercko/ko mice were used not as a model of aging, but rather as a model specifically for Terc deletion. The old WT mice function as a control cohort to observe possible common but also deviating effects between aging and Terc deletion. In our sequencing data, we observe that uninfected young WT mice are very similar to uninfected Tercko/ko mice. Other studies have also reported this lack of major differences between uninfected WT and Tercko/ko mice in the G3 knockout mice (Kang et al., 2018). Conversely, uninfected young WT and Tercko/ko mice exhibited great differences, for instance, regarding the numbers of differentially expressed genes (Supplemental Figure 1H). Thus, differences between naturally aged mice and young G3 Tercko/ko mice are not surprising. To clarify this aspect we reconstructed the paragraph discussing the Tercko/ko mice (lines 126-134). Additionally we added a paragraph explaining the purpose of the naturally aged mice to the lines 134 to 138:

“As control cohort age-matched young WT mice were utilized. To investigate whether Terc deletion, beyond critical telomere shortening, impacts the pulmonary immune response, we used young Tercko/ko mice. Additionally, naturally aged mice (2 years old) were infected to explore the potential link to a fully developed aging phenotype.”

(3) Related to #2, group design for comparisons lacks a clear rationale. The authors stipulate that TercKO will mimic natural aging, but in fact, the only significant differences seen between groups in susceptibility to S. aureus are, contrary to the authors' expectation, between young Terc-KO and naturally old mice (Figures 1A and B, no difference between young Terc-KO and young wt); or there are no significant differences at all between groups (Figures 1, C, D,).

We thank the reviewer for this essential comment. As mentioned above the Tercko/ko mice in this study are not selected to model natural aging. To model telomerase dysfunction and accelerated aging selection of later generation or aged Tercko/ko mice would have been more suitable. 

The lack of statistical significance in some figures is likely due to the heterogeneity of disease phenotype of S. aureus infection in mice, which is a limitation of our study that we discuss in our discussion section in lines 576-582. The phenotype of S. aureus infection can vary greatly within a mouse population, highlighting the limitations of mice as a model for S. aureus infections. To account for this heterogeneity we divided the infected Tercko/ko mice cohort into different degrees of severity based on the clinical score and the presence of bacteria in organs other than the lung (mice with systemic infection). 

Despite the heterogeneity especially within the Tercko/ko mice cohort the differences between the knockout and young as well as old WT mice were striking. Including the fatal infections, 80% of the Tercko/ko mice had a severe course of disease, while none of the WT mice displayed a severe course (Figure 1A, B and Supplemental Figure 1A, B). This hints towards a clear role of Terc in the response to S. aureus infection in mice. Thus while in some figures the differences are not significant, strong trends towards a more severe phenotype of S. aureus infection in the Tercko/ko mice regarding bacterial load, score and inflammatory response could be observed in our study. 

Another example of inadequate group design is when the authors begin dividing their Terc-KO groups by clinical score into animals with or without "systemic infection" (the condition where a bacterium spreads uncontrollably across the many organs and via blood, which should be properly called sepsis), and then compare this sepsis group to other groups (Supplementary Figures 1G; Figure 2; lines 374-376 and 389391). This gives them significant differences in several figures, but because they did not clearly indicate where they applied this stratification in the figure legends, the data are somewhat confusing. Most importantly, methodologically it is highly inappropriate to compare one mouse with sepsis to another one without. If Terc-KO mice with sepsis are a comparator group, then their controls have to be wild-type mice with sepsis, who are dealing with the same high bacterial load across the body and are presumably forced to deploy the same set of immune defenses.

We sincerely appreciate the significant time and effort you have invested in reviewing our manuscript. However, with all due respect, we must point out that the definition of sepsis you have referenced is considered outdated. According to the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3), sepsis is defined as "a life-threatening organ dysfunction caused by a dysregulated host response to infection" (Marvin Singer, 2016, JAMA). Given this fundamental misunderstanding of our findings, we find the comment regarding the inadequacy of our groups to be both dismissive and lacking in scientific merit. We would like to emphasize that the group size used in our study is consistent with accepted standards in infection research. We strongly reject any insinuations of inadequacy that have been repeatedly mentioned throughout the review.

In order to provide a nuanced investigation of disease severity in Tercko/ko mice, we added the term “systemic infection” to the figures whenever the mice were divided into groups of mice with and without systemic infection. This is the case for Figure 2A and Supplemental Figure 1C-E. The division into mice with and without systemic infection is also mentioned in the figure legend of Figure 2A in lines 932 to 935 and for Supplemental Figure 1 in lines 1052-1053. We agree that Supplemental Figure 1G is somewhat confusing as the mice with systemic infection are highlighted in this graph but not included as a separate group within our sequencing analysis. We added a sentence to the figure legend clarifying this (lines 1042-1044):

“Nevertheless, the infected Tercko/ko mice were considered one group for the expression analysis and not split into separate groups for the subsequent analysis.”

Additionally, we revised the section regarding this grouping in different degrees of severity in our Material and Methods section to clarify that this division was only performed for specific analysis (line 191):

“…for the indicated analysis.”

Furthermore, the mice which were classified as systemically infected mice were not septic mice, as mentioned above. Those mice were classified by us as systemically infected based on their clinical score and the presence of bacteria in other organs than the lung as stated in the lines 188-191 and 377-381. Bacteremia is a symptom of very severe cases of hospital-acquired pneumonia with a very high mortality (De la Calle et al., 2016).

Therefore, the systemically infected mice or rather mice with bacteremia display an especially severe pneumonia phenotype, which is distinct from sepsis. The presence of this symptom in our Tercko/ko mice further highlights the clinical relevance of our study. This aspect was added to the manuscript in the lines 568-570.

“The detection of bacteria in extra pulmonary organs is of particular interest, as bacteremia is a symptom of severe pneumonia and is associated with high mortality (De la Calle et al., 2016).”

(4) The authors conclude that disregulated inflammation and T-cell dysfunction play a major role in S. aureus susceptibility. This may or may not be an important observation, because many KO mice are abnormal for a variety of reasons, and until such reasons are mechanistically dissected, the physiological importance of the observation will remain unclear.

Two points are important here. First, there is no natural counterpart to a Terc-KO, which is a complete loss of a key non-enzymatic component of the telomerase complex starting in utero. 

Second, the authors truly did not examine the key basic features of their model, including the features of basic and induced inflammatory and immune responses. This analysis could be done either using model antigens in adjuvants, defined innate immune stimuli (e.g. TLR, RLR, or NLR agonists), or microbial challenge. The only data provided along these lines are the baseline frequencies of total T cells in the spleen of the three groups of mice examined (not statistically significant, Figure 4B). We do not know if the composition of naïve to memory T cell subsets may have been different, and more importantly, we have no data to evaluate whether recruitment of the immune response (including T cells) to the lung upon microbial challenge is similar or different. So, what are the numbers and percentages of T cells and alveolar macrophages in the lung following S. aureus challenge and are they even comparable or are there issues in mobilizing the T cell response to the site of infection? If, for example, Terc-KO mice do not mobilize enough T cells to the lung during infection, that would explain the paucity in many T-cellassociated genes in their transcriptomic set that the authors report. That in turn may not mean dysfunction of T cells but potentially a whole different set of defects in coordinating the response in Terc-KO mice.

We thank the reviewer for highlighting these important aspects. Regarding the first point, indeed there is no naturally occurring deletion of Terc in humans. However, studies reported reduced expression of Terc and Tert in the tissues of aged mice and rats (Tarry-Adkins et al., 2021; Zhang et al., 2018). Terc itself has been found to have several important immunomodulatory functions such as the activation of the NFκB or PI3-kinase pathway (Liu et al., 2019; Wu et al., 2022). As those aforementioned pathways are relevant for the immune response to S. aureus infections, the authors were interested in exploring the impact of Terc deletion on the pulmonary immune response. The potential immunomodulatory functions of Terc are discussed in lines 106-121. To further clarify our rationale we added a sentence to the introduction in lines 121-125.

“Interestingly, downregulation of Terc and Tert expression in tissues of aged mice and rats has been found (Tarry-Adkins, Aiken, Dearden, Fernandez-Twinn, & Ozanne, 2021; Zhang et al., 2018). Therefore, as a potential immunomodulatory factor reduced Terc expression could be connected to agerelated pathologies.”

Regarding the second point, as we focused on the effect of Terc deletion in the lung and its role in S. aureus infection, we investigated inflammatory and immune response parameters relevant to this setting. For instance, inflammation parameters in the lungs of all three mice cohorts were measured to investigate differences in the inflammatory response in the non-infected and infected mice (Figure 2A). Those measurements showed no baseline difference in key inflammatory parameters between young WT and Tercko/ko mice, which is consistent with previous findings (Kang et al., 2018). The inflammatory response to infection with S. aureus in the Tercko/ko mice cohort differed significantly from the other cohorts (Figure 2A), hinting towards a dysregulated inflammatory response due to Terc deletion. Furthermore, we investigated general immune cell frequencies such as dendritic cells, macrophages, and B cells in the spleen of all three mice cohorts to gather a baseline understanding of the general immune cell populations. In our manuscript only total T cell frequencies were included due to its relevance for our data regarding T cells (Figure 4B). This data could show that there was no difference of total amount of T cells in the spleen of all three mice cohorts. For a more detailed insight into our analysis we added the frequencies of the other immune cell populations analyzed in the spleen as a Supplemental Figure 3B-F. Additionally, a figure legend for the graphs was added to lines 1075-1094.

Therefore, while we did not analyze baseline frequencies of specific populations of T cells, we analyzed and characterized the inflammatory and immune response of our model in a way relevant to our research question. 

The differences observed in T cell marker and TCR gene expression was also partly present between the uninfected and infected Tercko/ko mice such as the complete absence of CD247 expression in infected Tercko/ko, which is however expressed in uninfected mice of this cohort (Figure 4A, C and D). Thus, this effect cannot be solely attributed to an inadequate mobilization of T cells to the lung after infectious challenge. However, we agree that a more detailed insight into recruited immune cells to the lung or frequencies of different T cell populations could contribute to a better understanding of the proposed mechanism and would be an interesting experiment to conduct in further studies. We accept this as a limitation of our study and included it in our discussion section in lines 719-723:

“As total CD4+ T cells were analyzed in this study, it would be useful to investigate specific T cell populations such as memory and effector T cells to elucidate the potential mechanism leading to T cell dysfunctionality in further detail. Additionally, analysis of differences in immune cell recruitment to the lungs between young WT and Tercko/ko mice would be relevant.”

(5) Related to that, immunological analysis is also inadequate. First, the authors pull signatures from the total lung tissue, which is both imprecise and potentially skewed by differences, not in gene expression but in types of cells present and/or their abundance, a feature known to be affected by aging and perhaps by Terc deficiency during infection. Second, to draw any conclusions about immune responses, the authors would have to track antigen-specific T cells, which is possible for a wide range of microbial pathogens using peptide-MHC multimers. This would allow highly precise analysis of phenomena the authors are trying to conclude about. Moreover, it would allow them to confirm their gene expression data in populations of physiological interest

We thank the reviewer for highlighting this important and relevant point. In our study, we aimed to investigate the role of Terc expression in modulating inflammation and the immune response to S. aureus infection in the lung. To address this, we examined the overall impact of age, genotype, and infection on lung inflammation and gene expression. Therefore, sequencing of total lung tissue was essential for addressing the research question posed. Our findings demonstrate that Tercko/ko mice exhibit a more severe phenotype following S. aureus infection, characterized by an increased bacterial load and heightened lung inflammation (Figures 1 and 2). Furthermore, our data suggest that Terc plays a role in regulating inflammation through activation of the NLRP3 inflammasome, along with the dysregulation of several T cell marker genes (Figures 2, 4, and 5). However, this study lacks a detailed analysis of distinct T cell populations, including antigen-specific T cells, as noted earlier. Investigating these aspects in future studies would be valuable to validate and expand upon our findings. We have incorporated these suggestions into the discussion section (lines 719-723)

“As total CD4+ T cells were analyzed in this study, it would be useful to investigate specific T cell populations such as memory and effector T cells to elucidate the potential mechanism leading to T cell dysfunctionality in further detail. Additionally, analysis of differences in immune cell recruitment to the lungs between young WT and Tercko/ko mice would be relevant.”

Nevertheless, our study provides first evidence of a potential connection between T cell functionality and Terc expression.  

Third, the authors co-incubate AM and T cells with S. aureus. There is no information here about the phenotype of T cells used. Were they naïve, and how many S. aureus-specific T cells did they contain? Or were they a mix of different cell types, which we know will change with aging (fewer naïve and many more memory cells of different flavors), and maybe even with a Terc-KO? Naïve T cells do not interact with AM; only effector and memory cells would be able to do so, once they have been primed by contact with dendritic cells bringing antigen into the lymphoid tissues, so it is unclear what the authors are modeling here. Mature primed effector T cells would go to the lung and would interact with AM, but it is almost certain that the authors did not generate these cells for their experiment (or at least nothing like that was described in the methods or the text).

Thank you for bringing up this important question. For the co-cultivation experiment of T cells and alveolar macrophages, total CD4+ T cells of both young WT and Tercko/ko were used. We did not select for a specific population of T cells. Our sequencing data indicated the complete downregulation of CD247 expression, which is an important part of the T cell receptor, in the lungs of infected Tercko/ko mice (Figure 4A, C and D). Given that this factor is downregulated under chronic inflammatory conditions, we investigated the impact of the inflammatory response in alveolar macrophages on the expression of various T cell-derived cytokines, as well as CD247 expression (Figure 5D, E) (Dexiu et al., 2022). This aspect is also highlighted in the discussion in lines 622-636. Therefore, a co-cultivation model of T cells and alveolar macrophages was established and confronted with heat-killed S. aureus to elicit an inflammatory response of the macrophages. To emphasize this purpose, we have revised our statement about the model setup in lines 516-518 of the manuscript: 

“An overactive inflammatory response could be a potential explanation for the dysregulated TCR signaling.”

The authors hope this will clarify the intent behind the model setup.

(6) Overall, the authors began to address the role of Terc in bacterial susceptibility, but to what extent that specifically involves inflammation and macrophages, T cell immunity, or aging remains unclear at present.

We thank the reviewer for the helpful and relevant comments. The authors accept the limitations of the presented study such as the reduced number of Tercko/ko mice and the limitations of murine models for S. aureus infection itself and discuss those in the discussion section in the lines 558-560; 576-582; 688-690 and 719-725. However, we hope that our responses have provided sufficient evidence to convince the reviewer that our data supports a clear role for Terc expression in regulating the immune response to bacterial infections, particularly with respect to inflammation and its potential connection to T cell functionality.  

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

The good element first:

I read this paper with genuine interest and applaud the authors for investigating the posited question. I consider it by all means scientifically relevant in the context of physiological/pathophysiological aging and reaction to a disease (here pneumonia). The Terc deletion model looks very appropriate for the question and the methodology is very advanced/in-depth. The data flow/selection of endpoints and assays is very logical to me. Moreover, I like the breakdown of pneumonia into varying levels of severity.

We thank the reviewer for their time and effort taken to revise our manuscript. Additionally, we are grateful to receive your positive feedback regarding our study design and research question.

The weaknesses:

(1) I cannot help but notice that the study is heavily underpowered. As such, it is inadmissible. The key reason is that it is the first of its kind and seminal findings must be strongly propped by the evidence. It is apparent to me that the data scatter presented in the figures tends to be abnormally distributed (e.g. obvious bimodal distribution in some groups). Therefore, the presented comparisons (even if stat. sign) can be heavily misleading in terms of: i) the true magnitude of the observed effects and ii) possible type 2 error in some cases of p value >0.05. Solution: repeat the study to ensure reasonable power/reliability. This will also make it stronger as it will immediately demonstrate its reproducibility (or lack of it).

Thank you for bringing up this extremely relevant point. We acknowledge the issue of the small sample size of Tercko/ko mice as a major limitation of our study. This limitation is also included in our discussion section in the lines 558-560. Thus we fully agree with this limitation and transparently discuss this in our manuscript. However, due to the strict German animal welfare regulations it is not possible to obtain more Tercko/ko mice, as mentioned above. Furthermore, since fatal infections occurred in the Tercko/ko mice cohort we had a reduced number of mice available. 

However, the differences between the Tercko/ko and WT mice were striking. Including the fatal infections 80% of the Tercko/ko mice had a severe course of disease, while none of the WT mice displayed a severe course. This hints towards a clear role of Terc in the response to S. aureus infection in mice.  

(2) In the stat analysis section of M&Ms, the authors feature only 1 sentence. This cannot be. A detailed stats workup needs to be included there. This is very much related to the above weakness; e.g. it is impossible to test for normality (to choose an appropriate post-hoc test) with n=3. Back to square one: study underpowered.

We thank the reviewer for highlighting this important aspect. We carefully revised the method section in lines 357-360 to include all relevant information: 

“Data are presented as mean ± SD, or as median with interquartile range for violin and box plots, with up to four levels of statistical significance indicated. P-values were calculated using Kruskal-Wallis test. Individual replicates are represented as single data points.”

(3) Pneumonia severity. While I noted that as a strength, I also note it as weakness here. It looks to me like the authors stopped halfway with this. I totally support testing a biological effect(s) such as the one investigated here across a spectrum of a given disease severity. The authors mention that they had various severity phenotypes produced in their model but this is not visible in the data figs. I strongly suggest including that as well; i.e., to study the posited question in the severe and mild pneumonia phenotype. This is a very smart path and previous preclinical research clearly demonstrated that this severe/mild distinction is very relevant in the context of the observed responses (their presence/absence, longevity, dynamics, etc). I realize this is challenging, thus, I would probably use this approach in the Terc k/o model as sort of a calibrator to see whether the exacerbation observed in the current setup (severe?) will be also present in a mild pneumonia phenotype. S. aureus can be effectively titrated to produce pneumonia of varying severity.

We thank the reviewer for bringing up this relevant point. 

In our study, we could observe heterogeneity within the infected Tercko/ko cohort. Therefore as pointed out by the reviewer we assigned different degrees of severity to those groups based on clinical scores, the fatal outcome of the disease (fatal subgroup), and the presence of bacteria in organs other than the lungs (systemic infection subgroup) as stated in our materials and methods part in the lines 188-191 (Supplemental Figure 1A and B). Moreover, we highlighted this difference in a number of our figures. For example, when categorizing the mice into groups with and without systemic infection, we noticed that the mice with systemic infection demonstrated a higher bacterial load, significant body weight loss, and increased lung weight (see Supplemental Figure 1C-E). Interestingly, the two mice with systemic infection clustered separately from the other mice, indicating that the mice with systemic infection are transcriptomically distinct from the other mice cohorts (Supplemental Figure 1G). Additionally, the inflammatory response was exclusively elevated in the lungs of mice with systemic infection (Figure 2C). Thus, we included this distinction in several figures and attempted to study the differences between those subgroups but also their similarities. For instance, we could observe that some changes in the transcriptome were present in all three infected Tercko/ko mice such as the complete absence of CD247 expression at 24 hpi (Figure 4D). This distinction therefore provided a more detailed insight into the underlying mechanisms of disease severity in Tercko/ko mice and is lacking in other studies. We agree with the reviewer, that a study investigating mild and severe pneumonia phenotypes would be clinically relevant. However, as noted above, due to ethical considerations related to animal welfare and sustainability, as well as compliance with German animal welfare regulations, it is not possible to obtain additional Tercko/ko mice to carry out the proposed experiment. 

(4) Please read ARRIVE guidelines and note the relevant info in M&Ms as ARRIVE guidelines point out.

Thank you for emphasizing this crucial aspect. We revised our materials and methods section according to the ARRIVE guidelines (lines 179-206).

“Tercko/ko mice aged 8 weeks, were used for infection studies (n = 8; non-infected = 3; infected = 5). Female young WT (age 8 weeks) and old WT (age 24 months) C57Bl/6 mice (both n = 10; non-infected = 5; infected = 5) were purchased from Janvier Labs (Le Genest-Saint-Isle, France). All infected mouse cohorts were compared to their respective non-infected controls, as well as to the infected groups from other cohorts. Additionally, comparisons were made between the non-infected cohorts across all groups.

All mice were anesthetized with 2% isoflurane before intranasal infection with S. aureus USA300 (1x108 CFU/20µl) per mouse. After 24 hours, the mice were weighed and scored as previously described (Hornung et al., 2023). Infected Tercko/ko mice were grouped into different degrees of severity based on their clinical score, fatal outcome of the disease (fatal) and the presence of bacteria in organs other than the lung (systemic infection) for the indicated analysis. Mice with fatal infections were excluded from subsequent analyses, with only their final scores being reported. The mice were sacrificed via injection of an overdose of xylazine/ketamine and bleeding of axillary artery after 24 hpi. BAL was collected by instillation and subsequent retrieval of PBS into the lungs. Serum and organs were collected. Bacterial load in the BAL, kidney and liver was determined by plating of serially diluted sample as described above. For this organs were previously homogenized in the appropriate volume of PBS. Gene expression was analyzed in the right superior lung lobe. Lobes were therefore homogenized in the appropriate amount of TriZol LS reagent (Thermo Fisher Scientific, Waltham, MA, US) prior to RNA extraction. The left lung lobe was embedded into Tissue Tek O.C.T. (science services, Munich, Germany) and stored at 80°C until further processing for histological analysis. Cytokine measurements were performed using the right inferior lung lobe. Lobes were previously homogenized in the appropriate volume of PBS. Remaining organs were stored at -80°C until further usage. Mouse studies were conducted without the use of randomization or blinding.“

(5) There are also some other descriptive deficits but they are of a much smaller caliber so I do not list them.

We thank the reviewer for their valuable and insightful suggestions for improving our manuscript. We hope that our responses and the corresponding revisions address these suggestions satisfactorily.

Concluding: the investigative idea is great/interesting and the methodological flow is adequate but the low power makes this study of low reliability in its current form. I strongly urge the authors to walk the extra mile with this work to make it comprehensive and reliable. Best of luck!

Reviewer #2 (Recommendations For The Authors):

(1) Many legends are uninformative and do not contain critical information about the experiments. For example, Figure 2A with cytokine measurements (in lung homogenates?) is likely showing data from an ELISA or Luminex test, but there is no mention of that in the legend. It stands next to Figure 2B, which is a gene expression map, again, likely from the lung (prepared how, normalized how, etc?) lacking even the most basic information. Further, Figure 2D has no information on the meaning/effect size of gene ratios on the x-axis. Figures 3 and 4 are presumably the subsets of their transcriptome data set (whole lung, harvested on d ?? post-infection), but that is just a guess on my part. Even in the main text, the timing and the controls for the transcriptomic study are not stated (ln. 398 and onwards). The authors really need to revise the figure legends and provide all the details that an average reader would need to be able to interpret the data.

We thank the reviewer for bringing up this important point. The figure legends of all figures including supplemental figures were revised to ensure they include all relevant data necessary for accurate interpretation of the graphs. Additionally, we clarified the sequenced samples in lines 427-429:

“We performed mRNA sequencing of the murine lung tissue of infected and non-infected mice at 24 hpi to elucidate potential differentially expressed genes that contribute to the more severe illness of Tercko/ko mice.”

(2) Telomere shortening affects differentially different cells and its role in aging is nuanced - different in mesenchymal cells with no telomerase induction, in non-replicating cells, and in hematopoietic cells that can readily induce telomerase. The authors should be mindful of that in setting up their introduction and discussion.

Thank you for mentioning this essential aspect. We revised our introduction and discussion to reflect the nuanced role of telomerase shortening in different tissues (lines 83-92 and 690-695):

“Telomerase activity is restricted to specific tissues and cell types, largely dependent on the expression of Tert. While Tert is highly expressed in stem cells, progenitor cells, and germline cells, its expression is minimal in most differentiated cells (Chakravarti, LaBella, & DePinho, 2021). Consequently, the impact of telomerase dysfunction on tissues varies according to their self-renewal rate. (Chakravarti et al., 2021). One important aspect of telomere dysfunction is the impact of telomere shortening on the immune system as well as the hematopoietic system. Tissues or organ systems that are highly replicative, such as the skin or the hematopoietic system, are affected first by telomere shortening (Chakravarti et al., 2021).”

“It is important to note that telomere shortening has a significant impact on the immune system. Although young Tercko/ko mice were used in this study, telomere shortening is still likely to be a contributing factor. Therefore, further experiments investigating the role of T cell senescence in this model should therefore be conducted.”

(3) Syntax and formulations need to be improved and made more scientifically precise in several spots. Specifically, in 62-63, the authors say that the aged immune system "is also discussed to be more irritable", please change to reflect the common notion that the reaction to infection is dysregulated; in many cases inflammation itself is initially blunted, misdirected, and of different type (e.g. for viruses, the key IFN-I responses are not increased but decreased). In lines 114-117, presumably, the two sentences were supposed to be connected by a comma, although some editing for clarity is probably needed regardless. Line 252, please change "unspecific" to "non-specific". Line 264, please capitalize German.

We thank the reviewer for bringing these important points to our attention. We revised our introduction regarding the aged immune response in lines 61-69:

“Age-related dysregulation of the immune response is also characterized by inflammaging, defined as the presence of elevated levels of pro-inflammatory cytokines in the absence of an obvious inflammatory trigger (Franceschi et al., 2000; Mogilenko, Shchukina, & Artyomov, 2022). Additionally, immune cells, such as macrophages, exhibit an activated state that alters their response to infection (Canan et al., 2014). In contrast, the immune response of macrophages to infectious challenges has been shown to be initially impaired in aged mice (Boe, Boule, & Kovacs, 2017). Thus aging is a relevant factor impacting the pulmonary immune response.”

Sentences were edited to provide more clarity in lines 131-134:

“Although G3 Tercko/ko mice with shortened telomeres were used in this study, they were infected at a young age (8 weeks). This approach allowed for the investigation of Terc deletion effects rather than telomere dysfunction.”

“Unspecific was changed to “non-specific” in line 282 and “German” was capitalized in line 293 and 558.

We appreciate and thank you for your time spent processing this manuscript and look forward to your response.

References

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Dexiu, C., Xianying, L., Yingchun, H., & Jiafu, L. (2022). Advances in CD247. Scand J Immunol, 96(1), e13170. https://doi.org/10.1111/sji.13170  

Herrera, E., Samper, E., Martín-Caballero, J., Flores, J. M., Lee, H. W., & Blasco, M. A. (1999). Disease

states associated with telomerase deficiency appear earlier in mice with short telomeres. Embo j, 18(11), 2950-2960. https://doi.org/10.1093/emboj/18.11.2950  

Hornung, F., Schulz, L., Köse-Vogel, N., Häder, A., Grießhammer, J., Wittschieber, D., Autsch, A., Ehrhardt, C., Mall, G., Löffler, B., & Deinhardt-Emmer, S. (2023). Thoracic adipose tissue contributes to severe virus infection of the lung. International Journal of Obesity, 47(11), 10881099. https://doi.org/10.1038/s41366-023-01362-w  

Kang, Y., Zhang, H., Zhao, Y., Wang, Y., Wang, W., He, Y., Zhang, W., Zhang, W., Zhu, X., Zhou, Y., Zhang, L., Ju, Z., & Shi, L. (2018). Telomere Dysfunction Disturbs Macrophage Mitochondrial Metabolism and the NLRP3 Inflammasome through the PGC-1α/TNFAIP3 Axis. Cell Reports, 22(13), 3493-3506. https://doi.org/https://doi.org/10.1016/j.celrep.2018.02.071  

Khan, A. M., Babcock, A. A., Saeed, H., Myhre, C. L., Kassem, M., & Finsen, B. (2015). Telomere dysfunction reduces microglial numbers without fully inducing an aging phenotype. Neurobiology of Aging, 36(6), 2164-2175. https://doi.org/https://doi.org/10.1016/j.neurobiolaging.2015.03.008  

Lee, H.-W., Blasco, M. A., Gottlieb, G. J., Horner, J. W., Greider, C. W., & DePinho, R. A. (1998). Essential role of mouse telomerase in highly proliferative organs. Nature, 392(6676), 569-574. https://doi.org/10.1038/33345  

Liu, H., Yang, Y., Ge, Y., Liu, J., & Zhao, Y. (2019). TERC promotes cellular inflammatory response independent of telomerase. Nucleic Acids Research, 47(15), 8084-8095. https://doi.org/10.1093/nar/gkz584  

Matthe, D. M., Thoma, O. M., Sperka, T., Neurath, M. F., & Waldner, M. J. (2022). Telomerase deficiency reflects age-associated changes in CD4+ T cells. Immun Ageing, 19(1), 16. https://doi.org/10.1186/s12979-022-00273-0  

Rudolph, K. L., Chang, S., Lee, H. W., Blasco, M., Gottlieb, G. J., Greider, C., & DePinho, R. A. (1999). Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell, 96(5), 701-712. https://doi.org/10.1016/s0092-8674(00)80580-2  

Tarry-Adkins, J. L., Aiken, C. E., Dearden, L., Fernandez-Twinn, D. S., & Ozanne, S. (2021). Exploring Telomere Dynamics in Aging Male Rat Tissues: Can Tissue-Specific Differences Contribute to Age-Associated Pathologies? Gerontology, 67(2), 233-242. https://doi.org/10.1159/000511608  

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Zhang, M. W., Zhao, P., Yung, W. H., Sheng, Y., Ke, Y., & Qian, Z. M. (2018). Tissue iron is negatively correlated with TERC or TERT mRNA expression: A heterochronic parabiosis study in mice. Aging (Albany NY), 10(12), 3834-3850. https://doi.org/10.18632/aging.101676

I found this intresting because the Master of Life created everything and that is what they saw.

Juan 13:2-5, 12-17

Una lección de humildad 14 Por eso, si yo, el Señor y Maestro, les he lavado los pies a ustedes,+ ustedes también deben lavarse los pies unos a otros.

Juan 1:9-18 10 En realidad, él ya estaba en el mundo,+ y el mundo llegó a existir por medio de él,+ pero el mundo no lo conoció.*

Mateo 13:10-15 Porque el corazón de este pueblo se ha hecho insensible. Se han tapado los oídos y han cerrado los ojos,

Proverbios 8:22-31 Jesús reflejo a la perfección la sabiduría de Jehová, por ser su primogénito y mediante quien se hizo todo lo demás.

Mateo 13:34

explicó cómo mejorar las relaciones personales (5:23-26, 38-42; 7:1-5, 12), cómo mantener la pureza moral (5:27-32) y cómo llevar una vida con sentido (6:19-24; 7:24-27). Pero no solo dijo cómo conseguir sabiduría, sino que lo demostró dando explicaciones, razones y pruebas.

Me resulta interesante la comparación que realiza la autora entre Dios y la maquinaria moderna ya que, según entiendo, quiere mostrar como la idea de omnipresencia de Dios, se puede aplicar a la tecnología actual. Señala que esta invade de manera imperceptible nuestras vidas, de una manera u otra

La autora nos presenta la figura del ciborg como una herramienta que va más allá de las construcciones sociales que existen en la actualidad. No se puede identificar con ningún género, ni sigue unos patrones relacionales como los que están instaurados en la sociedad. Por ello, nos puede hacer plantearnos si este nuevo concepto podría cambiar nuestra forma de entender las identidades, haciendo que sean híbridas, libres y mutables.

Author response:

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

Reviewer 1:

Comment 1: IPA analysis was performed after scRNA-seq. Although it is knowledge-based software with convenient graphic utilities, it is questionable whether an unbiased genome-level analysis was performed. Therefore, it is not convincing if WNT is the only and best signal for the branching-off marker. Perhaps independent approaches, such as GO, pathway, or module analyses, should be performed to validate the finding.

Thanks for your comment. We agree with the reviewer that IPA is a knowledge-based and a hypothesis-driven method. Our hypothesis was that WNT/BMP pathways, among others, are heavily involved in the development of mesenchymal tissues in general and differentiation of tendons specifically. Therefore, we have looked at differentially expressed genes between clusters from a broad array of pathways featured in IPA that could point us towards molecular function that could make a difference. We further corroborated this hypothesis by using WNT inhibitors in subsequent experiments. To address this point, we have supplemented the discussion section with the following remark:

“This study is not without limitations. The IPA network analysis is a knowledge-based and hypothesis driven platform. We have specifically targeted known pathways to be involved in syndetome differentiation. However, WNT signaling stood out with very specific affinity to the off-target populations and we have verified our findings with experiments proving this hypothesis.”

Per the reviewer’s suggestion, we also performed a non-biased GO analysis (Supp. Fig. 6). Multiple pathways were detected in the three clusters of interest (Supp. Fig. 6A-C), including integrin-related and TGFβ-related pathways. However, in these three clusters of interest, WNT signaling was also detected as a prominent pathway. Therefore, we could conclude that it plays a pivotal role in the differentiation process. This hypothesis was later corroborated with WNT inhibitor experiments.

Comment 2: According to the method section, two iPSC lines were used for the study. However, throughout the manuscript, it is not clearly described which line was used for which experiment. Did they show similar efficiency in differentiation and in responses to WNTi? It is also worrisome if using only two lines is the norm in the stem cell field. Please provide a rationale for using only two lines, which will restrict the observation of individual-specific differential responses throughout the study.

Thanks for your comment. This proof-of-concept study is the first investigation that compares data of an in vitro tenogenic induction protocol that has been tested in more than one human iPSC lines. We agree that line-specific phenomena are difficult to interpret and reproduce. Therefore, it is critical to provide data supporting that the findings can be reproduced in more than one line. Some early studies used one line as proof of concept, however now we realize the need to show that the protocol works in at least one additional line.

Here we used the GMP-ready iPSC line CS0007iCTR-n5 for all optimization experiments. This newer low passage feeder-free line was generated from PBMCs and was designated as GMP-ready in the manuscript because it has been derived and cultured using cGMP xeno-free components (mTESR plus medium and rhLaminin-521 matrix substrate instead of Matrigel). We then wanted to confirm the application of the optimized protocol using the reference control line CS83iCTR-22n1 which has already been more widely used by our group1-5 and others.6 This line has been derived from fibroblasts and has been grown and expanded using MatrigelTM and mTESR1, followed by mTESR plus media. 

The question of number of lines needed is stage-dependent. In our opinion at the proof-of-concept level, two lines, one of which has been generated in GMP-like conditions is sufficient. Confirmation with multiple lines becomes more pertinent as we move towards scale-up/manufacturing, where considerations regarding robustness and consistency are raised. However, at this stage, it is crucial to understand the developmental processes that are involved in cell differentiation to ensure a more robust protocol can be modified and adapted later. In future studies, as we move towards clinical translation, it is warranted that the approach presented in this work will be further optimized and subsequently evaluated using at least 3 different cell lines that have been generated from various sources.

Comment 3: How similar are syndetome cells with or without WNTi? It would be interesting to check if there are major DEGs that differentiate these two groups of cells.

Thanks for your comment. Single cell RNAseq analysis revealed that treatment with WNTi upregulated tenogenic markers. In SYNWNTi, the expression levels of stage-specific markers COL1A1, COL3A1, SCX, MKX, DCN, BGN, FN1, and TNMD were higher compared to the untreated SYN group, as shown in Figure 5C. Density plots depicted an increase in the number of cells expressing COL1A1, COL3A1, SCX and TNMD in SYNWNTi compared to the SYN group, as illustrated in Figure 5D. Trajectory analysis of the WNTi-treated group revealed the absence of bifurcations observed in the untreated group (Fig. 5E). Therefore, it can be conjured that syndetome cells with and without WNTi are different.

Comment 4: Please discuss the improvement of the current study compared to previous ones (e.g., PMID 36203346 my study, 35083031- Tsutsumi, 35372337- Yoshimoto).

Thanks for your comment. In Papalamprou et al (2023)3, we differentiated iPSCs to mesenchymal stromal-like cells (iMSCs), which were then cultured into a 2D dynamic bioreactor for 7 days. In that study, we examined the impact of simultaneous overexpression of the tendon transcription factor Scleraxis (SCX) using a lentiviral vector and mechanical stimulation on the process of tenogenic differentiation. Following 7 days of uniaxial cyclic loading, we observed notable modifications in the morphology and cytoskeleton organization of iPSC-derived MSCs (iMSCs) overexpressing SCX. Additionally, there was an increase in extracellular matrix (ECM) deposition and alignment, along with upregulation of early and late tendon markers. This proof-of-concept study showed that iPSC-derived MSCs could be a viable cell candidate for cell therapy applications and that mechanical stimulation is contributing to the differentiation of iMSCs towards the tenogenic lineage.

Similarly, Tsutsumi et al7 overexpressed the tendon transcription factor Mohawk (MKX) stably in iPSC-derived MSCs using lentiviral vectors. These cells were then used to seed collagen hydrogels which were mechanically stimulated in a cyclic stretch 3D culture bioreactor for 15 days to create artificial tendon-like tissues, which the authors termed “bio-tendons”. Bio-tendons were then decellularized to remove cellular remnants from the xenogeneic human iPSC-derived cells and were subsequently transplanted in an in vivo Achilles tendon rupture mouse model. The authors reported improved histological and biomechanical properties in the Mkx-bio-tendon mice vs. the GFP-bio-tendon controls, providing another proof-of-concept study in favor of the utilization of iPSC-derived MSCs for tendon cell therapies, while also addressing the immunogenicity of cells of allogeneic/xenogeneic origin. Therefore, the above two studies used tendon transcription factor overexpression and mechanical loading either in 2D or 3D to differentiate MSCs towards the tendon/ligament lineage.

Yoshimoto et al8 optimized a stepwise iPSC to tenocyte induction protocol using a SCX-GFP transgenic mouse iPSC line, by monitoring GFP expression over time. The group performed scRNA-seq to characterize the induction of mesodermal progenitors towards the tenogenic lineage and to shed light into their developmental trajectory. That study unveiled that Retinoic Acid (RA) signaling activation enhanced chondrogenic differentiation, which was in contrast to the study of Kaji et al (2021), which also used a SCX-GFP mouse iPSC line. Kaji et al inhibited TGF and BMP signaling during the process of mesodermal induction and reported that RA signaling eliminated SCX induction entirely and promoted a switch to neural fate. Yoshimoto et al suggested that variations in mesodermal cell identity could be due to the different methods used for mesodermal differentiation. In contrast to the Kaji et al study, Yoshimoto et al opted to stimulate WNT and block the Hedgehog pathway during mesoderm induction. Loh et al (2016) identified the branchpoint from the primitive streak to either the paraxial mesoderm (PSM) or the lateral plate mesoderm (LPM) as the result of two mutually exclusive signaling conditions. Specifically, they reported that induction of PSM was achieved through BMP suppression and WNT stimulation, while the specification of lateral mesoderm was accomplished by BMP stimulation and WNT suppression, all with concurrent TGFβ suppression/FGF stimulation. Lastly, a similar approach towards PSM induction from primitive streak (TGF off/BMP off/ WNT on/FGF on) has been used by many subsequent studies Matsuda et al (2020),9 Wu et al (2021)10 and Nakajima et al (2021).11 The diversity of the above-mentioned approaches points to the plasticity of mesodermal progenitors and the need for additional studies to better understand mesodermal specification and subsequent induction towards sclerotome and syndetome.   

In the current study we optimized a stepwise differentiation protocol using xeno-free cGMP ready media and two different cell lines, one of which was cGMP-ready. We used scRNA-seq to characterize the differentiation, which led us to identify off-target cells that were closer to a neural phenotype. We performed pathway analyses and hypothesized that WNT signaling activity might have contributed to the emergence of the off-target cells. To test this, we used a WNT inhibitor (PORCN) to block WNT activity at the SCL stage and at the SYN stage. We found that blockade of WNT signaling at the end of the SM stage and during SCL and SYN induction resulted in a more homogeneous population, while eliminating the neural-like cell cluster. This is the first study that utilized scRNA-seq to shed light into the developmental trajectory of stepwise iPSC to tendon differentiation of human iPSCs and provided a proof-of-concept for the generation of a more homogeneous syndetome population. Further studies are needed to further fine-tune both the process and the final product, as well as elucidate the functionality of iPSC-derived syndetome cells in vitro and in vivo.

Reviewer 2:

General concerns: The authors demonstrated the efficiency of syndetome induction solely by scRNA-seq data analysis before and after pathway inhibition, without using e.g. FACS analysis or immunofluorescence (IF)-staining based assessment. A functional assessment and validation of the induced cells is also completely missing.

We appreciate and agree with the reviewer’s critique regarding further analyses of differentiated iPSC-derived syndetome-like cells, including functional assessment of the differentiated cells. Immunofluorescence was used at all timepoints of induction for phenotype confirmation (Fig. 2,4). Flow cytometry for DLL1 was utilized to benchmark efficient differentiation to PSM (Loh et al,12 Nakajima et al11. Specifically, DLL1 expression was assessed with flow cytometry after 4 days of induction, and was used to optimize the parameter of initial iPSC aggregate seeding density, which has been previously found to be crucial for in vitro differentiation protocols (Loh et al12). Unfortunately, this parameter is usually not reported although it could be critical to establish protocol replication between different lines.

The function of tendon progenitors is usually reported as response to mechanical cues and the ability to regenerate tendon injuries. In future studies we intend to assess the functionality of the generated syndetome and tendon progenitors and their response to in vitro biomechanical stimulation as previously reported to iMSCSCX+ cells3, 13 and in vivo in a critical tendon defect  similarly to what has been previously reported.2 

Comment 1: Notably, in Figure 1D, certain PSM markers (TBXT, MSGN1, WNT3A) show higher expression on day 3. If the authors initiate SM induction on day 3 instead of day 4, could this potentially enhance the efficiency of syndetome-like cell induction?

Thanks for your comment. In the current work, we initially optimized differentiation to PSM via expression of DLL1, whose gene expression peaked at d4. We found that this was influenced by the initial iPSC aggregate seeding density. We wanted to generate a homogeneous DLL1+ population which we assessed via gene expression, flow cytometry, IF and scRNA-seq (Fig. 1D, 2C, 3C and Suppl. Fig.1). Given the fact that different lines might display a diverse developmental timeline, we also confirmed reproducibility of the protocol with a second cell line. We appreciate the reviewer’s suggestion to investigate additional protocol iterations, such as the proposed one at the PSM stage, as we move towards a better understanding of key developmental events during in vitro induction.

Comment 2:  In the third paragraph of the result section the authors note, "Interestingly, SCX, a prominent tenogenic transcription factor, was significantly downregulated at the SCL stage compared to iPSC, but upregulated during the differentiation from SCL to SYN." Despite this increase, the expression level of SCX in SYN remains lower than that in iPSCs in Fig.1G and Fig.3C. Can the authors provide an explanation for this? Can the authors provide IF data using iPSCs and compare it with in vitro-induced SYN cells? Can the authors provide e.g. additional scRNA-seq data which could support this statement?

Thank you for your comment. In Fig. 1G, SCX expression in SYN was upregulated compared to SCL, however, it was shown to be similar to iPSCs. This suggests a baseline stochastic expression of SCX possibly stemming from spontaneous differentiation of iPSCs in culture (Fig. 3C). Previous research has shown that tenogenic marker gene expression tends to reduce during postnatal tendon maturation (Yin et al., 2016b14 Grinstein et al., 2019.15 Yoshimoto et al (2022) utilized a transgenic mouse iPSC-SCX-GFP line  to track SCX expression. It was shown that SCX expression peaked after 7d of tenogenic induction and was then decreased at day 14, which marked the end of tenogenic induction. The authors postulated that this pattern of gene expression could either indicate further maturation of tenocytes at subsequent time points, or that the number of non-tenogenic cells increased from T7 to T14.

In the present work, we showed SCX gene expression upregulation in SYN compared to SCL, as well as significant upregulation of TNMD, EGR1, COL1A1 and COL3A1 (Fig.1G). Supp. Fig.8 has been added to show feature plots of SCX and TNMD expression from SCL, SYN and SYNWNTi.  The significant upregulation of later markers of tenogenic differentiation suggests that the 21 days of tenogenic induction might have matured the cells. Since gene expression analysis only conveys a snapshot of the transcriptional profile of a cell population, it is likely that we might have missed the peak of SCX upregulation (Supp. Fig. 5). Following treatment with the WNT inhibitor, the SYNWNTi group displayed increased SCX expression (% cells expressing SCX) compared to SYN, which might also be due to a more homogeneous population of syndetome-like cells following treatment with WNTi. In the SYNWNTi group, TNMD was shown to be expressed in the SYN cluster, whereas SCX was mostly found in the cluster that was labelled as fibrocartilage (FC) cluster based on the expression of COL2A1/SOX9/FN1/BGN/COL1A1 markers. Due to the fact that SCX+/SOX9+ progenitor cells are able to give rise to both tendon and cartilage (Sugimoto 2013)16, it could be postulated that this cluster contains tendon progenitors. Interestingly, the FC cluster was not observed in the second iPSC line that we tested, which resulted in a more homogeneous induction to syndetome (78.5% vs. 66.9% SYN cells, Supp. Table 1 & Supp. Fig.3). This slight discrepancy between the two lines and more specifically the presence of the FC cluster only in the 007i line, warrants further investigation. Taken together, these data indicate that the tenogenic induction duration could likely be shortened. Further work to assess the time course of SCX expression over the entire tenogenic induction could be used to further optimize the in vitro induction. For instance, a human edited iPSCSCX-GFP+ line could be generated and used to track SCX expression during the entire induction.

Comment 3: In the fourth paragraph of the result section the authors state, "SM markers (MEOX1, PAX3) and SCL markers (PAX1, PAX9, NKX3.2, SOX9) were upregulated in a stepwise manner." However, the data for MEOX1 and NKX3.2 seems to be missing from Figure 3B-C. The authors should provide this data and/or additional support for their claim.

Thanks for your comment. Feature plots for MEOX1 and NKX3.2 have been added to the Supplemental information (Supp. Fig. 9).

Comment 4: In Figures 2B and 2E, the background of the red channel seems extremely high. Are there better images available, particularly for MEOX1? Given the expected high expression of MEOX1 in SM cells, the authors should observe a strong signal in the nucleus of the stained somitic mesoderm-like cells, but that is not the case in the shown figure. The authors should provide separate channel images instead of merged ones for clarity. The antibody which the authors used might not be specific. Can the authors provide images using an antibody which has been shown to work previously e.g. antibody by ATLAS (Cat#: HPA045214)?

As requested by the reviewer, we have provided separate channels for those images in the Supplement (Supp. Fig. 7). The images show relatively high expression of these markers in SM cells.

Comment 5: In Fig. 2C and Supplementary Fig. 1, the authors present data from immunofluorescence (IF) staining and FACS analysis using a DLL1 antibody. While FACS analysis indicates an efficiency of 96.2% for DLL1+ cells, this was not clearly observed in their IF data. How can the authors explain this discrepancy? Could the authors quantify their IF data and compare it with the corresponding FACS data?

Thanks for your comment. We performed flow cytometric analysis of DLL1 expression to optimize cell seeding density using the 007i line. In the present study, we used IF only in a qualitative manner, that is to confirm protein expression of selected markers. It could be noted that the use of poly-lysine coated coverslips, which are needed for IF, might have slightly altered the density of the cells on the coverslip vs. the plate. Lastly, it cannot be ruled out that the different substrate could have influenced their phenotype differentially through matrix interactions and signaling. On the other hand, flow cytometry by nature is a quantitative and single cell approach, whereas IF staining is qualitative. Therefore, for the purpose of this proof-of-concept work, we tend to trust the quantitative data from the flow cytometry results more than semi-quantitative confirmation achieved through IF staining using coverslips. 

Comment 6: In Fig. 2G, PAX9 is expected to be expressed in the nucleus, but the shown IF staining does not appear to be localized to the nucleus. Could the authors provide improved or alternative images to clarify this? The authors should use antibodies shown to work with high specificity as already reported by other groups.

Thanks for your comment. Indeed, the staining seems to be mostly cytoplasmic. We have used antibodies that were previously reported3 and repeated the staining, however, the same results were replicated. We can speculate that this transcription factor has additional role in the iPSC-derived cells and might be traveling to the cytoplasm. Unfortunately, we have no evidence to this phenomenon.  

Comment 7: Why did the authors choose to display day 10 data for SYN induction in Fig. 4A? Could they provide information about the endpoint of their culture at day 21?

Thank you for your comment. In Fig. 1G we provided gene expression analyses results for several selected early and later tendon markers for the endpoint of our culture, that is day 21. Following scRNA-seq at each stage of the differentiation (iPSC at d0, PSM at d4, SM at d8, SCL at d11 and the endpoint day 32 for SYN), we performed DEG analysis using the IPA platform. We identified activation of genes associated with the WNT signaling pathway in the off-target clusters. We hypothesized that WNT pathway inhibition might block the formation of unwanted fates and induce a more homogeneous differentiation outcome. We thus tested a WNT inhibitor and compared the inhibitor-treated group with a non-treated group. We then assessed selected neural markers during the course of the inhibitor application. In Fig. 4A we presented gene expression of key selected markers at day 21 using qPCR, which was approximately in the middle of the syndetome induction. Since we observed that the inhibitor downregulated the selected neural markers, we then applied the inhibitor until the endpoint of the initial induction and proceeded to analyze the results using scRNA-seq (Fig. 5). Lastly, it should be acknowledged that this was a proof-of-concept study, and additional optimizations are needed regarding the application of the inhibitor (timing, duration, concentration, etc).

Comment 8: In Supplementary Fig. 5, the authors depicted the expression level of SCX, a SYN marker, which peaked at day 14 and then decreased. By day 21, it reached a level comparable to that of iPSCs. Given this observation, could the authors provide a characterization of the cells at day 21 during SYN induction using IF? What was the rationale behind selecting 21 days for SYN induction? The authors also need to show 'n numbers'; how many times were the experiments repeated independently (independent experiments)?

Thanks for your comment. During the optimization process, we initially used RT-qPCR to track gene expression of selected tenogenic markers using the 007i line. We found that after 21 days of tenogenic induction there was upregulation of the few established tendon markers, that is COL1A1, COL3A1, EGR1 and quite importantly, the more definitive later tendon marker, TNMD. Thus, we decided to proceed with this protocol prior to testing other compounds including the WNT inhibitor WNT-C59. However, as has been discussed in the manuscript, this extended tenogenic induction resulted in cell attrition without the application of the WNT inhibitor. This phenomenon was ameliorated following WNT inhibition. Thus, it could be postulated that the protocol could be further optimized by shortening tenogenic induction to less than 21 days.

The experiments that were conducted to optimize the differentiation process were repeated independently at least n=3 times using qPCR and IF using two lines, that is the 007i and the 83i line as described in the manuscript. The scRNAseq analysis represents a population of cells from in vitro differentiation that originated from the same donor line, therefore it was performed on n=1 sample at each stage. However, the effects of inhibitor application (sample SYNWNTi) were also confirmed using a second cell line (83i), thus a total of n=2 independent samples were analyzed.  

Comment 9: Overall the shown immunofluorescence (IF) data does not appear convincing. Could the authors please provide clearer images, including separate channel images, a bright field image, and magnified views of each staining?

Thanks for your comment. The separate channels images were added to the supplemental data (Supp. Fig. 7). We agree with the reviewer regarding the limitations of IF staining, especially with the added confounding factor of using poly-lysine coated coverslips. We would like to point out, that in the current work IF staining is not the main finding or the primary outcome measure, and that it is only used to further support the differentiation by providing a qualitative assessment of protein presence and localization. We describe in this paper our thesis regarding the limitations of IF and the need for more high-throughput unbiased approaches to quantification when using IF staining. For instance, spatial transcriptomics combined with mass cytometry or flow cytometry could be used for a more unbiased approach. Thus, in the present manuscript we based our conclusion on the quantitative gene expression, single cell sequencing and flow cytometry.

Comment 10: As stated by the authors in the manuscript, another research group performed FACS analysis to assess the efficiency of syndetome induction using SCX antibody, and/or quantification of immunofluorescence (IF) with SCX, MKX, COL1A1, or COL2A1 antibodies. Could the authors conduct a comparative analysis of syndetome induction efficiency both before and after protocol optimization, utilizing FACS analysis in conjunction with an SCX reporter line or antibody staining, e.g. quantifying induction efficiency via immunofluorescence (IF) staining with syndetome-specific marker genes?

Thank you for your comment. As discussed in a previous comment, we agree with the reviewer that the generation of a human iPSC-SCX-GFP line would shed light into SCX expression over the entire course of induction. In the current work we used IF as qualitative confirmation of specific marker expression and we showed the presence of SCX, MKX, COL1 and COL3 in SYNWNTi as well as the absence of neuronal markers. As we also pointed it out in the present manuscript, IF can only be considered as a semi-quantitative assessment burdened with several technical limitations as well as operator bias and lower sensitivity and accuracy compared to flow cytometry or scRNA-seq, unless performed in a more unbiased manner. To further clarify this point, firstly, using poly-lysine coated coverslips for IF staining, results in a different substrate environment compared to the Geltrex-coated plates that were used for the induction. Additionally, we noticed that cells grew overconfluent at the edges of the coverslips. This is an important point, since as we have observed in this work, seeding density is critical for the reproducibility of the protocol. It could further be postulated that a different cell substrate stiffness might also have an effect on this process. In our opinion, in this context IF should rather be used qualitatively and a combination of flow cytometry with scRNAseq should be utilized to draw quantitative conclusions such as induction efficiencies of a certain cell type. Since we also observed inconsistencies with the SCX antibodies we tested, the generation of edited human iPSC lines (such as SCX-GFP, MKX-GFP and TNMD-GFP) would be the preferred approach to further explore the efficiency of differentiation.

Comment 11: To enhance the paper's significance, the authors should conduct functional validation experiments and proper assessment of their induced syndetome-like cells. They could perform e.g. xeno-transplantation experiments with syndetome cells into SCID-mice or injury models. They could also assess whether the in vitro induced cells could be applied for in vitro tendon/ligament formation.

Thanks for your comment. For the purpose of this proof-of-concept in vitro study, our primary goal was to initially evaluate a stepwise tenogenic induction protocol using GMP-ready cell lines and chemically defined media. Then, we wanted to utilize the analytical power of scRNA-seq in order to characterize and optimize the protocol, thus focusing on one developmental stage that is not well understood, that of syndetome specification from sclerotome, and hypothesized that by fine-tuning the WNT pathway we would be able to generate a more homogeneous syndetome cell population. We fully agree with the reviewer that the warranted next steps should be to conduct several functional validation experiments, such as in vitro 2D/3D tendon/ligament formation and in vivo transplantation in allogeneic or xenogeneic injury models.

Comment 12: The authors should also compare their scRNA-seq data with actual human embryo data sets, something which could be done given the recent increase in available human embryo scRNA-seq data sets.

This is a great idea and intriguing study. Unfortunately, not all data sets are available at the moment and specifically embryonic and MSK scRNA-seq data is very scarce, although growing. We have no access to data sets from human tendon development, and thus will have to leave this comparison for future studies.

Reviewer 3:

Comment 1: The data outlining the differences between the differentiation outcome of the two tested iPSCs is intriguing, but the authors fail to comment on potential differences between the two iPSC lines that could result in drastically different cell outputs from the same differentiation protocol. This is a critically important point, as the majority of the SCX+ cells generated from the 007i cells using their WNTi protocol were found in the FC subpopulation that failed to form from the 83i line under the same protocol. From the analysis of only these 2 cell lines in vitro, it is difficult to assess whether this WNTi protocol can be broadly used to generate tenogenic cells.

Thanks for your comment. This proof-of-concept study is the first investigation that compares data of an in vitro tenogenic induction protocol that has been tested into more than one cell lines. Using unsupervised clustering we identified 11 clusters, which were classified into 6 cell subpopulations. The only observed difference between the two lines was a small subset that was labeled as fibrocartilage (FC), which displayed expression of both tenogenic and chondrogenic markers. This subpopulation was observed in 007i line but not in the 83i line at the end of the SYN induction. Importantly, DEG analysis also showed that it was enriched for SCX. It has been shown that SCX+/SOX9+ progenitors are a distinct multipotent cell group, responsible for the development of SCX−/SOX9+ chondrocytes and SCX+/SOX9− tenocytes/ligamentocytes (Sugimoto 2013)16. As noted in a previous comment (Comment 2 from Reviewer 1), we might have missed SCX upregulation during the 21-day syndetome induction. This can be further supported by Fig. 5E trajectory analysis which shows that this subpopulation (FC) precedes the SYN cell subpopulation. The fact that this subpopulation was present in one line but not the other, might indicate that 83i line resulted in a more mature tendon population. Therefore, we would rather posit that in the case of 83i line, it might not be that the FC subpopulation failed to form, but rather that it was missed in our scRNAseq endpoint analysis which showed that a more homogeneous SYN population was formed (8.7 % in 007i vs. 0.26 % in 83i, Supp. Table 1 & Supp. Fig. 3B). Future studies are warranted to characterize the SYN induction timeline as it pertains to SCX expression followed up by maturation from tenogenic progenitor to tenocytes.

Comment 2: The authors make claims to changes in protein expression but fail to quantify either fluorescence intensity or percent cell expression from their immunofluorescence analyses to substantiate these claims. These claims are not fully supported by the data as presented as it is unclear whether there is increased expression of tendon markers at the protein level or more cells surviving the protocol. Additionally, in images where 3 channels are merged, it would be helpful to show individual channels where genes are shown in similar spectra (ie. Fig 2I SCX/MKX). Furthermore, the current layout and labelling scheme of Figure 4 makes it very difficult to compare conditions between SYN and SYNWNTi protocols.

Thanks for your comment. Protein expression at each stage was verified with immunofluorescence cytochemistry whereby cells were cultured onto poly-lysine coated coverslips, which were then fixed, stained and imaged (Fig. 2). However, prior to WNT inhibitor application, we noticed gradual cell attrition in the cultures at the end of differentiation (Fig. 1B, 2I). The images show qualitative differences with and without the WNT inhibitor. This could be attributed to the heterogeneity of the cell population at SCL stage, which was confirmed by scRNA-seq (Fig. 3A). As it has been discussed previously (Reviewer 2 comments 5 & 9), in the current paper we didn’t provide any IF quantitative analysis because of the qualitative nature of the staining technique. In future work another high-resolution imaging modality will be considered like single cell proteomics and flow cytometry or mass cytometry in order to perform a more unbiased quantitative single cell analysis across different stages and samples. Furthermore, we have added single channel images in the supplemental information.

Comment 3: Individual data points should also be presented for all qPCR experiments (ie. Fig 4A). Biological replicate information is missing from several experiments, particularly the immunofluorescence data, and it is unclear whether the qPCR data was generated from technical or biological replicates.

Thanks for your comment. We have added additional information regarding replicates in each figure legend. We have also changed Fig. 4A.

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Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The authors want to elucidate which are the mechanisms that regulate the immune response in physiological conditions in cortical development. To achieve this goal, authors used a wide range of mutant mice to analyse the consequences of immune activation in the formation of cortical ectopia in mice.

Strengths:

The authors demonstrated that Abeta monomers are anti-inflammatory and inhibit microglial activation. This is a novel result that demonstrates the physiological role of APP in cortical development.

Weaknesses:

-On the other hand, cortical ectopia has been already described in mouse models in which the amyloid signalling has been disrupted (Herms et al., 2004; Guenette et al., 2006), making the current study less novel.

We agree these previous studies have implicated amyloid precursor protein in cortical ectopia. However, since these studies use whole-body knockouts, they have not implicated the functional roles of specific cell types.  Nor have they identified the specific mechanisms underlying the formation of this unique class of cortical ectopia. In contrast, our studies show that the disruption of a novel Abeta-regulated signaling pathway in microglia is the primary cause of ectopia formation in this class of ectopia mutants. This is the first time that microglia have been specifically implicated in the development of cortical ectopia. We further show that elevated MMP activity and resulting cortical basement membrane degradation is the underlying mechanism leading to ectopia formation.  This is also the first time that MMP activity and basement membrane degradation (instead of maintenance) have been implicated in cortical ectopia development. As such, our results have provided novel insights into the diverse mechanisms underlying cortical ectopia formation in developmental brain disorders.

One of the molecules analysed is Ric8a, a GTPase activator involved in neuronal development. Authors used the conditional mutant mice Emx1-Ric8a to delete Ric8a from early progenitors and glutamatergic neurons in the pallium. Emx1-Ric8a mutant mice present cortical ectopias and authors attributed this malformation to the increase in inflammatory response due to Ric8a deletion in microglia. Several discordances do not fit this interpretation:

- The role of Ric8a in cortical development and function has been already described in several papers, but none of them has been cited in the current manuscript (Kask et al., 2015, 2018; Ruisu et al., 2013; Tonissoo et al., 2006).

We have included reference to the published works on ric8a in cortical development in revision.

- Ectopia formation in the cortex has been already described in Nestin-Ric8a cKO mice (Kask et al., 2015). In the current manuscript, authors analyzed the same mutant mice (Nestin-Ric8a), but they did not detect any ectopia. Authors should discuss this discordance.

The expression pattern of nestin-cre is known to vary dependent on factors including transgene insertion site, genetic background, and sex. Early studies show, for example, that the nestin gene promoter drives cre expression in many non-neural tissues in another transgenic line in the FVB/N genetic background (Dubois et al Genesis. 2006 Aug;44(8):355-60. doi: 10.1002/dvg.20226).  The specific nestin-cre line used in Kask et al 2015 has also been shown to be active in brain microglia and lead to increased microglia pro-inflammatory activity upon breeding to a conditional allele of a cholesterol transporter gene (Karasinska et al., Neurobiol Dis. 2013 Jun:54:445-55; Karasinska et al.,  J Neurosci. 2009 Mar 18; 29(11): 3579–3589; Takampri et al., Brain Res. 2009 May 13:1270:10-8). These factors may in part underlie the apparent discrepancy.  We have now incorporated this discussion into the revision.

- Authors claim that microglia express Emx1, and therefore, Ric8a is deleted in microglia cells. However, the arguments for this assumption are very weak and the evidence suggests that this is not the case. This is an important point considering that authors want to emphasise the role of Ric8a in microglia activation, and therefore, additional experiments should demonstrate that Ric8a is deleted in microglia in Emx1-Ric8a mutant mice.

We have observed altered mRNA expression of several genes in purified microglia cultured from the emx1-cre mutants (Supplemental Fig. 8), which indicates that ric8a is deleted from microglia and suggests a role of microglial ric8a deficiency in ectopia formation.  This interpretation is further strengthened by the observation that deletion of ric8a from microglia using a microglia-specific cx3cr1-cre results in similar ectopia (Fig. 2). We also have other data supporting this interpretation, including data showing induction of the expression of a cre reporter in brain microglia by emx1-cre and loss of ric8a gene expression in microglia cells isolated from emx1-cre mutants. These data have now been incorporated into the text and in revised Supplemental Fig. 8 (new panels c-c” & d).

Reviewer #2 (Public Review):

Kwon et al. used several conditional KO mice for the deletion of ric8a or app in different cell types. Some of them exhibited pial basement membrane breaches leading to neuronal ectopia in the neocortex.

They first investigated ric8a, a Guanine Nucleotide Exchange Factor for Heterotrimeric G Proteins. They observed the above-mentioned phenotype when ric8a is deleted from microglia and neural cells (ric8a-emx1-cre or dual deletion with cre combination cx3cr1 (in microglia) and nestin (in neural cells)) but not in microglia alone or neural cells alone (whether it is in CR cells (ric8a-Wnt3a-cre), post-mitotic neurons (nex-cre or dlx5/6-cre), or in progenitors and their progeny (nestin-cre or foxg1-cre). They also show that ric8a KO mutant microglia cells stimulated in vitro by LPS exhibit an increased TNFa, IL6 and IL1b secretion compared to controls (Fig 2). They therefore injected LPS in vivo and observed the neuronal ectopia phenotype in the ric8a-cx3cr1-cre (microglial deletion) cortices at P0 (Fig 2). They suggest that ric8a KO in neuronal cells mimics immune stimulation (but we have no clue how ric8a KO in neural cells would induce immune stimulation).

We agree we do not currently know the precise mechanisms by which mutant microglia are activated in the mutant brain.  However, this does not affect the conclusion that deficiency in the Abeta monomer-regulated APP/Ric8a pathway in microglia is the primary cause of cortical ectopia in these mutants, since we have shown that genetic disruption of this pathway in microglia alone by targeting different pathway components, using cell type specific cre, in several different approaches, all results in similar cortical ectopia phenotypes.  Regarding the source of the immunogens, there are several possibilities which we plan to investigate in future studies. For example, the clearance of apoptotic cells and associated cellular debris is an important physiological process and deficits in this process have been linked to inflammatory diseases throughout life (Doran et al., Nat Rev Immunol. 2020 Apr;20(4):254-267; Boada-Romero et al., Nat Rev Mol Cell Biol. 2020 Jul;21(7):398-414.).  In the embryonic cortex, studies have shown that large numbers of cell death take place starting as early as E12 (Blaschke et al., Development. 1996 Apr;122(4):1165-74; Blaschke et al., J Comp Neurol. 1998 Jun 22;396(1):39-50).  Studies have also shown that radial glia and neuronal progenitors play critical roles in the clearance of apoptotic cells and associated cellular debris in the brain (Lu et al., Nat Cell Biol. 2011 Jul 31;13(9):1076-83; Ginisty et al., Stem Cells. 2015 Feb;33(2):515-25; Amaya et al., J Comp Neurol. 2015 Feb 1;523(2):183-96). Moreover, Ric8a-dependent heterotrimeric G proteins have been found to specifically promote the phagocytic activity of both professional and non-professional phagocytic cells (Billings et al., Sci Signal. 2016 Feb 2;9(413):ra14; Preissler et al., Glia. 2015 Feb;63(2):206-15; Pan et al. Dev Cell. 2016 Feb 22;36(4):428-39; Flak et al. J Clin Invest. 2020 Jan 2;130(1):359-373; Zhang et al., Nat Commun. 2023 Sep 14;14(1):5706).  Thus, it is probable that the failure to promptly clear up apoptotic cells and debris by mutant radial glia may play a role in triggering mutant microglial activation in ric8a-emx1-cre mutants. We have now included these possibilities in the text of the revised manuscript. However, the precise mechanisms remain to be determined in future studies, which, however, do not affect the conclusion of the current study.

The authors then turned their attention on APP. They observed neuronal ectopia into the marginal zone when APP is deleted in microglia (app-cxcr3-cre) + intraperitoneal LPS injection (they did not show it, but we have to assume there would not be a phenotype without the injection of LPS) (Fig 3). (The phenotype is similar but not identical to ric8a-cx3cr1-cre + LPS. They suggest that the reason is because they had to inject 3 times less LPS due to enhanced immune sensitivity in this genetic background but it is only a hypothesis). After in vitro stimulation by LPS, app mutant microglia show a reduced secretion of TNFa and IL6 but not IL1b (this is the opposite to ric8a-cx3cr1-cre microglia cells) while peritoneal macrophages in culture show increased secretion of TNFa, IL1, IL6 and IL23 (fig 3 and Suppl. Fig 9).

We have data showing that that app-cxcr3-cre mutants without LPS injection do not show ectopia, which has now been included in the revised supplemental Fig. 9 (new panels c-d).  The reason we employ LPS injection is, in the first place, that we do not see a phenotype without the injection. We agree, and have also stated in the text, that the phenotype of the app mutants is not as severe as that of the ric8a mutant.  Besides the low LPS dosage used, we also suggest that other app family members may compensate since the ectopia in the app family gene mutants reported previously were only observed in app/aplp1/2 triple knockouts, not even in any of the double knockouts (Herms et al., 2004). We have further clarified this point in the text. These possibilities are also not mutually exclusive. Nonetheless, the results clearly show that microglia specific app mutation causes cortical ectopia upon embryonic immune stimulation. They have thus implicated a specifical role of microglial APP in cortical ectopia formation.

The different response of ric8a and app mutant microglia to LPS results from in vitro culturing of microglia. We have shown that, when acutely isolated macrophages are used, these mutants show changes in the same direction (both increased cytokine secretion) (Fig. 4).  This demonstrates without culturing app mutant microglial lineage cells indeed behave in the same way as ric8a mutant cells.

The microglia used for analysis in in vitro assays in this study have all been cultured for two weeks before assay. They have thus been under chronic stimulation exposed to dead cells and debris in the culture dish through this period.  Previous studies have shown that dependent on the degree of perturbation to the inflammation-regulating pathways, such exposures can differentially affect microglial cytokine expression, sometimes in an opposite direction from expected.  For example, under chronic immune stimulation, while the trem2+/- microglia, which are heterozygous mutant for the anti-inflammatory Trem2, show elevated pro-inflammatory cytokine expression (as is expected), trem2-/- (null) microglia under the same conditions instead not only do not show increases but for some pro-inflammatory cytokines, actually show decreases in expression (Sayed et al.,, Proc Natl Acad Sci U S A. 2018 Oct 2;115(40):10172-10177).  In several systems, Ric8a-dependent heterotrimeric G proteins have been shown to act downstream of APP and mediate one of the branches of the signaling activated by APP (Milosch et al., Cell Death Dis. 2014 Aug 28;5(8):e1391; Fogel et al,, Cell Rep. 2014 Jun 12;7(5):1560-1576; Ramaker et al., J Neurosci. 2013 Jun 12;33(24):10165-81; Nishimoto et al., Nature. 1993 Mar 4;362(6415):75-9).  Indeed, APP cytoplasmic domain is known to also bind to and signalig through several other proteins including FE65, Mena, and TIP60 (Cao & Sudhof, Science 2001. 293:115-120).  It is likely that in microglia Ric8a-dependent heterotrimeric G proteins may also mediate only a subset of the signaling downstream of APP.  As such, app knockout in microglia may have more severe effects on microglial anti-inflammatory regulation than ric8a knockout.  As a result, upon chronic immune activation, app knockout may lead to a microglial phenotype similar to the trem2 null mutation phenotype as discussed above, while ric8a knockout leads to a phenotype similar to trem2+/- phenotype). This may explain the subdued TNF and IL6 secretion by cultured app (but not ric8a) mutant microglia.

Amyloid beta (Ab) being one of the molecules binding to APP, the authors showed that Ab40 monomers (they did not test Ab40 oligomers) partially inhibit cytokines (TNFa, IL6, IL1b, MCP-1, IL23a, IL10) secretion in vitro by microglia stimulated by LPS but does not affect secretion by microglia from app-cx3cr1-cre (tested for TNFa, IL6, IL1b, IL23a, IL10) (Fig 4, Suppl fig 10) (but still does it in aplp2-cx3cr1-cre) and does not affect secretion by ric8a-cx3cr1-cre microglia (tested for TNFa and IL6 but still suppress IL1b) (Therefore here is another difference between app and ric8a KO microglia).

We have tested the effects of Abeta40 oligomers, which induce instead of suppressing microglial cytokine secretion, and have included the data (new panel j in supplemental Fig. 10).  As mentioned above, in several systems, Ric8a-dependent heterotrimeric G proteins have been shown to act downstream of APP and mediate one of the branches of the signaling activated by APP (Milosch et al., Cell Death Dis. 2014 Aug 28;5(8):e1391; Fogel et al,, Cell Rep. 2014 Jun 12;7(5):1560-1576; Ramaker et al., J Neurosci. 2013 Jun 12;33(24):10165-81; Nishimoto et al., Nature. 1993 Mar 4;362(6415):75-9).  We assume that this is likely also true in microglia and that Ric8a-dependent heterotrimeric G proteins may mediate a subset and only a subset of the signaling downstream of APP.  This may explain the difference in the effects of app and ric8a knockout mutation in abolishing the anti-inflammatory effects of Abeta monomers on IL-1b vs TNF/IL-6.  This difference also suggests that TNF/IL-6 and IL-1b secretion must be regulated by different mechanisms in microglia. Indeed, it is well established in immunology that the secretion of IL1b, but not of TNF or IL6, is regulated by inflammasome-dependent mechanisms (see, for example, Proz & Dixit. Nat Rev Immunol. 2016 Jul;16(7):407-20. doi: 10.1038/nri.2016.58).

The authors injected inhibitors of Akt or Stat3 in the ric8a-emx1-cre cortex and found it suppressed neuronal ectopia (Fig 5, Suppl fig 11). It is not clear whether it suppresses immune stimulation from neuronal cells or immune reaction from microglia cells.

We agree at present the pharmacological approaches we have taken are not able to distinguish these possibilities.  However, no matter which is the case, our results still implicate a role of excessive microglial activation in the formation of cortical ectopia and support the conclusion of the study.  Thus, while worthwhile of further investigation, this question does not impact the conclusion of the current study. Furthermore, as mentioned, we plan to determine the mechanisms of how ric8a mutation in neural cells induces immune activation in future studies. These results will likely enable us to more specifically address this question.

Finally, the authors examined the activities of MMP2 and MMP9 in the developing cortex using gelatin gel zymography. The activity and protein levels of MMP9 but not MMP2 in the ric8a-emx1-cre cortex were claimed significantly increased (Fig 5, Suppl fig 12). Unfortunately, they did not show it in the app-cx3cr1-cre +LPS mouse. They make a connection between ric8a deletion and MMP9 but unfortunately do not make the connection between app deletion and MMP9, which is at the center of the pathway claimed to be important here). Then they injected BB94, a broad-spectrum inhibitor of MMPs or an inhibitor specific for MMP9 and 13. They both significantly suppress the number and the size of the ectopia in ric8a mutants (Fig5).

For all the gelatin gel zymography analysis, we quantify protein concentrations in the cortical lysates using the Bio-Rad Bradford assay kit and load the same amounts of proteins per lane. The results across lanes are all directly comparable. From the quantification, our results clearly show that MMP9 activity levels are increased in the mutants (we have now included whole gel images and quantification in a new supplemental Figure 13).  The similar levels of MMP2 in all lanes also provide an internal control further supporting the observation of a specific change in MMP9.  For this analysis, we focus on the ric8a-emx1-cre mutants since the app-cx3cr1-cre +LPS animals show ectopia only in only subsets of mutants and in most cases only in one of the hemispheres.  Experiments examining potential changes in MMP9 are therefore unlikely to yield meaningful results.  On the other hand, we have clearly shown that the administration of different classes of MMP inhibitors significantly eliminate ectopia in ric8a-emx1-cre mutants. This has strongly implicated a functional contribution of MMPs.

After reading the manuscript, I still do not know how ric8a in neural cells is involved in the immune inhibition. Is it through the control of Ab monomers? In addition, the authors did not show in vivo data supporting that Ab monomers are the key players here. As the authors said, this is not the only APP interactor. Finally, I still do not know how ric8a is linked to APP in microglia in the model.

As detailed above, there are several possibilities including potential deficits in the clearance of apoptotic cells and associated debris that may trigger microglial activation in ri8ca-emx1-cre mutants. We will investigate these possibilities in future studies.  We have now incorporated these possibilities in the revised text.  As for the role of Abeta monomers, we have indicated that we currently do not have evidence that in the developing cortex Abeta monomers play a role in inhibiting microglia.  We have also indicated in the manuscript that our conclusion is that a microglial signaling pathway that is activated by Abeta monomers in vitro regulates normal brain development in vivo, not that Abeta monomers themselves regulate brain development.  Regarding the link between Ric8a and APP, the reviewer has missed several major lines of supporting evidence. For example, we have shown that Abeta monomers activate a pathway in microglia that inhibits the secretion of several proinflammatory cytokines including TNF, IL-6, IL-10, and IL-23 (Figure 4 and Supplemental Figures 8-10).  This inhibition is abolished when either app or ric8a gene is deleted from microglia.  This clearly indicates that app and ric8a act in the same genetic pathway (the pathway activated by Abeta monomers) in microglia. We also show that this Abeta monomer-activated pathway also inhibits the transcription of several cytokines in microglia.  This inhibition is also abolished when either app or ric8a gene is deleted from microglia.  This reinforces the conclusion that app and ric8a act in the same pathway in microglia.  Furthermore, cell type specific deletion of app or ric8a from microglia in vivo also results in similar phenotypes of cortical ectopia. Together, these results strongly support the conclusion that app and ric8a act in the same pathway that is activated by Abeta monomers in vitro in microglia. This conclusion is also consistent with published findings that Ric8a dependent heterotrimeric G proteins bind to APP and mediate subsets of APP signaling across different species (Milosch et al., Cell Death Dis. 2014 Aug 28;5(8):e1391; Fogel et al,, Cell Rep. 2014 Jun 12;7(5):1560-1576; Ramaker et al., J Neurosci. 2013 Jun 12;33(24):10165-81; Nishimoto et al., Nature. 1993 Mar 4;362(6415):75-9).         

While several of the findings presented in this manuscript are of potential interest, there are a number of shortcomings. Here are some suggestions that could improve the manuscript and help substantiate the conclusions:

(1) As the title suggests it, the focus is on Ab and APP functions in microglia. However, the analysis is more focused on ric8a. The connection between ric8a and APP in this study is not investigated, besides the fact that their deletion induces somewhat similar but not identical phenotypes. Showing a similar phenotype is not enough to conclude that they are working on the same pathway. The authors should find a way to make that connection between ric8a and app in the cells investigated here.

As discussed above, the reviewer misses several major lines of evidence showing that APP and Ric8a acts in the same pathway in microglia.  Besides the similarity of the ectopia phenotypes, for example, we have shown that Abeta monomers activates a pathway in microglia that inhibits the secretion of several proinflammatory cytokines including TNF, IL-6, IL-10, and IL-23 (Figure 4 and Supplemental Figures 8-11).  These inhibitory effects are abolished when either app or ric8a gene is deleted from microglia.  This clearly indicates that app and ric8a act in the same genetic pathway, a pathway that is activated by Abeta monomers in vitro, in microglia. We also show that this Abeta monomer-activated pathway inhibits the transcription of several cytokine genes in microglia.  These effects are again abolished when either app or ric8_a gene is deleted from microglia.  This further reinforces the conclusion that _app and ric8a act in the same pathway in microglia.  Not only so we also show that the same results are true in macrophages.  Thus, these results strongly support the conclusion that app and ric8a act in the same genetic pathway in microglia. This conclusion is also consistent with published findings that Ric8a dependent heterotrimeric G proteins biochemically bind to APP and mediate subsets of APP signaling across different species (Milosch et al., Cell Death Dis. 2014 Aug 28;5(8):e1391; Fogel et al,, Cell Rep. 2014 Jun 12;7(5):1560-1576; Ramaker et al., J Neurosci. 2013 Jun 12;33(24):10165-81; Nishimoto et al., Nature. 1993 Mar 4;362(6415):75-9).  

(2) This would help to show the appearance of breaches in the pial basement membrane leading to neuronal ectopia; to investigate laminin debris, cell identity, Wnt pathway for app-cxcr3-cre + LPS injection as you did for ric8a-emx1-cre.

We have now provided further data on pial basement membrane breaches in the app-cxcr3-cre + LPS animals (new panels e-f” in supplemental Fig 9).  We have not observed any changes in cell identity or Wnt pathway activity in ric8a-emx1-cre mutants.  It is thus of limited value to examine potential changes in these areas in the app-cxcr3-cre + LPS animals.   

(3) As a control, this would help to show that app-cxcr3-cre without the LPS injection does not display the phenotype.

We have the data on app-cx3cr1-cre mutants without LPS injection, which show no ectopia.  We have now included the data in the revised supplemental Fig. 9 (new panels c-d).

(4) This would help to show the activity and protein levels of MMP9 and MMP2 and perform the rescue experiments with the inhibitors in the app-cx3cr1-cre cortex +LPS.

As discussed above, we focus analysis on the ric8a-emx1-cre mutants since app-cx3cr1-cre +LPS animals show ectopia in only a subset of mutants and in most cases only in one of the hemispheres.  Determining potential changes in MMP9 levels and effects of MMP inhibitors are therefore not likely to yield meaningful data.  On the other hand, we have shown that MMP9 levels are increased and administration of different classes of MMP inhibitors eliminate cortical ectopia in ric8a-emx1-cre mutants.  We have also shown a similar break in the basement membrane in app-cx3cr1-cre +LPS animals (new panels e-f” in supplemental Fig 9). These results together strongly implicates a role played by MMPs.

(5) Is MMP9 secreted by microglia cells or neural cells?

Our in situ hybridization data show MMP9 is most highly expressed in a sparse microglia-like cell population in the embryonic cortex, suggesting that microglia may be a major source of MMP9. We have incorporated these data in a new supplemental Fig. 12 (panel a). The precise identity of these cells, however, requires further validation.

(6) The in vitro evidence indicates that one of the multiple APP interactors, ie Ab40 monomers, is less effective in suppressing the expression of some cytokines by microglia cells mutants for ric8a (TNFa and IL6 but still suppress IL1b) or APP (TNFa, IL6, IL1b, IL23a, IL10) when compared to WT. But there are other interactors for APP. In order to support the claim, it seems crucial to have in vivo data to show that Ab40 monomers are the molecules involved in preventing the breach in the pial basement membrane.

As addressed in detail above, we have indicated that our conclusion is that a microglial signaling pathway that is activated by Abeta monomers in vitro regulates normal brain development in vivo, not that Abeta monomers themselves regulate brain development in vivo.  We currently do not have evidence that the Abeta monomers play a role in inhibiting microglia during cortical development.  There are candidate ligands for the pathway in the developing cortex, the functional study of which, however, is a major undertaking beyond the scope of the current study.

(7) In order to claim that this is specific to Ab40 monomers and not oligomers, it is necessary to show that the Ab40 oligomers do not have the same effect in vitro and in vivo. Also, an assay should be done to show that your Ab preparations are pure monomers or oligomers.

We have tested the effects of Abeta40 oligomers, which induce instead of suppressing microglial cytokine secretion, and have included these data in revision in a new panel j in supplemental Fig. 10. The protocols we use in preparing the monomers and oligomers are standard protocols employed in the field of Alzheimer’s disease research. They have been repeatedly optimized and validated over the past decades.  

(8) Most of the cytokine secretion assays used microglia cells in culture. Two results draw my attention. Ric8a deletion increases TNFa and IL6 secretion after LPS stimulation in vitro on microglia cells while app deletion decreases their secretion. Then later, papers show that the decrease in IL1b induced by Ab on microglia cells is prevented by APP deletion but not ric8a deletion. Those two pieces of data suggest that ric8a and APP might not be in the same pathway. In addition, the phenotype from app-cxcr3-cre + LPS injection and ric8a-cxcr3-cre + LPS injection are not exactly the same. It could be due to the level of LPS as the author suggests or it might not be. More experiments are needed to prove they are in the same pathway.

As discussed above, the reviewer misses several major lines of evidence, which strongly support the conclusion that APP and Ric8a act in the same pathway activated by Abeta monomers in microglia (see detailed discussion in point 1 above).  The differential response of TNFa/IL-6 of app and ric8a mutant microglia likely results from chronic immune stimulation during in vitro culturing, which is known to alter microglial cytokine response (see detailed discussion in point 9 below). We have demonstrated that this is indeed the case by showing that, without culturing, acutely isolated app and ric8a mutant macrophages both display elevated TNFa/IL-6 secretion (Figure 4). 

Regarding the different regulation of TNF/IL-6 vs IL-1b by APP and Ric8a, as discussed above, in several systems, Ric8a-dependent heterotrimeric G proteins (which are degraded in ric8a mutant cortices, see new supplemental Fig. 9) have been shown to act downstream of APP and mediate one of the branches of the signaling activated by APP (Milosch et al., Cell Death Dis. 2014 Aug 28;5(8):e1391; Fogel et al,, Cell Rep. 2014 Jun 12;7(5):1560-1576; Ramaker et al., J Neurosci. 2013 Jun 12;33(24):10165-81; Nishimoto et al., Nature. 1993 Mar 4;362(6415):75-9).  This is likely also the case in microglia and Ric8a-dependent heterotrimeric G proteins may mediate only a subset of the anti-inflammatory signaling activated by APP.  As such, app, mutation may abolish all the inhibitory effects of Abeta monomers (both those on TNF/IL-6 and those on IL-1b), but ric8a mutation may abolish only a subset only those on TNF/IL-6 but not those on IL-1b).  This also suggests that the secretion of TNF/IL-6 and IL-1b must be regulated by different mechanisms in microglia.  Indeed, it is well established in immunology that the secretion of IL1b, but not that of TNF or IL6, is regulated by inflammasome-dependent mechanisms (see, for example, Proz & Dixit. Nat Rev Immunol. 2016 Jul;16(7):407-20. doi: 10.1038/nri.2016.58).

(9) How do the authors reconcile the reduced TNFa and IL6 secretion upon stimulation of app mutant microglia with the model where app is attenuating immune response in vivo? Line 213 says that microglia exhibit attenuated immune response following chronic stimulation but I don't know if 3 hours of LPS in vitro is a chronic stimulation.

The reviewer has misunderstood.  The microglia used in this study have all been cultured in vitro for approximately two weeks before assay. They have thus been under chronic stimulation exposed to dead cells and debris in the culture dish.  Dependent on the degree of perturbation to the inflammation-regulating pathways, such exposures are known to change microglial cytokine expression, sometimes in an opposite direction than expected.  For example, under chronic immune stimulation, while the trem2+/- microglia, which are heterozygous mutant for the anti-inflammatory Trem2, show elevated pro-inflammatory cytokine expression, trem2-/- (null) microglia under the same conditions instead not only do not show increases but for some pro-inflammatory cytokines, actually show decreases in expression (Sayed et al.,, Proc Natl Acad Sci U S A. 2018 Oct 2;115(40):10172-10177).  As mentioned, in several systems, Ric8a-dependent heterotrimeric G proteins have also been shown to bind to APP and mediate one of the branches of the signaling activated by APP (Milosch et al., Cell Death Dis. 2014 Aug 28;5(8):e1391; Fogel et al,, Cell Rep. 2014 Jun 12;7(5):1560-1576; Ramaker et al., J Neurosci. 2013 Jun 12;33(24):10165-81; Nishimoto et al., Nature. 1993 Mar 4;362(6415):75-9). Thus, it is likely that in microglia, Ric8a-dependent heterotrimeric G proteins also mediate only a subset of the anti-inflammatory signaling activated by APP.  As such, app knockout in microglia may have more severe effects than ric8a knockout on microglial immune activation, resembling the relationship between trem2 null vs heterozygous mutation discussed above. As such, it is predicted that chronic immune stimulation such as in vitro culturing will result in attenuated pro-inflammatory cytokine expression in app mutant microglia but elevated cytokine expression in ric8a mutant microglia. This may explain why TNF and IL6 secretion by cultured app mutant microglia is subdued, but acutely isolated _a_pp mutant macrophages instead show increased cytokine secretion. The latter may be more representative of the response of app mutant microglia in the absence of chronic stimulation.

(10) Line 119: In their model, the authors suggest that there is a breach in pial basement membrane but that the phenotype is different from the retraction of the radial fibers due to reduced adhesion. So, could the author discuss to what substrate the radial fibers are attached to, in their model where the pial surface is destroyed?

Radial glial endfeet normally bind to the basement membrane via cell surface receptors including the integrin and the dystroglycan protein complexes. We observe free radial glial endfeet at the breach sites, apparently without attachment to any basement membrane.  However, we cannot exclude the possibility that there may be residual, broken-off basement membrane components bound to the endfeet that are not detected by the methodology employed. 

(11) The authors should show that the increased cytokine secretion observed in vitro is also happening in vivo in ric8a-emx1-cre compared to WT mice and compared to ric8a-nestin-cre mice. Or when app is deleted in microglia (app-cxcr3-cre) + LPS injection compared to WT mice +LPS.

Unfortunately, this is not technically feasible since it is not possible to extract the extracellular (secreted) fractions of cytokines from an embryonic brain without causing cell lysis and the release of the intracellular pool.  This, however, does not affect our conclusion that the Abeta monomer-regulated microglia pathway plays a key role in regulates normal brain development since its genetic disruption, by different approaches, clearly results in brain malformation.

(12) The authors injected inhibitors of Akt or Stat3 in the ric8a-emx1-cre cortex and found that it suppressed neuronal ectopia (Fig 5, Suppl fig 11). Does it suppress immune stimulation from neuronal cells or immune reaction from microglia cells?

As discussed above, we agree at present the pharmacological approaches we have taken are not able to distinguish these two possibilities.  However, whichever is true, it does not affect our conclusion.  Also, we plan to determine the mechanisms of how ric8a mutation in neural cells induce immune activation in future studies. These results will likely enable us to adopt specific approaches to address this question.

(13) Fig 5 and Supplementary fig 12: Please show a tubulin loading control in Fig 5i as you did in suppl fig 12 d (gel zymography). Please provide a gel zymography showing side by side Control, mutant and mutant +DM/S3I treatment. The same request for the MMP9 staining. Please provide statistics for control vs mutant for suppl fig 12c and d..

We have now included whole gel zymography images with four control and four mutant individual samples as well as quantification in a new supplemental Fig.13 (panels b-c). This clearly shows increases in MMP9, while the MMP2 levels appear similar between controls and mutants. For all of the experiments of gelatin gel zymography, we quantify protein concentrations in the cortical lysates using the Bio-Rad Bradford assay kit and load the same amounts of proteins per lane. The results across lanes are thus all comparable.  The MMP9 staining images for the controls and mutants have also all been taken with the same parameters on the microscope and can be directly compared.  The statistics have now been provided as suggested.

(14) Please provide the name and the source of the MMP9/13 inhibitor used in this study.

This inhibitor is MMP-9/MMP-13 inhibitor I (CAS 204140-01-2), from Santa Cruz Biotechnology. This information has been included in revision.

(15) The results show that deletion of ric8a in microglia and neural cells induced pia membrane breaches but no phenotype is apparent in ric8a deletion in microglia or neural cells alone. Then, the results showed that intraperitoneal injection of LPS induced the phenotype in ric8a-cxcr3-cre mutants. It would be beneficial as a control supporting the model to show that the insult induced by LPS injection does not induce the phenotype in the ric8a-foxg1-cre mice.

We agree it may potentially be useful to show that LPS injection does not induce ectopia in ric8a-foxg1-cre mice.  Unfortunately, since the ric8a-foxg1-cre mutation shows no phenotype, we are no longer in possession of this line.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

- The information in the abstract and the introduction is only related to app. So, it is very abrupt how authors start the manuscript studying the role of Ric8a, with no information at all about this protein and why the authors want to investigate this role in microglial activation. Later in the manuscript, the authors tried to link Ric8a with app to study the role of app in the inflammatory response and ectopia formation. This link is quite weak as well.

In the last paragraph of the Introduction, we explain the use of the ric8a mutant and how it leads to discovery of the Abeta monomer-regulated pathway. We have now improved the writing in revision to make these points especially the link between APP and Ric8a-regulated G proteins more clear.  In the Results section, we have also improved the writing on the potential link of Ric8a to APP by highlighting, among others, the fact that ric8a and app pathway mutants are among a unique group of a few mouse mutants (ric8a, app/aplp1/2, and apbb1/2) that show cortical ectopia exclusively in the lateral cortex, while all other cortical ectopia mutants also show severe ectopia are at the cortical midline.  This suggests that similar mechanisms may underlie the ectopia formation in this small group of mutants.

-In order to validate the mouse model, double immunofluorescence or immunofluorescence+in situ hybridization should be performed to show that microglia express ric8a and that is eliminated in the Emx1-Ric8a mutant mice.

As mentioned above, we have additional lines of evidence showing that ric8a is deleted from microglia in emx1-cre mutants. This includes data showing induction of the expression of a cre reporter in brain microglia by emx1-cre and loss of ric8a mRNA expression in microglia cells isolated from emx1-cre mutants.  These data have now been included in revised supplemental Fig. 8.

-In Supplemental Fig. 6, the authors claimed that cell proliferation is normal in Ric8a mutant mice without doing any quantification. They also quantified the angle of mitotic division of progenitors in the ventricular zone, but there are no images for the spindle orientation quantification, and no description of how they did it. In addition, this data is contrary to what has already been published in conditional Ric8a mutant mice (Kask et al., 2015). The Vimentin staining should be improved.

We have provided quantification of cell proliferation (phospho-histone 3 staining at the ventricular surface) in revised supplemental Fig. 6g, which shows no significant differences in the number of positive cells. We have also provided details on the definition of the angle of cleavage plane orientation in revised supplemental Fig. 6h and in the Methods section.  We are not sure why the results are different from the other study. We were indeed anticipating deficits in mitotic spindle orientation and spent major efforts in the analysis of this potential deficit.  However, based on the data, we could not draw the conclusion.     

-Analysis of the MMP9 expression should be done by western blot and not by immunofluorescence. In fact, the MMP9 expression shown in Figure 5g,h, does not correspond with RNA expression shown in gene expression atlas like genepaint or the allen atlas, doubting the specificity of the antibody. The expression of Mmp9 is quite low or absent in the cortex at E13.5-E14.5, making this protein very unlikely to be responsible for laminin degradation during development.

We have performed gelatin gel zymography on MMP2/9, which shows increased MMP9 activity levels in the mutant cortex. This is similar to Western blot analysis (all lanes are loaded with the same amounts of cortical lysates).  We have now included whole gel zymography images with four control and four mutant individual samples as well as quantification in a new supplemental Fig.13 (panels b-c).  The immunofluorescence staining of MMP9, a different type of analysis, was designed as a complementary approach, the results of which also support the interpretation of increases in MMP9 protein.  Regarding MMP9 RNA expression, please also note that MMP9 is secreted, and the protein expression pattern is expected to be different from that of RNA. We have performed wholemount in situ using dissected E13.5 mouse forebrains.  Our data (in new supplemental Fig.13a) show that MMP9 mRNA is strongly expressed in a sparse population of cells many of which appear to align along blood vessels. We suspect these are microglial lineage cells populating the embryonic cortex at this stage (see, for example, Squarzoni et al., Cell Rep. 2014 Sep 11;8(5):1271-9. doi: 10.1016/j.celrep.2014.07.042.).  Our control in situ using a Tnc5 probe also shows that the MMP9 signal is not a result of nonspecific probe binding.  Since the MMP9 expressing cells are very sparse even in the wholemount specimens while most database RNA in situ expression data are obtained using thin sections, we suspect this may be why the signal may have been missed in the databases.  As for functional contributions, we agree that we cannot rule roles played by other MMPs.  However, based on the ectopia suppression data, our results clearly indicate a critical contribution by MMP9/13.

For MMP9 activity, authors should show the whole membrane with a minimum of three control and three mutant individual samples and with the quantification.<br /> - The graphs should be improved, including individual values and titles of the Y axes.

We have included whole membrane zymography images with four control and four mutant individual samples as well as quantification in a new supplemental Fig.13b-c.  The graphs have also been improved as suggested.

Bilan des Thèmes et Idées Clés sur le Financement des Établissements Privés sous Contrat en France

Ce document synthétise les informations et analyses issues du rapport parlementaire "l16b2423_rapport-information.pdf" sur le financement des établissements d'enseignement privés sous contrat en France.

Thèmes Principaux:

1. Cadre juridique et historique:

Le rapport retrace l'évolution du cadre légal depuis la loi Falloux (1850) jusqu'à la loi Debré (1959) qui constitue le fondement du système actuel.

La loi Debré a instauré le contrat d'association permettant aux établissements privés de s'associer au service public d'éducation en contrepartie du financement des enseignants et des frais de fonctionnement par l'État.

Le rapport souligne l'accroissement progressif des financements publics alloués aux établissements privés suite à des lois ultérieures (loi Guermeur, loi Chevènement).

1. Financement des établissements privés sous contrat:

Le rapport détaille les sources de financement des établissements privés: l'État, les collectivités territoriales, les ménages et les entreprises.

L'État finance les salaires des enseignants et une partie des frais de fonctionnement via le programme budgétaire 139.

Les collectivités territoriales versent des forfaits d'externat pour les élèves de leur territoire scolarisés dans le privé et peuvent accorder des subventions d'investissement.

Le rapport met en lumière l'augmentation des financements publics alloués aux établissements privés ces dernières années.

1. Contrôles et dérives:

Le rapport met en évidence la faiblesse des contrôles exercés sur les établissements privés, notamment en ce qui concerne l'utilisation des fonds publics.

La Cour des comptes a pointé du doigt des "dérives" dans l'utilisation des fonds publics, notamment la fongibilité des produits et l'opacité de la gestion des forfaits d'externat.

Des exemples de détournement de fonds publics à d'autres fins que celles prévues sont cités, notamment l'utilisation des crédits du Pacte pour financer des heures supplémentaires non liées à l'enseignement.

1. Mixité sociale et scolaire:

Le rapport s'interroge sur l'impact du financement public des établissements privés sur la mixité sociale et scolaire.

Il constate un recul de la mixité sociale et scolaire dans les établissements privés sous contrat, avec des écarts croissants d'Indice de Position Sociale (IPS) entre le public et le privé.

Le rapport analyse les causes de cette ségrégation: stratégies d'évitement de l'enseignement public par certaines familles, concurrence entre établissements, absence de mécanismes incitatifs à la mixité dans le financement des établissements privés.

1. Propositions:

Le rapport formule des propositions pour renforcer la transparence et le contrôle de l'utilisation des fonds publics par les établissements privés.

Il recommande notamment de distinguer dans les budgets des collectivités les dépenses obligatoires des dépenses facultatives, de normer le modèle d'allocation des moyens de l'État, d'imposer aux établissements privés une comptabilité analytique plus détaillée.

Le rapport propose également des mesures pour favoriser la mixité sociale et scolaire: introduction d'un critère d'IPS dans l'allocation des moyens de l'État et dans le calcul du forfait d'externat, modulation des subventions facultatives en fonction de la mixité sociale.

Il suggère de renforcer les contrôles sur les pratiques d'éviction des élèves en difficulté et d'instaurer un système de pénalités financières pour les établissements qui s'y livrent. Idées et Faits Importants:

Caractère propre: Le rapport met en avant l'ambiguïté de la notion de "caractère propre" qui permet aux établissements privés de conserver leurs spécificités tout en bénéficiant de financements publics. "Il revient à chaque établissement de définir ce qui fait son « caractère propre », sans en délimiter le périmètre a priori."

Fongibilité des produits: La pratique de verser tous les financements publics dans une caisse commune sans traçabilité de l'affectation des fonds est critiquée.

"La gestion conduit, en pratique, à verser ces produits à une unique caisse commune et la comptabilité que tiennent les établissements ne permet pas systématiquement de garantir la traçabilité de l’affectation des bons produits aux bonnes dépenses."

Forfait d'externat: Le rapport souligne que les collectivités territoriales n'ont aucun contrôle sur l'utilisation du forfait d'externat versé aux établissements privés. "Les collectivités territoriales n’ont ainsi aucunement la capacité d’identifier la manière dont le forfait scolaire versé pour chaque enfant est ventilé, ou ce qu’il permet réellement de financer."

Manque de transparence et de contrôle: Le rapport déplore le manque de transparence sur les financements des établissements privés et l'insuffisance des contrôles sur l'utilisation des fonds publics. "Proposition n° 6 : Élaborer un « jaune budgétaire » annuel retraçant le financement alloué aux établissements d’enseignement privés, y compris hors programme 139."

Mixité sociale et scolaire: Le rapport s'alarme de la ségrégation sociale et scolaire croissante et appelle à des mesures pour la combattre. "Pour M. Vannier, il est aujourd’hui indispensable d’intégrer un mécanisme de malus diminuant la dotation de l’État en moyens d’enseignement des établissements privés sous contrat dont l’IPS est supérieur à la moyenne pondérée des établissements publics de même rang, situés dans le secteur de carte scolaire où ils sont implantés."

Conclusion

Le rapport met en lumière les enjeux complexes liés au financement public des établissements d'enseignement privés sous contrat en France. Il appelle à un renforcement de la transparence et du contrôle de l'utilisation des fonds publics, tout en proposant des mesures pour favoriser la mixité sociale et scolaire. Le débat sur l'équilibre entre liberté de l'enseignement et égalité des chances reste ouvert.

"theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it"

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Contents

• • [

    Designing a tympanum through stereographic projection

    ](https://en.wikipedia.org/wiki/Astrolabe#Designing_a_tympanum_through_stereographic_projection) - [

    The tropics and the equator define the tympanum
    
    ](https://en.wikipedia.org/wiki/Astrolabe#The_tropics_and_the_equator_define_the_tympanum)
    

    • [

      The horizon and the measurement of altitude

      ](https://en.wikipedia.org/wiki/Astrolabe#The_horizon_and_the_measurement_of_altitude) - [

      The meridians and the measurement of azimuth

      ](https://en.wikipedia.org/wiki/Astrolabe#The_meridians_and_the_measurement_of_azimuth) - [

  See also

  ](https://en.wikipedia.org/wiki/Astrolabe#See_also) - [

  References

  ](https://en.wikipedia.org/wiki/Astrolabe#References) - [

  Bibliography

  ](https://en.wikipedia.org/wiki/Astrolabe#Bibliography) - [

  External links

  ](https://en.wikipedia.org/wiki/Astrolabe#External_links)

Astrolabe

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From Wikipedia, the free encyclopedia

For other pages with a similar name, see Astrolabe (disambiguation). Not to be confused with Cosmolabe.

North African, 9th century AD, Planispheric Astrolabe. Khalili Collection.

A modern astrolabe made in Tabriz, Iran in 2013.

An astrolabe (Greek: ἀστρολάβος astrolábos, 'star-taker'; Arabic: ٱلأَسْطُرلاب al-Asṭurlāb; Persian: ستاره‌یاب Setāreyāb) is an astronomical instrument dating to ancient times. It serves as a star chart and physical model of visible heavenly bodies. Its various functions also make it an elaborate inclinometer and an analog calculation device capable of working out several kinds of problems in astronomy. In its simplest form it is a metal disc with a pattern of wires, cutouts, and perforations that allows a user to calculate astronomical positions precisely. It is able to measure the altitude above the horizon of a celestial body, day or night; it can be used to identify stars or planets, to determine local latitude given local time (and vice versa), to survey, or to triangulate. It was used in classical antiquity, the Islamic Golden Age, the European Middle Ages and the Age of Discovery for all these purposes.

The astrolabe, which is a precursor to the sextant,^[1]^ is effective for determining latitude on land or calm seas. Although it is less reliable on the heaving deck of a ship in rough seas, the mariner's astrolabe was developed to solve that problem.

Applications

16th-century woodcut of measurement of a building's height with an astrolabe

The 10th-century astronomer ʿAbd al-Raḥmān al-Ṣūfī wrote a massive text of 386 chapters on the astrolabe, which reportedly described more than 1,000 applications for the astrolabe's various functions.^[2]^ These ranged from the astrological, the astronomical and the religious, to navigation, seasonal and daily time-keeping, and tide tables. At the time of their use, astrology was widely considered as much of a serious science as astronomy, and study of the two went hand-in-hand. The astronomical interest varied between folk astronomy (of the pre-Islamic tradition in Arabia) which was concerned with celestial and seasonal observations, and mathematical astronomy, which would inform intellectual practices and precise calculations based on astronomical observations. In regard to the astrolabe's religious function, the demands of Islamic prayer times were to be astronomically determined to ensure precise daily timings, and the qibla, the direction of Mecca towards which Muslims must pray, could also be determined by this device. In addition to this, the lunar calendar that was informed by the calculations of the astrolabe was of great significance to the religion of Islam, given that it determines the dates of important religious observances such as Ramadan.^[citation needed]^

Etymology

The Oxford English Dictionary gives the translation "star-taker" for the English word astrolabe and traces it through medieval Latin to the Greek word ἀστρολάβος : astrolábos,^[3]^^[4]^ from ἄστρον : astron "star" and λαμβάνειν : lambanein "to take".^[5]^

In the medieval Islamic world the Arabic word al-Asturlāb (i.e., astrolabe) was given various etymologies. In Arabic texts, the word is translated as ākhidhu al-Nujūm (Arabic: آخِذُ ٱلنُّجُومْ, lit. 'star-taker'), a direct translation of the Greek word.^[6]^

Al-Biruni quotes and criticises medieval scientist Hamza al-Isfahani who stated:^[6]^ "asturlab is an arabisation of this Persian phrase" (sitara yab, meaning "taker of the stars").^[7]^ In medieval Islamic sources, there is also a folk etymology of the word as "lines of lab", where "Lab" refers to a certain son of Idris (Enoch). This etymology is mentioned by a 10th-century scientist named al-Qummi but rejected by al-Khwarizmi.^[8]^

History

Ancient era

An astrolabe is essentially a plane (two-dimensional) version of an armillary sphere, which had already been invented in the Hellenistic period and probably been used by Hipparchus to produce his star catalogue. Theon of Alexandria (c. 335 -- c. 405) wrote a detailed treatise on the astrolabe.^[9]^ The invention of the plane astrolabe is sometimes wrongly attributed to Theon's daughter Hypatia (born c. 350--370; died AD 415),^[10]^^[11]^^[12]^^[13]^ but it's known to have been used much earlier.^[11]^^[12]^^[13]^ The misattribution comes from a misinterpretation of a statement in a letter written by Hypatia's pupil Synesius (c. 373 -- c. 414),^[11]^^[12]^^[13]^ which mentions that Hypatia had taught him how to construct a plane astrolabe, but does not say that she invented it.^[11]^^[12]^^[13]^ Lewis argues that Ptolemy used an astrolabe to make the astronomical observations recorded in the Tetrabiblos.^[9]^ However, Emilie Savage-Smith notes "there is no convincing evidence that Ptolemy or any of his predecessors knew about the planispheric astrolabe".^[14]^ In chapter 5,1 of the Almagest, Ptolemy describes the construction of an armillary sphere, and it is usually assumed that this was the instrument he used.

Astrolabes continued to be used in the Byzantine Empire. Christian philosopher John Philoponus wrote a treatise (c. 550) on the astrolabe in Greek, which is the earliest extant treatise on the instrument.^[a]^ Mesopotamian bishop Severus Sebokht also wrote a treatise on the astrolabe in the Syriac language during the mid-7th century.^[b]^ Sebokht refers to the astrolabe as being made of brass in the introduction of his treatise, indicating that metal astrolabes were known in the Christian East well before they were developed in the Islamic world or in the Latin West.^[15]^

Medieval era

Astrolabes were further developed in the medieval Islamic world, where Muslim astronomers introduced angular scales to the design,^[16]^ adding circles indicating azimuths on the horizon.^[17]^ It was widely used throughout the Muslim world, chiefly as an aid to navigation and as a way of finding the Qibla, the direction of Mecca. Eighth-century mathematician Muhammad al-Fazari is the first person credited with building the astrolabe in the Islamic world.^[18]^

The mathematical background was established by Muslim astronomer Albatenius in his treatise Kitab az-Zij (c. AD 920), which was translated into Latin by Plato Tiburtinus (De Motu Stellarum). The earliest surviving astrolabe is dated AH 315 (AD 927--928). In the Islamic world, astrolabes were used to find the times of sunrise and the rising of fixed stars, to help schedule morning prayers (salat). In the 10th century, al-Sufi first described over 1,000 different uses of an astrolabe, in areas as diverse as astronomy, astrology, navigation, surveying, timekeeping, prayer, Salat, Qibla, etc.^[19]^^[20]^

An Arab astrolabe from 1208

The spherical astrolabe was a variation of both the astrolabe and the armillary sphere, invented during the Middle Ages by astronomers and inventors in the Islamic world.^[c]^ The earliest description of the spherical astrolabe dates to Al-Nayrizi (fl. 892--902). In the 12th century, Sharaf al-Dīn al-Tūsī invented the linear astrolabe, sometimes called the "staff of al-Tusi", which was "a simple wooden rod with graduated markings but without sights. It was furnished with a plumb line and a double chord for making angular measurements and bore a perforated pointer".^[21]^ The geared mechanical astrolabe was invented by Abi Bakr of Isfahan in 1235.^[22]^

The first known metal astrolabe in Western Europe is the Destombes astrolabe made from brass in the eleventh century in Portugal.^[23]^^[24]^ Metal astrolabes avoided the warping that large wooden ones were prone to, allowing the construction of larger and therefore more accurate instruments. Metal astrolabes were heavier than wooden instruments of the same size, making it difficult to use them in navigation.^[25]^

Spherical astrolabe

A depiction of Hermann of Reichenau with an astrolabe in a 13th-century manuscript by Matthew Paris

Herman Contractus of Reichenau Abbey, examined the use of the astrolabe in Mensura Astrolai during the 11th century.^[26]^ Peter of Maricourt wrote a treatise on the construction and use of a universal astrolabe in the last half of the 13th century entitled Nova compositio astrolabii particularis. Universal astrolabes can be found at the History of Science Museum in Oxford.^[27]^ David A. King, historian of Islamic instrumentation, describes the universal astrolobe designed by Ibn al-Sarraj of Aleppo (aka Ahmad bin Abi Bakr; fl. 1328) as "the most sophisticated astronomical instrument from the entire Medieval and Renaissance periods".^[28]^

English author Geoffrey Chaucer (c. 1343--1400) compiled A Treatise on the Astrolabe for his son, mainly based on a work by Messahalla or Ibn al-Saffar.^[29]^^[30]^ The same source was translated by French astronomer and astrologer Pélerin de Prusse and others. The first printed book on the astrolabe was Composition and Use of Astrolabe by Christian of Prachatice, also using Messahalla, but relatively original.

Front of an Indian astrolabe now kept at the Royal Museum of Scotland at Edinburgh.

In 1370, the first Indian treatise on the astrolabe was written by the Jain astronomer Mahendra Suri, titled Yantrarāja.^[31]^

A simplified astrolabe, known as a balesilha, was used by sailors to get an accurate reading of latitude while at sea. The use of the balesilha was promoted by Prince Henry (1394--1460) while navigating for Portugal.^[32]^

The astrolabe was almost certainly first brought north of the Pyrenees by Gerbert of Aurillac (future Pope Sylvester II), where it was integrated into the quadrivium at the school in Reims, France, sometime before the turn of the 11th century.^[33]^ In the 15th century, French instrument maker Jean Fusoris (c. 1365--1436) also started remaking and selling astrolabes in his shop in Paris, along with portable sundials and other popular scientific devices of the day.

Astronomical Instrument Detail by Ieremias Palladas 1612

Thirteen of his astrolabes survive to this day.^[34]^ One more special example of craftsmanship in early 15th-century Europe is the astrolabe designed by Antonius de Pacento and made by Dominicus de Lanzano, dated 1420.^[35]^

In the 16th century, Johannes Stöffler published Elucidatio fabricae ususque astrolabii, a manual of the construction and use of the astrolabe. Four identical 16th-century astrolabes made by Georg Hartmann provide some of the earliest evidence for batch production by division of labor. In 1612, Greek painter Ieremias Palladas incorporated a sophisticated astrolabe in his painting depicting Catherine of Alexandria. The painting was entitled Catherine of Alexandria and featured a device called the System of the Universe (Σύστημα τοῦ Παντός). The device featured the planets with the names in Greek: Selene (Moon), Hermes (Mercury), Aphrodite (Venus), Helios (Sun), Ares (Mars), Zeus (Jupiter), and Chronos (Saturn). The device also featured celestial spheres following the Ptolemaic model and Earth was depicted as a blue sphere with circles of geographic coordinates. A complex line representing the axis of the Earth covered the entire instrument.^[36]^

Medieval astrolabes

• 

  A treatise explaining the importance of the astrolabe by Nasir al-Din al-Tusi, Persian scientist

• 

  Astrolabe of Jean Fusoris, made in Paris, 1400

• 

  An 18th-century Persian astrolabe

• 

  Disassembled 18th-century astrolabe

• 

  Exploded view of an astrolabe

• 

  Animation showing how celestial and geographic coordinates are mapped on an astrolabe's tympan through a stereographic projection. Hypothetical tympan (40° north latitude) of a 16th-century European planispheric astrolabe.

• 

  Astrolabe manual from the Alfonso X of Castile work Libros del saber de astronomía, 1276.

• 

  A page from the 1575 book "Astrolabium" depicting an astrolabe. Masha'Allah Public Library Bruges [nl] Ms. 522

Astrolabes and clocks

Amerigo Vespucci observing the Southern Cross by looking over the top of an armillary sphere bizarrely held from the top as if it were an astrolabe; however, an astrolabe cannot be used by looking over its top. The page inexplicably contains the word astrolabium. By Jan Collaert II. Museum Plantin-Moretus, Antwerp, Belgium.

Mechanical astronomical clocks were initially influenced by the astrolabe; they could be seen in many ways as clockwork astrolabes designed to produce a continual display of the current position of the sun, stars, and planets. For example, Richard of Wallingford's clock (c. 1330) consisted essentially of a star map rotating behind a fixed rete, similar to that of an astrolabe.^[37]^

Many astronomical clocks use an astrolabe-style display, such as the famous clock at Prague, adopting a stereographic projection (see below) of the ecliptic plane. In recent times, astrolabe watches have become popular. For example, Swiss watchmaker Ludwig Oechslin designed and built an astrolabe wristwatch in conjunction with Ulysse Nardin in 1985.^[38]^ Dutch watchmaker Christaan van der Klauuw also manufactures astrolabe watches today.^[39]^

Construction

An astrolabe consists of a disk, called the mater (mother), which is deep enough to hold one or more flat plates called tympans, or climates. A tympan is made for a specific latitude and is engraved with a stereographic projection of circles denoting azimuth and altitude and representing the portion of the celestial sphere above the local horizon. The rim of the mater is typically graduated into hours of time, degrees of arc, or both.^[40]^

Above the mater and tympan, the rete, a framework bearing a projection of the ecliptic plane and several pointers indicating the positions of the brightest stars, is free to rotate. These pointers are often just simple points, but depending on the skill of the craftsman can be very elaborate and artistic. There are examples of astrolabes with artistic pointers in the shape of balls, stars, snakes, hands, dogs' heads, and leaves, among others.^[40]^ The names of the indicated stars were often engraved on the pointers in Arabic or Latin.^[41]^ Some astrolabes have a narrow rule or label which rotates over the rete, and may be marked with a scale of declinations.

The rete, representing the sky, functions as a star chart. When it is rotated, the stars and the ecliptic move over the projection of the coordinates on the tympan. One complete rotation corresponds to the passage of a day. The astrolabe is, therefore, a predecessor of the modern planisphere.

On the back of the mater, there is often engraved a number of scales that are useful in the astrolabe's various applications. These vary from designer to designer, but might include curves for time conversions, a calendar for converting the day of the month to the sun's position on the ecliptic, trigonometric scales, and graduation of 360 degrees around the back edge. The alidade is attached to the back face. An alidade can be seen in the lower right illustration of the Persian astrolabe above. When the astrolabe is held vertically, the alidade can be rotated and the sun or a star sighted along its length, so that its altitude in degrees can be read ("taken") from the graduated edge of the astrolabe; hence the word's Greek roots: "astron" (ἄστρον) = star + "lab-" (λαβ-) = to take. The alidade had vertical and horizontal cross-hairs which plots locations on an azimuthal ring called an almucantar (altitude-distance circle).

An arm called a radius connects from the center of the astrolabe to the optical axis which is parallel with another arm also called a radius. The other radius contains graduations of altitude and distance measurements.

A shadow square also appears on the back of some astrolabes, developed by Muslim astrologists in the 9th Century, whereas devices of the Ancient Greek tradition featured only altitude scales on the back of the devices.^[42]^ This was used to convert shadow lengths and the altitude of the sun, the uses of which were various from surveying to measuring inaccessible heights.^[43]^

Devices were usually signed by their maker with an inscription appearing on the back of the astrolabe, and if there was a patron of the object, their name would appear inscribed on the front, or in some cases, the name of the reigning sultan or the teacher of the astrolabist has also been found to appear inscribed in this place.^[44]^ The date of the astrolabe's construction was often also signed, which has allowed historians to determine that these devices are the second oldest scientific instrument in the world. The inscriptions on astrolabes also allowed historians to conclude that astronomers tended to make their own astrolabes, but that many were also made to order and kept in stock to sell, suggesting there was some contemporary market for the devices.^[44]^

Construction of astrolabes

• 

  The Hartmann astrolabe in Yale collection. This instrument shows its rete and rule.

• 

  Celestial Globe, Isfahan (?), Iran 1144. Shown at the Louvre Museum, this globe is the third oldest surviving in the world.

• 

  Computer-generated planispheric astrolabe

Mathematical basis

The construction and design of astrolabes are based on the application of the stereographic projection of the celestial sphere. The point from which the projection is usually made is the South Pole. The plane onto which the projection is made is that of the Equator.^[45]^

Designing a tympanum through stereographic projection

Parts of an Astrolabe tympanum

The tympanum captures the celestial coordinate axes upon which the rete will rotate. It is the component that will enable the precise determination of a star's position at a specific time of day and year.

Therefore, it should project:

1. The zenith, which will vary depending on the latitude of the astrolabe user.
2. The horizon line and almucantar or circles parallel to the horizon, which will allow for the determination of a celestial body's altitude (from the horizon to the zenith).
3. The celestial meridian (north-south meridian, passing through the zenith) and secondary meridians (circles intersecting the north-south meridian at the zenith), which will enable the measurement of azimuth for a celestial body.
4. The three main circles of latitude (Capricorn, Equator, and Cancer) to determine the exact moments of solstices and equinoxes throughout the year.

The tropics and the equator define the tympanum

Stereographic projection of Earth's tropics and equator from the South Pole.

On the right side of the image above:

1. The blue sphere represents the celestial sphere.
2. The blue arrow indicates the direction of true north (the North Star).
3. The central blue point represents Earth (the observer's location).
4. The geographic south of the celestial sphere acts as the projection pole.
5. The celestial equatorial plane serves as the projection plane.
6. Three parallel circles represent the projection on the celestial sphere of Earth's main circles of latitude:
   • In orange, the celestial Tropic of Cancer.
   • In purple, the celestial equator.
   • In green, the celestial Tropic of Capricorn.

When projecting onto the celestial equatorial plane, three concentric circles correspond to the celestial sphere's three circles of latitude (left side of the image). The largest of these, the projection on the celestial equatorial plane of the celestial Tropic of Capricorn, defines the size of the astrolabe's tympanum. The center of the tympanum (and the center of the three circles) is actually the north-south axis around which Earth rotates, and therefore, the rete of the astrolabe will rotate around this point as the hours of the day pass (due to Earth's rotational motion).

The three concentric circles on the tympanum are useful for determining the exact moments of solstices and equinoxes throughout the year: if the sun's altitude at noon on the rete is known and coincides with the outer circle of the tympanum (Tropic of Capricorn), it signifies the winter solstice (the sun will be at the zenith for an observer at the Tropic of Capricorn, meaning summer in the southern hemisphere and winter in the northern hemisphere). If, on the other hand, its altitude coincides with the inner circle (Tropic of Cancer), it indicates the summer solstice. If its altitude is on the middle circle (equator), it corresponds to one of the two equinoxes.

The horizon and the measurement of altitude

Stereographic projection of an observer's horizon at a specific latitude

On the right side of the image above:

1. The blue arrow indicates the direction of true north (the North Star).
2. The central blue point represents Earth (the observer's location).
3. The black arrow represents the zenith direction for the observer (which would vary depending on the observer's latitude).
4. The two black circles represent the horizon surrounding the observer, which is perpendicular to the zenith vector and defines the portion of the celestial sphere visible to the observer, and its projection on the celestial equatorial plane.
5. The geographic south of the celestial sphere acts as the projection pole.
6. The celestial equatorial plane serves as the projection plane.

When projecting the horizon onto the celestial equatorial plane, it transforms into an ellipse upward-shifted relatively to the center of the tympanum (both the observer and the projection of the north-south axis). This implies that a portion of the celestial sphere will fall outside the outer circle of the tympanum (the projection of the celestial Tropic of Capricorn) and, therefore, won't be represented.

Stereographic projection of the horizon and an almucantar.

Additionally, when drawing circles parallel to the horizon up to the zenith (almucantar), and projecting them on the celestial equatorial plane, as in the image above, a grid of consecutive ellipses is constructed, allowing for the determination of a star's altitude when its rete overlaps with the designed tympanum.

The meridians and the measurement of azimuth

Stereographic projection of the north-south meridian and a meridian 40° E on the tympanum of an astrolabe

On the right side of the image above:

1. The blue arrow indicates the direction of true north (the North Star).
2. The central blue point represents Earth (the observer's location).
3. The black arrow represents the zenith direction for the observer (which would vary depending on the observer's latitude).
4. The two black circles represent the horizon surrounding the observer, which is perpendicular to the zenith vector and defines the portion of the celestial sphere visible to the observer, and its projection on the celestial equatorial plane.
5. The five red dots represent the zenith, the nadir (the point on the celestial sphere opposite the zenith with respect to the observer), their projections on the celestial equatorial plane, and the center (with no physical meaning attached) of the circle obtained by projecting the secondary meridian (see below) on the celestial equatorial plane.
6. The orange circle represents the celestial meridian (or meridian that goes, for the observer, from the north of the horizon to the south of the horizon passing through the zenith).
7. The two red circles represent a secondary meridian with an azimuth of 40° East relative to the observer's horizon (which, like all secondary meridians, intersects the principal meridian at the zenith and nadir), and its projection on the celestial equatorial plane.
8. The geographic south of the celestial sphere acts as the projection pole.
9. The celestial equatorial plane serves as the projection plane.

When projecting the celestial meridian, it results in a straight line that overlaps with the vertical axis of the tympanum, where the zenith and nadir are located. However, when projecting the 40° E meridian, another circle is obtained that passes through both the zenith and nadir projections, so its center is located on the perpendicular bisection of the segment connecting both points. In deed, the projection of the celestial meridian can be considered as a circle with an infinite radius (a straight line) whose center is on this bisection and at an infinite distance from these two points.

If successive meridians that divide the celestial sphere into equal sectors (like "orange slices" radiating from the zenith) are projected, a family of curves passing through the zenith projection on the tympanum is obtained. These curves, once overlaid with the rete containing the major stars, allow for determining the azimuth of a star located on the rete and rotated for a specific time of day.

See also

• Astronomy in the medieval Islamic world
• Equatorium
• Hamburg Planetarium
• List of astronomical instruments
• Philippe Danfrie, designer and maker of mathematical instruments, globes and astrolabes
• Planetarium
• Yantraraja
• Zeiss-Planetarium Jena

References

Footnotes

1.

1. Savage-Smith, Emilie (1993). "Book Reviews". Journal of Islamic Studies. 4 (2): 296--299. doi:10.1093/jis/4.2.296. There is no evidence for the Hellenistic origin of the spherical astrolabe, but rather evidence so far available suggests that it may have been an early but distinctly Islamic development with no Greek antecedents.

Notes

1.

1. Gentili, Graziano; Simonutti, Luisa; Struppa, Daniele C. (2020). "The Mathematics of the Astrolabe and Its History". Journal of Humanistic Mathematics. 10: 101--144. doi:10.5642/jhummath.202001.07. hdl:2158/1182616. S2CID 211008813.

Bibliography

• Evans, James (1998), The History and Practice of Ancient Astronomy, Oxford University Press, ISBN 0-19-509539-1
• Stöffler, Johannes (2007) [First published 1513], Stoeffler's Elucidatio -- The Construction and Use of the Astrolabe [Elucidatio Fabricae Ususque Astrolabii], translated by Gunella, Alessandro; Lamprey, John, John Lamprey, ISBN 978-1-4243-3502-2
• King, D. A. (1981), "The Origin of the Astrolabe According to the Medieval Islamic Sources", Journal for the History of Arabic Science, 5: 43--83
• King, Henry (1978), Geared to the Stars: the Evolution of Planetariums, Orreries, and Astronomical Clocks, University of Toronto Press, ISBN 978-0-8020-2312-4
• Krebs, Robert E.; Krebs, Carolyn A. (2003), Groundbreaking Scientific Experiments, Inventions, and Discoveries of the Ancient World, Greenwood Press, ISBN 978-0-313-31342-4
• Laird, Edgar (1997), Carol Poster and Richard Utz (ed.), "Astrolabes and the Construction of Time in the Late Middle Ages", Constructions of Time in the Late Middle Ages, Evanston, Illinois: Northwestern University Press: 51--69
• Laird, Edgar; Fischer, Robert, eds. (1995), "Critical edition of Pélerin de Prusse on the Astrolabe (translation of Practique de Astralabe)", Medieval & Renaissance Texts & Studies, Binghamton, New York, ISBN 0-86698-132-2
• Lewis, M. J. T. (2001), Surveying Instruments of Greece and Rome, Cambridge University Press, ISBN 978-0-511-48303-5
• Morrison, James E. (2007), The Astrolabe, Janus, ISBN 978-0-939320-30-1
• Neugebauer, Otto E. (1975), A History of Ancient Mathematical Astronomy, Springer, ISBN 978-3-642-61912-0
• North, John David (2005), God's Clockmaker: Richard of Wallingford and the Invention of Time, Continuum International Publishing Group, ISBN 978-1-85285-451-5

External links

Wikimedia Commons has media related to:\ Astrolabe (category)

Wikisource has the text of the 1911 Encyclopædia Britannica article "Astrolabe".

Look up astrolabe in Wiktionary, the free dictionary.

• Interactive digital astrolabe by Alex Boxer
• A digital astrolabe (HTML5 and javascript)
• Astrolabe Tech Made ... Not So Easy
• paper astrolabe generator, from the ESO
• "Hello World!" for the Astrolabe: The First Computer Video of Howard Covitz's Presentation at Ignite Phoenix, June 2009. Slides for Presentation Licensed as Creative Commons by-nc-nd.
• Video of Tom Wujec demonstrating an astrolabe. Archived 2012-03-23 at the Wayback Machine Taken at TEDGlobal 2009. Includes clickable transcript. Licensed as Creative Commons by-nc-nd.
• Archive of James E. Morrison's extensive website on Astrolabes
• A working model of the Dr. Ludwig Oechslin's Astrolabium Galileo Galilei watch
• Ulysse Nardin Astrolabium Galilei Galileo: A Detailed Explanation
• Fully illustrated online catalogue of world's largest collection of astrolabes
• Mobile astrolabe and horologium
• Medieval equal hour horary quadrant
• A Beginner's Guide to Basic Construction and Use of the Astrolabe (using ruler, protractor and compasses) (PDF), archived from the original (PDF) on 2015-06-17, retrieved 2018-10-26

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  ](https://en.wikipedia.org/wiki/Astrolabe#Construction)- [

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  ](https://en.wikipedia.org/wiki/Astrolabe#Mathematical_basis)- Modern editions of John Philoponus' treatise on the astrolabe are De usu astrolabii eiusque constructione libellus (On the Use and Construction of the Astrolabe), ed. Heinrich Hase, Bonn: E. Weber, 1839, OCLC 165707441 (or id. Rheinisches Museum für Philologie 6 (1839): 127--71); repr. and translated into French by Alain Philippe Segonds, Jean Philopon, traité de l'astrolabe, Paris: Librairie Alain Brieux, 1981, OCLC 10467740; and translated into English by H.W. Green in R.T. Gunther, The Astrolabes of the World, Vol. 1/2, Oxford, 1932, OL 18840299M repr. London: Holland Press, 1976, OL 14132393M pp. 61--81.- O'Leary, De Lacy (1948). How Greek Science Passed to the Arabs. Routledge and Kegan Paul. "The most distinguished Syriac scholar of this later period was Severus Sebokht (d. 666--7), Bishop of Kennesrin. [...] In addition to these works [...] he also wrote on astronomical subjects (Brit. Mus. Add. 14538), and composed a treatise on the astronomical instrument known as the astrolabe, which has been edited and published by F. Nau (Paris, 1899)."\ Severus' treatise was translated by Jessie Payne Smith Margoliouth in R.T. Gunther, Astrolabes of the World, Oxford, 1932, pp. 82--103.- "HISTORIANS' HOME YIELDS RICH LODE; New York Society Searches Its Own Building for Items to Mark Anniversary; SHOW OPENS THURSDAY; Portrait of Stuyvesant and Champlain's Astrolabe Will Be on Display". The New York Times. May 18, 1964. Retrieved February 4, 2024.- Bean, Adam L. (2009). "Astrolabes". In Birx, H. James (ed.). Encyclopedia of Time: Science, Philosophy, Theology, & Culture. Vol. 1. SAGE. pp. 59--60. ISBN 978-1-4129-4164-8.- "Astrolabe". Oxford English Dictionary (2nd ed.). 1989.- "Astrolabe". Oxford Dictionaries. Archived from the original on October 22, 2013.- "Online Etymology Dictionary". Etymonline.com. Retrieved 2013-11-07.- King 1981, p. 44.- King 1981, p. 51.- King 1981, p. 45.- Lewis 2001.- Michael Deakin (August 3, 1997). "Ockham's Razor: Hypatia of Alexandria". ABC Radio. Retrieved July 10, 2014.- Theodore, Jonathan (2016). The Modern Cultural Myth of the Decline and Fall of the Roman Empire. Manchester, England: Palgrave, Macmillan. p. 183. ISBN 978-1-137-56997-4.- Deakin, Michael A. B. (2007). Hypatia of Alexandria: Mathematician and Martyr. Amherst, New York: Prometheus Books. pp. 102--104. ISBN 978-1-59102-520-7.- Bradley, Michael John (2006). The Birth of Mathematics: Ancient Times to 1300. New York City, New York: Infobase Publishing. p. 63. ISBN 9780816054237.- Savage-Smith, Emilie (1992). "Celestial Mapping" (PDF). In Harley, J. B.; Woodward, David (eds.). The History of Cartography, Volume 2, Book 1: Cartography in the Traditional Islamic and South Asian Societies. The History of Cartography. Vol. 2. Chicago, Illinois: University of Chicago Press. ISBN 0226316351.- Sebokht, Severus. "Description of the astrolabe". Tertullian.org.- See p. 289 of Martin, L. C. (1923), "Surveying and navigational instruments from the historical standpoint", Transactions of the Optical Society, 24 (5): 289--303, Bibcode:1923TrOS...24..289M, doi:10.1088/1475-4878/24/5/302, ISSN 1475-4878.- Berggren, J. Lennart (2007), "Mathematics in Medieval Islam", in Katz, Victor J. (ed.), The Mathematics of Egypt, Mesopotamia, China, India, and Islam: a Sourcebook, Princeton University Press, p. 519, ISBN 978-0-691-11485-9- Richard Nelson Frye: Golden Age of Persia. p. 163- Nizamoglu, Cem (2005-08-10). "Using an Astrolabe". Muslim Heritage. Retrieved 2023-10-16.- Lachièz-Rey, Marc; Luminet, Jean-Pierre (2001). Celestial Treasury: From the Music of Spheres to the Conquest of Space. Translated by Joe Laredo. Cambridge, United Kingdom: Cambridge University Press. p. 74. ISBN 978-0-521-80040-2.- O'Connor, John J.; Robertson, Edmund F., "Sharaf al-Din al-Muzaffar al-Tusi", MacTutor History of Mathematics Archive, University of St Andrews- Bedini, Silvio A.; Maddison, Francis R. (1966). "Mechanical Universe: The Astrarium of Giovanni de' Dondi". Transactions of the American Philosophical Society. 56 (5): 1--69. doi:10.2307/1006002. JSTOR 1006002.- "Qantara -- 'Carolingian' astrolabe". Qantara-med.org. Retrieved 2013-11-07.- Nancy Marie Brown (2010), "The Abacus and the Cross". p. 140. Basic Books. ISBN 978-0-465-00950-3- Boyle, David (2011). Toward the Setting Sun: Columbus, Cabot, Vespucci, and the Race for America. Bloomsbury Publishing USA. p. 253. ISBN 9780802779786..- Northrup, Cynthia Clark, ed. (2015). Encyclopedia of world trade: from ancient times to the present (Enhanced Credo ed.). Armonk, New York: Routledge. pp. 72. ISBN 978-0765680587. OCLC 889717964.- "Introduction". The Astrolabe: an Online Resource. 2006. Retrieved 2020-05-15.- Harley, J. B.; Woodward, David (1992). The history of cartography. Chicago, Illinois: University of Chicago Press. p. 31. ISBN 0-226-31635-1.- Kunitzsch, Paul (1981). "On the authenticity of the treatise on the composition and use of the astrolabe ascribed to Messahalla". Archives Internationales d'Histoire des Sciences Oxford. 31 (106): 42--62.- Selin, Helaine (2008-03-12). Encyclopaedia of the History of Science, Technology, and Medicine in Non-Western Cultures. Springer Science & Business Media. p. 1335. ISBN 978-1-4020-4559-2. Paul Kunitzsch has recently established that the Latin treatise on the astrolabe long ascribed to Ma'sh'allah and translated by John of Seville is in fact by Ibn al-Saffar, a disciple of Maslama al-Majriti.- Glick, Thomas; et al., eds. (2005), Medieval Science, Technology, and Medicine: An Encyclopedia, Routledge, p. 464, ISBN 0-415-96930-1- Northrup, Cynthia Clark, ed. (2015). Encyclopedia of world trade : from ancient times to the present ([Enhanced Credo edition] ed.). Armonk, New York: Routledge. pp. 460. ISBN 978-0765680587. OCLC 889717964.- Nancy Marie Brown (2010), "The Abacus and the Cross". p. 143. basic Books. ISBN 978-0-465-00950-3- Hockey, Thomas (2009). The Biographical Encyclopedia of Astronomers. Springer Publishing. ISBN 978-0-387-31022-0. Retrieved August 22, 2012.- Ralf Kern (2010), Wissenschaftliche Instrumente in ihrer Zeit. Band 1: Vom Astrolab zum mathematischen Besteck. Cologne, S. 204. ISBN 978-3-86560-865-9- Vafea, Flora (2017). "The Astronomical Instruments in Saint Catherine's Iconography at the Holy Monastery of Sinai The Almagest Volume 8, Issue 2". Almagest. 8 (2). Paris: University of Paris: 87. doi:10.1484/J.ALMAGEST.5.114932.- North 2005.- "Astrolabium G. Galilei". Ulysse Nardin. Archived from the original on 2 January 2011.- "Christaan van der Klauuw".- Stephenson, Bruce; Bolt, Marvin; Friedman, Anna Felicity (2000). The Universe Unveiled: Instruments and Images through History. Cambridge, UK: Cambridge University Press. pp. 108--109. ISBN 0-521-79143-X.- "Star Names on Astrolabes". Ian Ridpath. Retrieved 2016-11-12.- King, David A. Some Medieval Astronomical Instruments and Their Secrets, in Mazzolini, R. G. (ed.), Non-Verbal Communication in Science prior to 1900. Florence. p. 30.- King, David A. (2018). The Astrolabe: What it is & what it is not. Frankfurt, Germany: Frankfurt.- Mayer, L. A. (1956). Islamic astrolabists and their works*. A. Kunding. Bibcode:[1956iatw.book.....M](https://ui.a Main menu

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From Wikipedia, the free encyclopedia This article is about the sextant as used for navigation. For other uses, see Sextant (disambiguation). Not to be confused with Sexton (disambiguation). A sextant

A sextant is a doubly reflecting navigation instrument that measures the angular distance between two visible objects. The primary use of a sextant is to measure the angle between an astronomical object and the horizon for the purposes of celestial navigation.

The estimation of this angle, the altitude, is known as sighting or shooting the object, or taking a sight. The angle, and the time when it was measured, can be used to calculate a position line on a nautical or aeronautical chart—for example, sighting the Sun at noon or Polaris at night (in the Northern Hemisphere) to estimate latitude (with sight reduction). Sighting the height of a landmark can give a measure of distance off and, held horizontally, a sextant can measure angles between objects for a position on a chart.[1] A sextant can also be used to measure the lunar distance between the moon and another celestial object (such as a star or planet) in order to determine Greenwich Mean Time and hence longitude.

The principle of the instrument was first implemented around 1731 by John Hadley (1682–1744) and Thomas Godfrey (1704–1749), but it was also found later in the unpublished writings of Isaac Newton (1643–1727).

In 1922, it was modified for aeronautical navigation by Portuguese navigator and naval officer Gago Coutinho. Navigational sextants

Like the Davis quadrant, the sextant allows celestial objects to be measured relative to the horizon, rather than relative to the instrument. This allows excellent precision. Also, unlike the backstaff, the sextant allows direct observations of stars. This permits the use of the sextant at night when a backstaff is difficult to use. For solar observations, filters allow direct observation of the Sun.

Since the measurement is relative to the horizon, the measuring pointer is a beam of light that reaches to the horizon. The measurement is thus limited by the angular accuracy of the instrument and not the sine error of the length of an alidade, as it is in a mariner's astrolabe or similar older instrument.

A sextant does not require a completely steady aim, because it measures a relative angle. For example, when a sextant is used on a moving ship, the image of both horizon and celestial object will move around in the field of view. However, the relative position of the two images will remain steady, and as long as the user can determine when the celestial object touches the horizon, the accuracy of the measurement will remain high compared to the magnitude of the movement.

The sextant is not dependent upon electricity (unlike many forms of modern navigation) or any human-controlled signals (such as GPS). For these reasons it is considered to be an eminently practical back-up navigation tool for ships. Design

The frame of a sextant is in the shape of a sector which is approximately 1⁄6 of a circle (60°),[2] hence its name (sextāns, sextantis is the Latin word for "one sixth"). Both smaller and larger instruments are (or were) in use: the octant, quintant (or pentant) and the (doubly reflecting) quadrant[3] span sectors of approximately 1⁄8 of a circle (45°), 1⁄5 of a circle (72°) and 1⁄4 of a circle (90°), respectively. All of these instruments may be termed "sextants". Marine sextant Using the sextant to measure the altitude of the Sun above the horizon Sextants can also be used by navigators to measure horizontal angles between objects.

Attached to the frame are the "horizon mirror", an index arm which moves the index mirror, a sighting telescope, Sun shades, a graduated scale and a micrometer drum gauge for accurate measurements. The scale must be graduated so that the marked degree divisions register twice the angle through which the index arm turns. The scales of the octant, sextant, quintant and quadrant are graduated from below zero to 90°, 120°, 140° and 180° respectively. For example, the sextant illustrated has a scale graduated from −10° to 142°, which is basically a quintant: the frame is a sector of a circle subtending an angle of 76° at the pivot of the index arm.

The necessity for the doubled scale reading follows from consideration of the relations of the fixed ray (between the mirrors), the object ray (from the sighted object) and the direction of the normal perpendicular to the index mirror. When the index arm moves by an angle, say 20°, the angle between the fixed ray and the normal also increases by 20°. But the angle of incidence equals the angle of reflection so the angle between the object ray and the normal must also increase by 20°. The angle between the fixed ray and the object ray must therefore increase by 40°. This is the case shown in the graphic.

There are two types of horizon mirrors on the market today. Both types give good results.

Traditional sextants have a half-horizon mirror, which divides the field of view in two. On one side, there is a view of the horizon; on the other side, a view of the celestial object. The advantage of this type is that both the horizon and celestial object are bright and as clear as possible. This is superior at night and in haze, when the horizon and/or a star being sighted can be difficult to see. However, one has to sweep the celestial object to ensure that the lowest limb of the celestial object touches the horizon.

Whole-horizon sextants use a half-silvered horizon mirror to provide a full view of the horizon. This makes it easy to see when the bottom limb of a celestial object touches the horizon. Since most sights are of the Sun or Moon, and haze is rare without overcast, the low-light advantages of the half-horizon mirror are rarely important in practice.

In both types, larger mirrors give a larger field of view, and thus make it easier to find a celestial object. Modern sextants often have 5 cm or larger mirrors, while 19th-century sextants rarely had a mirror larger than 2.5 cm (one inch). In large part, this is because precision flat mirrors have grown less expensive to manufacture and to silver.

An artificial horizon is useful when the horizon is invisible, as occurs in fog, on moonless nights, in a calm, when sighting through a window or on land surrounded by trees or buildings. There are two common designs of artificial horizon. An artificial horizon can consist simply of a pool of water shielded from the wind, allowing the user to measure the distance between the body and its reflection, and divide by two. Another design allows the mounting of a fluid-filled tube with bubble directly to the sextant.

Most sextants also have filters for use when viewing the Sun and reducing the effects of haze. The filters usually consist of a series of progressively darker glasses that can be used singly or in combination to reduce haze and the Sun's brightness. However, sextants with adjustable polarizing filters have also been manufactured, where the degree of darkness is adjusted by twisting the frame of the filter.

Most sextants mount a 1 or 3-power monocular for viewing. Many users prefer a simple sighting tube, which has a wider, brighter field of view and is easier to use at night. Some navigators mount a light-amplifying monocular to help see the horizon on moonless nights. Others prefer to use a lit artificial horizon.[citation needed]

Professional sextants use a click-stop degree measure and a worm adjustment that reads to a minute, 1/60 of a degree. Most sextants also include a vernier on the worm dial that reads to 0.1 minute. Since 1 minute of error is about a nautical mile, the best possible accuracy of celestial navigation is about 0.1 nautical miles (190 m). At sea, results within several nautical miles, well within visual range, are acceptable. A highly skilled and experienced navigator can determine position to an accuracy of about 0.25-nautical-mile (460 m).[4]

A change in temperature can warp the arc, creating inaccuracies. Many navigators purchase weatherproof cases so that their sextant can be placed outside the cabin to come to equilibrium with outside temperatures. The standard frame designs (see illustration) are supposed to equalise differential angular error from temperature changes. The handle is separated from the arc and frame so that body heat does not warp the frame. Sextants for tropical use are often painted white to reflect sunlight and remain relatively cool. High-precision sextants have an invar (a special low-expansion steel) frame and arc. Some scientific sextants have been constructed of quartz or ceramics with even lower expansions. Many commercial sextants use low-expansion brass or aluminium. Brass is lower-expansion than aluminium, but aluminium sextants are lighter and less tiring to use. Some say they are more accurate because one's hand trembles less. Solid brass frame sextants are less susceptible to wobbling in high winds or when the vessel is working in heavy seas, but as noted are substantially heavier. Sextants with aluminum frames and brass arcs have also been manufactured. Essentially, a sextant is intensely personal to each navigator, and they will choose whichever model has the features which suit them best.

Aircraft sextants are now out of production, but had special features. Most had artificial horizons to permit taking a sight through a flush overhead window. Some also had mechanical averagers to make hundreds of measurements per sight for compensation of random accelerations in the artificial horizon's fluid. Older aircraft sextants had two visual paths, one standard and the other designed for use in open-cockpit aircraft that let one view from directly over the sextant in one's lap. More modern aircraft sextants were periscopic with only a small projection above the fuselage. With these, the navigator pre-computed their sight and then noted the difference in observed versus predicted height of the body to determine their position. Taking a sight

A sight (or measure) of the angle between the Sun, a star, or a planet, and the horizon is done with the 'star telescope' fitted to the sextant using a visible horizon. On a vessel at sea even on misty days a sight may be done from a low height above the water to give a more definite, better horizon. Navigators hold the sextant by its handle in the right hand, avoiding touching the arc with the fingers.[5]

For a Sun sight, a filter is used to overcome the glare such as "shades" covering both index mirror and the horizon mirror designed to prevent eye damage. Initially, with the index bar set to zero and the shades covering both mirrors, the sextant is aimed at the sun until it can be viewed on both mirrors through the telescope, then lowered vertically until the portion of the horizon directly below it is viewed on both mirrors. It is necessary to flip back the horizon mirror shade to be able to see the horizon more clearly on it. Releasing the index bar (either by releasing a clamping screw, or on modern instruments, using the quick-release button), and moving it towards higher values of the scale, eventually the image of the Sun will reappear on the index mirror and can be aligned to about the level of the horizon on the horizon mirror. Then the fine adjustment screw on the end of the index bar is turned until the bottom curve (the lower limb) of the Sun just touches the horizon. "Swinging" the sextant about the axis of the telescope ensures that the reading is being taken with the instrument held vertically. The angle of the sight is then read from the scale on the arc, making use of the micrometer or vernier scale provided. The exact time of the sight must also be noted simultaneously, and the height of the eye above sea-level recorded.[5]

An alternative method is to estimate the current altitude (angle) of the Sun from navigation tables, then set the index bar to that angle on the arc, apply suitable shades only to the index mirror, and point the instrument directly at the horizon, sweeping it from side to side until a flash of the Sun's rays are seen in the telescope. Fine adjustments are then made as above. This method is less likely to be successful for sighting stars and planets.[5]

Star and planet sights are normally taken during nautical twilight at dawn or dusk, while both the heavenly bodies and the sea horizon are visible. There is no need to use shades or to distinguish the lower limb as the body appears as a mere point in the telescope. The Moon can be sighted, but it appears to move very fast, appears to have different sizes at different times, and sometimes only the lower or upper limb can be distinguished due to its phase.[5]

After a sight is taken, it is reduced to a position by looking at several mathematical procedures. The simplest sight reduction is to draw the equal-altitude circle of the sighted celestial object on a globe. The intersection of that circle with a dead-reckoning track, or another sighting, gives a more precise location.

Sextants can be used very accurately to measure other visible angles, for example between one heavenly body and another and between landmarks ashore. Used horizontally, a sextant can measure the apparent angle between two landmarks such as a lighthouse and a church spire, which can then be used to find the distance off or out to sea (provided the distance between the two landmarks is known). Used vertically, a measurement of the angle between the lantern of a lighthouse of known height and the sea level at its base can also be used for distance off.[5] Adjustment

Due to the sensitivity of the instrument it is easy to knock the mirrors out of adjustment. For this reason a sextant should be checked frequently for errors and adjusted accordingly.

There are four errors that can be adjusted by the navigator, and they should be removed in the following order.

Perpendicularity error This is when the index mirror is not perpendicular to the frame of the sextant. To test for this, place the index arm at about 60° on the arc and hold the sextant horizontally with the arc away from you at arm's length and look into the index mirror. The arc of the sextant should appear to continue unbroken into the mirror. If there is an error, then the two views will appear to be broken. Adjust the mirror until the reflection and direct view of the arc appear to be continuous. Side error This occurs when the horizon glass/mirror is not perpendicular to the plane of the instrument. To test for this, first zero the index arm then observe a star through the sextant. Then rotate the tangent screw back and forth so that the reflected image passes alternately above and below the direct view. If in changing from one position to another, the reflected image passes directly over the unreflected image, no side error exists. If it passes to one side, side error exists. Alternatively, the user can hold the sextant on its side and observe the horizon to check the sextant during the day. If there are two horizons there is side error. In both cases, adjust the horizon glass/mirror until respectively the star or the horizon dual images merge into one. Side error is generally inconsequential for observations and can be ignored or reduced to a level that is merely inconvenient. Collimation error This is when the telescope or monocular is not parallel to the plane of the sextant. To check for this you need to observe two stars 90° or more apart. Bring the two stars into coincidence either to the left or the right of the field of view. Move the sextant slightly so that the stars move to the other side of the field of view. If they separate there is collimation error. As modern sextants rarely use adjustable telescopes, they do not need to be corrected for collimation error. Index error This occurs when the index and horizon mirrors are not parallel to each other when the index arm is set to zero. To test for index error, zero the index arm and observe the horizon. If the reflected and direct image of the horizon are in line there is no index error. If one is above the other adjust the index mirror until the two horizons merge. Alternatively, the same procedure can be done at night using a star or the Moon instead of the horizon.

See also

Astrolabe
Bris sextant
Davis quadrant
Gago Coutinho
Harold Gatty
History of longitude
Intercept method
Latitude
Longitude
Longitude by chronometer
Mariner's astrolabe
Navigation
Octant (instrument)
Quadrant (instrument)
Sextant (astronomy)

Notes

Seddon, J. Carl (June 1968). "Line of Position from a Horizontal Angle". Journal of Navigation. 21 (3): 367–369. doi:10.1017/S0373463300024838. ISSN 1469-7785. A.), McPhee, John (John; NSW., Museums and Galleries (2008). Great Collections : treasures from Art Gallery of NSW, Australian Museum, Botanic Gardens Trust, Historic Houses Trust of NSW, Museum of Contemporary Art, Powerhouse Museum, State Library of NSW, State Records NSW. Museums & Galleries NSW. p. 56. ISBN 9780646496030. OCLC 302147838. This article treats the doubly reflecting quadrant, not its predecessor described at quadrant. Dutton's Navigation and Piloting, 12th edition. G.D. Dunlap and H.H. Shufeldt, eds. Naval Institute Press 1972, ISBN 0-87021-163-3

Dixon, Conrad (1968). "5. Using the sextant". Basic Astro Navigation. Adlard Coles. ISBN 0-229-11740-6.

References

Bowditch, Nathaniel (2002). The American Practical Navigator. Bethesda, MD: National Imagery and Mapping Agency. ISBN 0-939837-54-4. Archived from the original on 2007-06-24.
Chisholm, Hugh, ed. (1911). "Sextant" . Encyclopædia Britannica. Vol. 24 (11th ed.). Cambridge University Press. pp. 765–767.
Cutler, Thomas J. (December 2003). Dutton's Nautical Navigation (15th ed.). Annapolis, MD: Naval Institute Press. ISBN 978-1-55750-248-3.
Department of the Air Force (March 2001). Air Navigation (PDF). Department of the Air Force. Retrieved 2014-12-28.
Great Britain Ministry of Defence (Navy) (1995). Admiralty Manual of Seamanship. The Stationery Office. ISBN 0-11-772696-6.
Maloney, Elbert S. (December 2003). Chapman Piloting and Seamanship (64th ed.). New York: Hearst Communications. ISBN 1-58816-089-0.
Martin, William Robert (1911). "Navigation" . In Chisholm, Hugh (ed.). Encyclopædia Britannica. Vol. 19 (11th ed.). Cambridge University Press. pp. 284–298.

External links Look up sextant in Wiktionary, the free dictionary. Wikimedia Commons has media related to Sextant.

Her Majesty's Nautical Almanac Office Archived 2011-02-21 at the Wayback Machine
The History of HM Nautical Almanac Office Archived 2016-06-24 at the Wayback Machine
Chapter 17 from the online edition of Nathaniel Bowditch's American Practical Navigator
Understand difference in Antique & Replica Sextant Archived 2017-08-17 at the Wayback Machine
CD-Sextant - Build your own sextant Simple do-it-yourself project.
Lunars web site. online calculation
Complete celnav theory book, including Lunars

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dsabs.harvard.edu/abs/1956iatw.book.....M).*

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The emergence of Drosophila EM connectomes has revealed numerous neurons within the associative learning circuit. However, these neurons are inaccessible for functional assessment or genetic manipulation in the absence of cell-type-specific drivers. Addressing this knowledge gap, Shuai et al. have screened over 4000 split-GAL4 drivers and correlated them with identified neuron types from the "Hemibrain" EM connectome by matching light microscopy images to neuronal shapes defined by EM. They successfully generated over 800 split-GAL4 drivers and 22 split-LexA drivers covering a substantial number of neuron types across layers of the mushroom body associative learning circuit. They provide new labeling tools for olfactory and non-olfactory sensory inputs to the mushroom body; interneurons connected with dopaminergic neurons and/or mushroom body output neurons; potential reinforcement sensory neurons; and expanded coverage of intrinsic mushroom body neurons. Furthermore, the authors have optimized the GR64f-GAL4 driver into a sugar sensory neuron-specific split-GAL4 driver and functionally validated it as providing a robust optogenetic substitute for sugar reward. Additionally, a driver for putative nociceptive ascending neurons, potentially serving as optogenetic negative reinforcement, is characterized by optogenetic avoidance behavior. The authors also use their very large dataset of neuronal anatomies, covering many example neurons from many brains, to identify neuron instances with atypical morphology. They find many examples of mushroom body neurons with altered neuronal numbers or mistargeting of dendrites or axons and estimate that 1-3% of neurons in each brain may have anatomic peculiarities or malformations. Significantly, the study systematically assesses the individualized existence of MBON08 for the first time. This neuron is a variant shape that sometimes occurs instead of one of two copies of MBON09, and this variation is more common than that in other neuronal classes: 75% of hemispheres have two MBON09's, and 25% have one MBON09 and one MBON08. These newly developed drivers not only expand the repertoire for genetic manipulation of mushroom body-related neurons but also empower researchers to investigate the functions of circuit motifs identified from the connectomes. The authors generously make these flies available to the public. In the foreseeable future, the tools generated in this study will allow important advances in the understanding of learning and memory in Drosophila.

Strengths:

(1) After decades of dedicated research on the mushroom body, a consensus has been established that the release of dopamine from DANs modulates the weights of connections between KCs and MBONs. This process updates the association between sensory information and behavioral responses. However, understanding how the unconditioned stimulus is conveyed from sensory neurons to DANs, and the interactions of MBON outputs with innate responses to sensory context remains less clear due to the developmental and anatomic diversity of MBONs and DANs. Additionally, the recurrent connections between MBONs and DANs are reported to be critical for learning. The characterization of split-GAL4 drivers for 30 major interneurons connected with DANs and/or MBONs in this study will significantly contribute to our understanding of recurrent connections in mushroom body function.

(2) Optogenetic substitutes for real unconditioned stimuli (such as sugar taste or electric shock) are sometimes easier to implement in behavioral assays due to the spatial and temporal specificity with which optogenetic activation can be induced. GR64f-GAL4 has been widely used in the field to activate sugar sensory neurons and mimic sugar reward. However, the authors demonstrate that GR64f-GAL4 drives expression in other neurons not necessary for sugar reward, and the potential activation of these neurons could introduce confounds into training, impairing training efficiency. To address this issue, the authors have elaborated on a series of intersectional drivers with GR64f-GAL4 to dissect subsets of labeled neurons. This approach successfully identified a more specific sugar sensory neuron driver, SS87269, which consistently exhibited optimal training performance and triggered ethologically relevant local searching behaviors. This newly characterized line could serve as an optimized optogenetic tool for sugar reward in future studies.

(3) MBON08 was first reported by Aso et al. 2014, exhibiting dendritic arborization into both ipsilateral and contralateral γ3 compartments. However, this neuron could not be identified in the previously published Drosophila brain connectomes. In the present study, the existence of MBON08 is confirmed, occurring in one hemisphere of 35% of imaged flies. In brains where MBON08 is present, its dendrite arborization disjointly shares contralateral γ3 compartments with MBON09. This remarkable phenotype potentially serves as a valuable resource for understanding the stochasticity of neurodevelopment and the molecular mechanisms underlying mushroom body lobe compartment formation.

Weaknesses:

There are some minor weaknesses in the paper that can be clarified:

(1) In Figure 8, the authors trained flies with a 20s, weak optogenetic conditioning first, followed by a 60s, strong optogenetic conditioning. The rationale for using this training paradigm is not explicitly provided.

These experiments were designed to test if flies could maintain consistent performance with repetitive and intense LED activation, which is essential for experiments involving long training protocols or coactivation of other neurons inside a brain.

In Figure 8E, if data for training with GR64f-GAL4 using the same paradigm is available, it would be beneficial for readers to compare the learning performance using newly generated split-GAL4 lines with the original GR64f-GAL4, which has been used in many previous research studies. It is noteworthy that in previously published work, repeating training test sessions typically leads to an increase in learning performance in discrimination assays. However, this augmentation is not observed in any of the split-GAL4 lines presented in Figure 8E. The authors may need to discuss possible reasons for this.

As the reviewer pointed out, many previous studies including ours used the original Gr64f-GAL4 in olfactory conditioning. Figure 1H of Yamada et al., 2023 (https://doi.org/10.7554/eLife.79042) showed such a result, where the first and second-order olfactory conditioning were assayed. Indeed, the first-order conditioning scores were gradually augmented over repeated training. In this experiment, we used low red LED intensity for the optogenetic activation. In the Figure 8E of the present paper, the first memory test was after 3x pairing of 20s odor with five 1s red LED without intermediate tests. Therefore, flies were already sufficiently trained to show a plateau memory level in “Test1”. In the revision of another recent report (Figure 1C-F of Aso et al., 2023; https://doi.org/10.7554/eLife.85756), we included the learning curve data of our best Gr64f-split-GAL4, SS87269. Under a less saturated training conditioning, SS87269 did show learning augmentation over repeated training.

(2) In line 327, the authors state that in all samples, the β'1 compartment is arborized by MBON09. However, in Figure 11J, the probability of having at least one β'1 compartment not arborized is inferred to be 2%. The authors should address and clarify this conflict in the text to avoid misunderstanding.

The chance of visualizing MBON08 in MCFO images was 21/209 in total (Figure 11I). If we assume that each of four cells adopt MBON08 development fate at this chance, we can calculate the probability for each case of MBON08/09 cell type composition. From this calculation, we inferred approximately 2% of flies would lack innervations to β'1 compartment in at least one hemisphere. However, we didn't observe a lack of β'1 arborizations in 169 sample flies. If these MBONs independently develop into MBON08 at 21/209 odds, the chance of never observing two MBON08s in either hemisphere of all 169 samples is 3.29%. Therefore, some developmental mechanisms may prevent the emergence of two MBON08 in the same hemisphere.

In the revised manuscript, we displayed these estimated probability for each case separately, and annotated actual observation on the right side.

(3) In general, are the samples presented male or female? This sample metadata will be shown when the images are deposited in FlyLight, but it would be useful in the context of this manuscript to describe in the methods whether animals are all one sex or mixed sex, and in some example images (e.g. mAL3A) to note whether the sample is male or female.

The samples presented in this study are mixed sex, except for Figure 11I, where genders are specified. We provided metadata information of the presented images in Supplemental File 7, and we added a paragraph in the in the method section:

“Most samples were collected from females, though typically at least one male fly was examined for each driver line. While we noticed certain lines such as SS48900, exhibited distinct expression patterns in females and males, we did not particularly focus on sexual dimorphism, which is analyzed elsewhere (Meissner et al. 2024). Therefore, unless stated otherwise, the presented samples are of mixed gender.

Detailed metadata, including gender information and the reporter used, can be found in Supplementary File 7.”

Reviewer #2 (Public Review):

Summary:

The article by Shuai et al. describes a comprehensive collection of over 800 split-GAL4 and split-LexA drivers, covering approximately 300 cell types in Drosophila, aimed at advancing the understanding of associative learning. The mushroom body (MB) in the insect brain is central to associative learning, with Kenyon cells (KCs) as primary intrinsic neurons and dopaminergic neurons (DANs) and MB output neurons (MBONs) forming compartmental zones for memory storage and behavior modulation. This study focuses on characterizing sensory input as well as direct upstream connections to the MB both anatomically and, to some extent, behaviorally. Genetic access to specific, sparsely expressed cell types is crucial for investigating the impact of single cells on computational and functional aspects within the circuitry. As such, this new and extensive collection significantly extends the range of targeted cell types related to the MB and will be an outstanding resource to elucidate MB-related processes in the future.

Strengths:

The work by Shuai et al. provides novel and essential resources to study MB-related processes and beyond. The resulting tools are publicly available and, together with the linked information, will be foundational for many future studies. The importance and impact of this tool development approach, along with previous ones, for the field cannot be overstated. One of many interesting aspects arises from the anatomical analysis of cell types that are less stereotypical across flies. These discoveries might open new avenues for future investigations into how such asymmetry and individuality arise from development and other factors, and how it impacts the computations performed by the circuitry that contains these elements.

Weaknesses:

Providing such an array of tools leaves little to complain about. However, despite the comprehensive genetic access to diverse sensory pathways and MB-connected cell types, the manuscript could be improved by discussing its limitations. For example, the projection neurons from the visual system seem to be underrepresented in the tools produced (or almost absent). A discussion of these omissions could help prevent misunderstandings.

We internally distributed efforts to produce split-GAL4 lines at Janelia Research Campus. The recent preprint (Nern et al., 2024; doi: https://doi.org/10.1101/2024.04.16.589741) described the full collection of split-GAL4 driver lines in the optic lobe including the visual projection neurons to the mushroom body. We cited this preprint in the revised manuscript by adding a short paragraph of discussion.

“Although less abundant than the olfactory input, the MB also receives visual information from the visual projection neurons (VPNs) that originate in the medulla and lobula and are targeted to the accessory calyx (Vogt et al. 2016; Li et al. 2020). A recent preprint described the full collection of split-GAL4 driver lines in the optic lobe, which includes the VPNs to the MB (Nern et al. 2024).”

Additionally, more details on the screening process, particularly the selection of candidate split halves and stable split-GAL4 lines, would provide valuable insights into the methodology and the collection's completeness.

The details of our split-GAL4 design and screening procedures were described in previous studies (Aso et al., 2014; Dolan et al., 2019). Available data and tools to design split-GAL4 changed over time, and we took different approaches accordingly. Many of split-GAL4 lines presented in this study were designed and screened in parallel to the lines for MBONs and DANs in 2010-2014 when MCFO images of GAL4 drivers and EM connectome were not yet available. With knowledge of where MBONs and DANs project, I (Y.A.) manually examined and annotated thousands of confocal stacks (Jenett et al., 2012; https://doi.org/10.1016/j.celrep.2012.09.011) to find candidate cell types that may concat with them.

Later I used more advanced computational tools (Otsuna et al., 2018; doi: https://doi.org/10.1101/318006) and MCFO images aligned to the standard brain volume (Meissner et al., 2023; DOI: 10.7554/eLife.80660.). Now, if one needs to further generate split-GAL4 lines for cell type identified in EM connectome data, neuron bridge website (https://neuronbridge.janelia.org/) can be very helpful to provide a list of GAL4 drivers that may label the neuron of interest.

Reviewer #3 (Public Review):

Summary:

Previous research on the Drosophila mushroom body (MB) has made this structure the best-understood example of an associative memory center in the animal kingdom. This is in no small part due to the generation of cell-type specific driver lines that have allowed consistent and reproducible genetic access to many of the MB's component neurons. The manuscript by Shuai et al. now vastly extends the number of driver lines available to researchers interested in studying learning and memory circuits in the fly. It is an 800-plus collection of new cell-type specific drivers target neurons that either provide input (direct or indirect) to MB neurons or that receive output from them. Many of the new drivers target neurons in sensory pathways that convey conditioned and unconditioned stimuli to the MB. Most drivers are exquisitely selective, and researchers will benefit from the fact that whenever possible, the authors have identified the targeted cell types within the Drosophila connectome. Driver expression patterns are beautifully documented and are publicly available through the Janelia Research Campus's Flylight database where full imaging results can be accessed. Overall, the manuscript significantly augments the number of cell type-specific driver lines available to the Drosophila research community for investigating the cellular mechanisms underlying learning and memory in the fly. Many of the lines will also be useful in dissecting the function of the neural circuits that mediate sensorimotor circuits.

Strengths:

The manuscript represents a huge amount of careful work and leverages numerous important developments from the last several years. These include the thousands of recently generated split-Gal4 lines at Janelia and the computational tools for pairing them to make exquisitely specific targeting reagents. In addition, the manuscript takes full advantage of the recently released Drosophila connectomes. Driver expression patterns are beautifully illustrated side-by-side with corresponding skeletonized neurons reconstructed by EM. A comprehensive table of the new lines, their split-Gal4 components, their neuronal targets, and other valuable information will make this collection eminently useful to end-users. In addition to the anatomical characterization, the manuscript also illustrates the functional utility of the new lines in optogenetic experiments. In one example, the authors identify a specific subset of sugar reward neurons that robustly promotes associative learning.

Weaknesses:

While the manuscript succeeds in making a mass of descriptive detail quite accessible to the reader, the way the collection is initially described - and the new lines categorized - in the text is sometimes confusing. Most of the details can be found elsewhere, but it would be useful to know how many of the lines are being presented for the first time and have not been previously introduced in other publications/contexts.

We revised the text as below.

“Among the 828 lines, a subset of 355 lines, collectively labeling at least 319 different cell types, exhibit highly specific and non-redundant expression patterns are likely to be particularly valuable for behavioral experiments. Detailed information, including genotype, expression specificity, matched EM cell type(s), and recommended driver for each cell type, can be found in Supplementary File 1. A small subset of 40 lines from this collection have been previously used in studies (Aso et al., 2023; Dolan et al., 2019; Gao et al., 2019; Scaplen et al., 2021; Schretter et al., 2020; Takagi et al., 2017; Xie et al., 2021; Yamada et al., 2023). All transgenic lines newly generated in this study are listed in Supplementary File 2 (Aso et al., 2023; Dolan et al., 2019; Gao et al., 2019; Scaplen et al., 2021; Schretter et al., 2020; Takagi et al., 2017; Xie et al., 2021; Yamada et al., 2023).”

And where can the lines be found at Flylight? Are they listed as one collection or as many?

They are listed as one collection - “Aso 2021” release. It is named “2021” because we released the images and started sharing lines in December of 2021 without a descriptive paper. We added a sentence in the Methods section.

“All splitGAL4 lines can be found at flylight database under “Aso 2021” release, and fly strains can be requested from Janelia or the Bloomington stock center.”

Also, the authors say that some of the lines were included in the collection despite not necessarily targeting the intended type of neuron (presumably one that is involved in learning and memory). What percentage of the collection falls into this category?

We do not have a good record of split-GAL4 screening to calculate the chance to intersect unintended cell types, but it was rather rare. Those unintended cell types can still be a part of circuits for associative learning (e.g. olfactory projection neurons) or totally unrelated cell types. For instance, among a new collection of split-LexA lines using Gr43a-LexADBD hemidriver (Figure 7-figure supplement 2), one line specifically intersected T1 neurons in the optic lobe despite that the AD line was selected to intersect sugar sensory neurons. We suspect that this is due to ectopic expression of Gr43a-LexADBD. Nonetheless, we included it in the paper because cell-type-specific Split-LexA driver for T1 will be useful irrespective of whether the expression of Gr43a gene is expressed in T1 or not.

And what about the lines that the authors say they included in the collection despite a lack of specificity? How many lines does this represent?

For a short answer, there are about 100 lines in the collection that lack the specificity for behavioral experiments.

We ranked specificity of split-GAL4 drivers in the Supplementary File 1. Rank 2 are the ideal lines, Rank 1 are less ideal but acceptable, and Rank 0 is not suitable for activation screening in behavioral experiments. Out of the 828 split-GAL4 lines reported here, there are 413, 305 and 103 lines in rank2, rank1 and rank0 categories respectively. 7 lines are not ranked for specificity because only flipout expression data are available.

Recommendations for the authors:

Reviewer #2 (Recommendations For The Authors):

As mentioned elsewhere and in addition to the minor points below, it is advisable for the authors to elaborate on the details of the screening process. Furthermore, a discussion about the circuits not targeted by their research, such as the visual projection neurons, would be beneficial.

See the response above to Reviewer #2’s public review.

Line 32-33: The citations are very fly-centric. the authors might want to consider reviews on the MB of other insect species regarding learning and memory.

We additionally cited Rybak and Menzel 2017’s book chapter on honey bee mushroom body.

Line 43-44: Citations should be added, e.g. Séjourné et al. (2011), Pai et al. (2013), Plaçais et al. (2013).

Citation added

Line 50-52: Citation Hulse et al. (2021) should be added.

Citation added

Line 162: In this part, it might be valuable for the reader to understand which of these PNs are actually connecting with KCs.

A full list of cell types within the MB were provided in Supplementary File 4 of the revised manuscript. See also response to Reviewer 3, Lines 150-1.

Line 179: Citation Burke et al. (2012) should be mentioned.

Citation added

Line 181: Thermogenic might be thermogenetic.

Corrected

Line 189: Citations add Otto et al. (2020) and Felsenberg et al. (2018).

Citations added

Line 208ff: The authors should consider discussing why they did not use other GR and IR promoters. For example, Gr5a is prominent in sugar-sensing, while Ir76b could be a reinforcement signal related to yeast food (Steck et al., 2018; Ganguly et al., 2017; see also Corfas et al., 2019 for local search).

We focused on the Gr64f promoter because of its relatively broad expression and successful use of Gr64f-GAL4 for fictive reward experiment. We added the Split-LexA lines with Gr43a and Gr66a promoters (Figure 7-figure supplement 2). Other gustatory sensory neurons also have the potential to be reinforcement signals, but we just did not have the bandwidth to cover them all.

Line 319: Consider citing Linneweber et al. (2020) for a neurodevelopmental account of such individuality.

We added a sentence and cited this reference.

“On the other hand, the neurodevelopmental origin of neuronal morphology appeared to have functional significance on behavioral individuality (Linneweber et al. 2020).”

Line 352: Citation add Hulse et al. (2021).

Citations added

Line 356ff: The utility and value of Split-LexA may not be apparent to non-expert readers. Moreover, how were LexADBDs chosen for creating these lines?

We have added an introductory sentence at the beginning of the paragraph and explained that these split-LexA lines were a conversion of split-GAL4 lines that were published in 2014 and frequently used in studying the mushroom body circuit.

“Split-GAL4 lines enable cell-type-specific manipulation, but some experiments require independent manipulation of two cell types. Split-GAL4 lines can be converted into split-LexA lines by replacing the GAL4 DNA binding domain with that of LexA (Ting et al., 2011). To broaden the utility of the split-GAL4 lines that have been frequently used since the publication in 2014 (Aso et al., 2014a), we have generated over 20 LexADBD lines to test the conversions of split-GAL4 to split-LexA. The majority (22 out of 34) of the resulting split-LexA lines exhibited very similar expression patterns to their corresponding original split-GAL4 lines (Figure 12).”

Line 374: Italicize Drosophila melanogaster.

Revised as suggested.

Reviewer #3 (Recommendations For The Authors):

Major Comments:

As mentioned in the Public Review, the drivers are nicely classified in the various subsections of the manuscript, but the statements in the text summarizing how many lines there are in specific categories are often confusing. For example, line 129 refers to "drivers encompassing 111 cell types that connect with the DANs and MBONs", but Figure 1E indicates that 46 new cell types downstream of MBONs and upstream of DANs have been generated. This seems like a discrepancy.

The 46 cell types in Figure 1E consider only the CRE/SMP/SIP/SLP area, where MBON downstreams and DAN upstreams are highly enriched, while the 111 cell types include all. To avoid confusion, we removed the “MBON downstream and DAN upstream” counting in Figure 1E in the revised manuscript.

Also, at line 75 the MBON lines previously generated by Rubin and Aso (2023) are referred to as though they are separate from the 828 described "In this report." Supplementary file 1 suggests, however, that they are included as part of this report.

Twenty five lines generated in Rubin and Aso (2023) were initially included in Supplementary file 1 for the convenience of users, but they were not counted towards the 828 new lines described in this report. To avoid confusion, we removed these 25 lines in the revised manuscript. Now all lines listed in Supplementary file 1 were generated in this study (“Aso 2021” release), and if a line has been used in earlier studies, or introduced in other contexts, for example the accompanying omnibus preprint (Meissener 2024, doi: 10.1101/2024.01.09.574419), the citations are listed in the reference column.

More generally, in lines 94-102 "828 useful lines based on their specificity, intensity and non-redundancy" are referred to, but they are subsequently subdivided into categories of lines with lower specificity (i.e. with off-target expression) and lines that did not target intended cell types (presumably ones unlikely to be involved in learning and memory). It would be useful to know how many lines (at least roughly) fall into these subcategories.

See the response above to Reviewer #3’s public review.

Finally, Figures 3B & C indicate cell types connected to DANs and MBONs and the number for which Split-Gal4 lines are available. The text (lines 136-7) states that the new collection covers 30 of these major cell types (Figure 3C)," but Figure 3C clearly has more than 30 dots showing the drivers available. Presumably existing and new driver lines are being pooled, but this should either be explained or the two should be distinguished.

“(Figure 3C)” was replaced with “(Supplementaryl File 3)” in the revised manuscript to correct the reference. Figure 3B & C are plots of all MB interneurons, not just the major cell types.

Minor Comments:

Although the paper is generally well written there are minor grammatical errors throughout (e.g. dropped articles, odd constructions, etc.) that somewhat detract from an otherwise smooth and enjoyable reading experience. A quick editing pass by a native speaker (i.e. any of several of the authors) could clean up these and numerous other small mistakes. A few examples: line 138 "presented" should be present; line 204: "contain off-targeted expressions" should be "have off-target expression;" line 219: "usage to substitute reward" is awkward at best and could be something like "use in generating fictive rewards"; line 326 "arborize[s]"; l. 331 "Based on the likelihood" should be something like "based on these observations"'; line 349 "[is] likely to appear"; l. 352 "extensive connection[s]"; line 353 "has [a] strong influence;" l. 963 "Projections" should be singular; etc.

All the mentioned examples have been corrected, and we have asked a native speaker to edit through the revised manuscript.

Lines 81-3: Is the lookup table referred to Suppl. File 1? A reference is desirable.

Yes, the lookup table referred to “Supplementary File 1” and a reference was added.

Lines 111-2: what is a "non-redundant set of...cell types?" Cell types that are represented by a single cell (or bilateral pair)? Or does this sentence mean that of the 828 lines, 355 are specific to a single cell type, and in total 319 cell types are targeted? The statement is confusing.

We revised the text as below.

“Figure 1E provides an overview of the categories of covered cell types. Among the 828 lines, a subset of 355 lines, collectively labeling at least 319 different cell types, exhibit highly specific and non-redundant expression patterns are likely to be particularly valuable for behavioral experiments. Detailed information, including genotype, expression specificity, matched EM cell type(s), and recommended driver for each cell type, can be found in Supplementary File 1. A small subset of 40 lines from this collection have been previously used in studies (Aso et al.,

2023; Dolan et al., 2019; Gao et al., 2019; Scaplen et al., 2021; Schretter et al., 2020; Takagi et al., 2017; Xie et al., 2021; Yamada et al., 2023). All transgenic lines newly generated in this study are listed in Supplementary File 2 (Aso et al., 2023; Dolan et al., 2019; Gao et al., 2019; Scaplen et al., 2021; Schretter et al., 2020; Takagi et al., 2017; Xie et al., 2021; Yamada et al., 2023).”

Line 148: "MB major interneurons" is a confusing descriptor for postsynaptic partners of MBONs.

We added a sentence to clarify the definition of the “MB major interneurons”.

“In the hemibrain EM connectome, there are about 400 interneuron cell types that have over 100 total synaptic inputs from MBONs and/or synaptic outputs to DANs. Our newly developed collection of split-GAL4 drivers covers 30 types of these ‘major interneurons’ of the MB (Supplementary File 3).”

Lines 150-1: Not sure what is meant by "have innervations within the MB." Sounds like cells are presynaptic to KCs, DANS, and MBONs, but Figure 3 Figure Supplement 1 indicates they include neurons that both provide and receive innervation to/from MB neurons. Please clarify.

For clarification, in the revised manuscript we have included a full list of cell types within the MB in Supplementary File 4. Included are all neurons with >= 50 pre-synaptic connections or with >=250 post-synaptic connections in the MB roi in the hemibrain (excluding the accessory calyx). The cell types include KCs, MBONs, DANs, PNs, and a few other cell types. The coverage ratio was updated based on this list.

Also, in line 152, what does it mean that they "may have been overlooked previously?" this seems unnecessarily ambiguous. Were they overlooked or weren't they?

Changed the text to “These lines offer valuable tools to study cell types that previously are not genetically accessible. Notably, SS85572 enables the functional study of LHMB1, which forms a rare direct pathway from the calyx and the lateral horn (LH) to the MB lobes (Bates et al., 2020). ”

Line 158 refers to PN cells within the MB, which are not mentioned in any place else as MB components.

What are these PNs and how do they differ from MBONs?

See responses to Lines 150-1 for clarification of cell types within the MB.

Line 188: not clear what is meant by "more continual learning tasks".

We rephrase it as “more complex learning tasks” to avoid jargon.

Line 235: Not clear why "extended training with high LED intensity" wouldn't promote the formation of robust memories. Is this for some reason unexpected based on previous experiments? Please explain.

See responses to weakness #1 of the same reviewer

Lines 317-9: It would be useful to state here that MB0N08 and MB0N09 are the two neurons labeled by MB083C.

Revised as suggested.

Line 368: Presumably the "lookup table" referred to is Supplementary File 1, but a reference here would be useful.

Yes, Supplementary File 1 and a reference was added.

Comments on Figures:

Figure 1C The "Dopamine Neurons" label position doesn't align with the Punishment and Reward labels, which is a bit confusing.

They are intentionally not aligned, because dopamine neurons are not reward/punishment per se. We intend to use the schematic to show that the punishment and reward are conveyed to the MB through the dopamine neuron layer, just as the output from the MB output neuron layer is used to guide further integration and actions. To keep the labels of “Dopamine neurons” and “MB Output Neurons” in a symmetrical position, we decide to keep the original figure unchanged. But we thank the reviewer for the kind suggestion.

Figure 1F and Figure 1 - Figure Supplement 1: the light gray labels presumably indicate the (EM-identified) neuron labeled by each line, but this should be explicitly stated in the figure legends. It would also be useful in the legends to direct the reader to the key (Supplementary File 1) for decoding neuronal identities.

Revised as suggested.

Figure 2: For clarity, I'd recommend titling this figure "LM-EM Match of the CRE011-specific driver SS45245". This reduces the confusion of mixing and matching the driver and cell-type names. Also, it would be helpful to indicate (e.g. with labels above the figure parts) that A & B represent the MCFO characterization step and C & D represent the LM-EM matching step of the pipeline. Revised as suggested.

Figure 6: For clarity, it would be useful to separately label the PN and sensory neuron groups. Also, for the sensory neurons at the bottom, what is the distinction between the cell names in gray and black font?

Figure 6 was updated to separate the non-olfactory PN and sensory neuron groups. The gray was intended for olfactory receptor neuron cell types that are additionally labeled in the driver lines. To avoid confusion, the gray cell types were removed in the revised figure, and a clarification sentence was added to the legend.

“Other than thermo-/hygro-sensory receptor neurons (TRNs and HRNs), SS00560 and MB408B also label olfactory receptor neurons (ORNs): ORN_VL2p and ORN_VC5 for SS00560, ORN_VL1 and ORN_VC5 for MB408B.”

Figure 7A: It's unclear why the creation of 6 Gr64f-LexADBD lines is reported. Aren't all these lines the same? If not, an explanation would be useful.

These six Gr64f-LexADBD lines are with different insertion sites, and with the presence or absence of the p10 translational enhancer. Explanation was added to legend. Enhanced expression level with p10 can be helpful to compensate for the general tendency that split-LexA is weaker than split-GAL4. Different insertions will be useful to avoid transvections with split-GAL4s, which are mostly in attP40 and attP2.

Figure 8F: It would help to include in the legend a brief description of each parameter being measured-essentially defining the y-axis label on the graphs as in Figure Supplement 2. Also, how is the probability of return calculated and what behavioral parameter does the change of curvature refer to?

We added a brief description to the behavioral parameters in the legend of Figure 8F.

“Return behavior was assessed within a 15-second time window. The probability of return (P return) is the percentage of flies that made an excursion (>10 mm) and then returned to within 3 mm of their initial position. Curvature is the ratio of angular velocity to walking speed.”

Figure 9E: What are the parenthetical labels for lines SS49267, SS49300, and SS35008?

They are EM bodyIDs. Figure legend was revised.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This is an interesting and valuable study that uses multiple approaches to understand the role of bursting involving voltage-gated calcium channels within the mediodorsal thalamus in the sedative-hypnotic effects of alcohol. Given its unique functional roles and connectivity pattern, the idea that the mediodorsal thalamus may have a fundamental role in regulating alcohol-induced transitions in consciousness state would be both important for researchers investigating thalamocortical dynamics and more broadly interesting for understanding brain function. In addition, the author's examination of the role of the voltage-gated calcium channel Cav3.1 provides some evidence that burst-firing mediated by this channel in the thalamus is functionally important for behavioral-state transitions. While many previous studies have suggested an analogous role for sleep-state regulation, the evidence for an analogous role of this type of bursting in sedative-induced transitions is more limited. Despite the importance of these results, however, there is some concern that the manipulations and recording approaches employed by the authors may affect other thalamic nuclei adjacent to the MD, such as the central lateral nucleus, which has also been implicated in controlling state transitions. The evidence for a specific role of the mediodorsal thalamus is therefore somewhat incomplete, and so additional validation is needed.

Strengths:

This study employs multiple, complementary research approaches including behavioral assays, sh-RNAbased localized knockdown, single-unit recordings, and patterned optogenetic interventions to examine the role of activity in the mediodorsal thalamus in the sedative-hypnotic effects of alcohol. Experiments and analyses included in the manuscript generally appear well conceived and are also generally well executed. Sample sizes are sufficiently large and statistical analysis appears generally appropriate though in some cases additional quantification would be helpful. The findings presented are novel and provide some interesting insight into the role of the thalamus as well as voltage-gated calcium channels within this region in controlling behavioral state transitions induced by alcohol. In particular, the observed effects of selective knockout along with recordings in total knockout of the voltage-gated calcium channel, Cav3.1, which has previously been implicated in bursting dynamics as well as state transitions, particularly in sleep, together suggest that the transition of thalamic neurons to a bursting pattern of firing from a more constant firing is important for transition to the sedated state produced by ethanol intoxication. While previous studies have similarly implicated Cav3.1 bursting in behavioral state transitions, the direct optogenetic interventions and single-unit recordings provide valuable new insight. These findings may also have interesting implications for the relationship between sleep process disruption associated with ethanol dependence, although the authors do not appear to examine this directly or extensively discuss these implications of their findings.

Weaknesses:

A key claim of the study is that the mediodorsal thalamus is specifically important for the sedative-hypnotic effect of ethanol and that a transition to a bursting pattern of firing in this circuit facilitates these effects due to a loss of a more constant tonic firing pattern. Despite the generally clear observed effects across the included experiments, however, the evidence presented does not fully support that the mediodorsal thalamus, in particular, is involved. This distinction is important because some previous studies have suggested that another thalamic nucleus which is very close to the mediodorsal thalamus, the central-lateral thalamus, has previously been suggested to play a role in preventing sedative-induced transitions. Despite its proximity to the mediodorsal thalamus, the central-lateral thalamus has a substantially different pattern of connectivity so distinguishing which region is impacted is important for understanding the findings in the manuscript. While sh- RNA knockdown appears to be largely centered in the mediodorsal thalamus in the example shown, (Figure 2) this is rather minimal evidence and it is also not well explained (indeed, the relevant panels do not even appear to be referenced in the text of the manuscript) and the consistency of the knockdown targeting is not quantified. Additional evidence should be provided to validate this approach. Similarly, while an example is shown for the expression of ChR2 (Fig. 5) there seems to be some spread of expression outside of the mediodorsal thalamus even in his example raising a concern about how regionally specific this effect.

The recordings targeting the mediodorsal thalamus could provide evidence of a direct association between changes in activity specifically in this part of the thalamus with the behavioral measures but there are currently some issues with making this link. One difficulty is that, although lesions are shown in Figure S5 to validate recording locations, this figure is relatively unclear and the examples appear to be taken from a different anterior/posterior location compared to the reference diagram. A larger image and improved visualization of the overall set of lesion locations that includes multiple anterior/posterior coronal sections would be helpful. Moreover, even for these example images, it is difficult to evaluate whether these are in the mediodorsal thalamus, particularly given the small size of the image shown. Ideally, an example image that is more obviously in the mediodorsal thalamus would also be included. Finally, an assessment of the relationship between the approximate locations of recorded neurons across the tetrode arrays and the behavioral measures would be very helpful in supporting the unique role of the mediodorsal thalamus. The lack of these direct links, in combination with the histological issues, reduces the insight that can be gained from this study.

In addition to the key experimental issues mentioned above, there are often problems in the text of the manuscript with reasoning or at least explanation as well as numerous minor issues with editing. The most substantial such issue is the lack of clarity in discussing the mediodorsal thalamus and other adjacent thalamic nuclei, such as the central-lateral nucleus, in the author's discussion of previous findings. Given that at last one of the manuscripts cited by the authors (Saalman, Front. Sys. Neuro. 2014) has directly claimed that central-lateral, rather than the mediodorsal, thalamus is important for arousal regulation related to a conscious state, this distinction should be addressed clearly in the discussion rather than papered over by grouping multiple thalamic nuclei as being medial. As part of this discussion, it would be important to consider additional relevant literature including Bastos et al., eLife, 2021 and Redinbaugh et al., Neuron, 2020 which are quite critical but currently do not appear to be cited. Considering additional literature relevant to the function of the mediodorsal thalamus would also be beneficial. While the methods employed generally seem sound, the description in the methods section is lacking in detail and is often difficult to follow. Analysis methods such as the burst index appear to only be given a brief explanation in the text and appear not to be mentioned in the methods section. Similarly, the staining method used in Figure 2 does not appear to be described in the methods section. The most substantial case is for the UMAP approach used in Figure 4-E which does not appear to be described in the methods or even described in the main text. The lack of detailed descriptions makes it difficult to evaluate the applicability and quality of the experimental and analytical approaches. Citations justifying the use of methods such as the approach to separate regular spiking and narrow spiking neuron subtypes are also needed.

Beyond the problems with content and reasoning discussed above, there are also some relatively minor issues with the clarity of writing throughout the paper (for example, in the abstract the authors refer to "the ethanol resistance behavior in WT mice" but it is difficult to parse what they mean by this statement. Similarly, the next sentence "These results support that the maintenance..." while clearer, is not well phrased. Though individually minor, issues like this re-occur throughout the manuscript and sometimes make it difficult to follow so the text should be revised to correct them. There are also some problems with labels such as the labels of A1/A2 in Figure 4, which appear to be incorrect. Also, S7 has no label] on the B panels. Finally, some references are not included (only a label of [ref]).

Reviewer #2 (Public Review):

In the current study, Latchoumane and collaborators focus on the Cav3.1 calcium channels in the mediodorsal thalamic nucleus as critical players in the regulation of brain-states and ethanol resistance in mice. By combining behavioural, electrophysiological, and genetic techniques, they report three main findings. First, KO Cav3.1 mice exhibit resistance to ethanol-induced sedation and sustained tonic firing in thalamocortical units. Second, knocked-down Cav3.1 mice reproduce the same behaviour when the mediodorsal, but not the ventrobasal, thalamic nucleus is targeted. Third, either optogenetic or electric stimulation of the mediodorsal thalamus reduces ethanol-induced sedation in control animals.

Overall, the study is well designed and performed, correctly controlled for confounds, and properly analysed. Nonetheless, it is important to address some aspects of the report. The results support the conclusions of the study. These results are likely to be relevant in the field of systems neuroscience, as they increase the molecular evidence showing how the thalamus regulates brain states.

Reviewer #1 (Recommendations For The Authors):

Aside from the additional quantification and clarification of the analysis discussed in the weakness section, in general, the experiments included in the manuscript seem reasonable. However, I would suggest one additional experiment as well as one control, both of which are relatively straightforward optogenetic experiments, that I feel would be helpful to further improve the study. First, as the authors note, the optogenetic interventions used do not directly address the relevance of the changes in bursting patterns observed in the knockout (KO), which are by far the most robust effect, with the changes in alcohol sensitivity. One approach that could help address this would be to use patterned suppression via inhibitory opsins (e.g. halorhodopsin) to "rescue" the periods of inhibition associated with bursting in the KO. Localizing this inhibition to the mediodorsal thalamus would also lend further credence to their claim that this nuclei is the relevant circuit for their observed effects. For the control, tonic activation of the ventrobasal nucleus, as the authors did for the mediodorsal nucleus, would be beneficial to rule out the possibility that the observed effect would occur with any thalamic nucleus. In addition to these experiments, I did not note the strategy for sharing data obtained through this study so this should be added.

R1 – 1: A key claim of the study is that the mediodorsal thalamus is specifically important for the sedative-hypnotic effect of ethanol and that a transition to a bursting pattern of firing in this circuit facilitates these effects due to a loss of a more constant tonic firing pattern. Despite the generally clear observed effects across the included experiments, however, the evidence presented does not fully support that the mediodorsal thalamus, in particular, is involved. This distinction is important because some previous studies have suggested that another thalamic nucleus which is very close to the mediodorsal thalamus, the central-lateral thalamus, has previously been suggested to play a role in preventing sedative-induced transitions. Despite its proximity to the mediodorsal thalamus, the central-lateral thalamus has a substantially different pattern of connectivity so distinguishing which region is impacted is important for understanding the findings in the manuscript.

R1-A1: The reviewer is right that CL has been pointed as another candidate structure with causal influence on arousal and consciousness. We have focused our efforts in including only recording single units that were from tetrode located in the MD specifically using the lesion code we explain in the method section and in response to R1 question#3. We also produced a quantification of Cav3.1 knock-down that clearly demonstrates that the KD experiment was itself specific to MD, bilaterally, and that CL to CM were minimally impacted by the knock-down process (Fig. 2C and D). Moreover, the optogenetic  (fiber incidence was 30 degrees guaranteeing a central coverage rather than lateral; Fiber optic NA = 0.22) and electric stimulation (bipolar twisted electrodes, 50uA) experiments were also very selective and specific to the MD (Fig.S5). It remains clear that MD might not be the sole structure involved in the brain state control towards sedation and “anesthetic states”, and CL might be a significant contributor as well, however, we show that CL manipulations were rather irrelevant in our experiments  (Fig. 2, S5, S9 and S11).

R1-2: While sh-RNA knockdown appears to be largely centered in the mediodorsal thalamus in the example shown, (Figure 2) this is rather minimal evidence and it is also not well explained (indeed, the relevant panels do not even appear to be referenced in the text of the manuscript) and the consistency of the knockdown targeting is not quantified. Additional evidence should be provided to validate this approach.

R1-A2: In order to address this important question, we have created an additional panel quantification to fig2D. We have then quantified the intensity per area of Cav3.1 expression in sub zones of 4 regions of interest: MD (left, right; 2 subzones each), Centro Medial (CM; 1 subzones in total), Centrolateral/Paraventricular nucleus (CL/PCN; left, right; 2 subzones each) and the submedial nucleus (SMT; left, right; used as a control for the intensity normalization; 1 subzones in total). This panel clearly illustrates that MD was knocked-down bilaterally (p<0.001). Moreover, CM (p<0.05) and CL (p<0.01) were also partially and unilaterally knocked down, as well. This analysis confirms that our KD had a high specificity to MD.

We added the relevant figure caption and text:

[Result section, Cav3.1 silencing in the MD, but not VB, increased ethanol resistance in mice, paragraph 3]

“We then characterized the change in Cav3.1 expression following the shControl and shCav3.1 knockdown injections in three test regions MD (left and right), CM (centromedial nucleus) and CL (centrolateral nuclei, left and right side) and a negative control region SMT (submedial thalamic nuclei, left and right side). The average intensity was obtained from two coronal brain slices for each mice used in the experiment (see Methods sections, Cav3.1 Intensity quantification). Our results show that the targeting of the knockdown was very specific to the bilateral MD (p<0.001; Fig. 2D). We noted that the CM (p<0.05) and a marginal unilateral knock-down of the CL were also observed (p<0.01). Notably, we tested the correlation between the level of knock-down in MD and the total time in LOM and observed a significant association (Fig. 2D inset; R = 0.599, p = 0.018). This result highlights that the Cav3.1 knock-down was specific to MD and with an intensity associated with ethanol-induced loss of motion.”

R1-3: One difficulty is that, although lesions are shown in Figure S5 to validate recording locations, this figure is relatively unclear and the examples appear to be taken from a different anterior/posterior location compared to the reference diagram. A larger image and improved visualization of the overall set of lesion locations that includes multiple anterior/posterior coronal sections would be helpful. Moreover, even for these example images, it is difficult to evaluate whether these are in the mediodorsal thalamus, particularly given the small size of the image shown. Ideally, an example image that is more obviously in the mediodorsal thalamus would also be included. Finally, an assessment of the relationship between the approximate locations of recorded neurons across the tetrode arrays and the behavioral measures would be very helpful in supporting the unique role of the mediodorsal thalamus.

R1-A3: Related to fig.S5, we re-distributed the position of the recordings from the tetrode electrode burned positions over 3 representative coronal planes that best represent the implant positions. We also provided additional snapshots of tetrode location. To identify the positions of four tetrodes in each animal, we encoded the positions with different electrical lesion strategies as follows: 1 lesion(tetrode 1), 2 lesions while we redrew the tetrode with 100 um interval (tetrode 2), 3 lesions with 200um interval (tetrode 3), 4 lesions with 50um intervals (tetrode4). Tetrodes that were found outside of the MD delimited region were discarded post analysis. A straight relationship between the closeness of the electrode is unfortunately not possible for tetrode recording, a straight silicone probe which maintains the spatial spacing in recording would have been a better approach in that case, but unfortunately, it was not performed in our study.

R1-4: In addition to the key experimental issues mentioned above, there are often problems in the text of the manuscript with reasoning or at least explanation as well as numerous minor issues with editing. The most substantial such issue is the lack of clarity in discussing the mediodorsal thalamus and other adjacent thalamic nuclei, such as the central-lateral nucleus, in the author's discussion of previous findings. Given that at last one of the manuscripts cited by the authors (Saalman, Front. Sys. Neuro. 2014) has directly claimed that central-lateral, rather than the mediodorsal, thalamus is important for arousal regulation related to a conscious state, this distinction should be addressed clearly in the discussion rather than papered over by grouping multiple thalamic nuclei as being medial. As part of this discussion, it would be important to consider additional relevant literature including Bastos et al., eLife, 2021 and Redinbaugh et al., Neuron, 2020 which are quite critical but currently do not appear to be cited. Considering additional literature relevant to the function of the mediodorsal thalamus would also be beneficial.

R1-A4: We thank the reviewer for his comments and suggestions. We agree that the added references mentioned by the reviewers are highly relevant and should be integrated in the manuscript. We have integrated the above-mentioned references and further developed on the discussion on the role of MD relative to other thalamic nuclei (ILN and CL in particular). We believe that this better-referenced and clarified text does improve the manuscript greatly.

[introduction section, paragraph 3]

“The centrolateral (CL) thalamic nucleus has been implicated in the modulation of arousal, behavior arrest 31, and improvement of level of consciousness during seizures 32. Notably, the direct electrical stimulation of the intralaminar nuclei (ILN) and, in particular CL, promoted hallmarks of arousal and awakening in primate under propofol and ketamine propofol anesthesia.”

[Discussion section, paragraph 1]

“In this work, we identified that the neural activity in MD plays a causal role in the maintenance of consciousness. Whole body Cav3.1 KO and MD-specific Cav3.1 KD mice showed resistance to loss of consciousness induced by hypnotic dose of ethanol. In WT mice, MD neurons demonstrated a reduced firing rate in natural (sleep) and ethanol-induced unconscious states compared to awake states. This neural activity reduction was impaired in KO mice. In particular, transition to an unconscious state was accompanied with a switch of firing mode from tonic firing to burst firing in WT mice whereas this modeshift disappeared in KO mice. Finally, optogenetic or electric stimulations of the MD after ethanol injection were sufficient to induce a resistance to loss of motion, supporting that the level of neural firing in the MD is critical to maintain conscious state and delay unconscious state. We showed that the expression of Cav3.1 t-type calcium channels in MD is a cellular modulator associated with this effect.”

[Discussion section, MD is a modulator of consciousness, paragraph 2 and 3]

“The MD is known to innervate limbic region, basal ganglia and medial prefrontal cortex 50 and increased activity in MD might modulate the stability of cortical UP states (e.g. awaken, aroused and attentive states) and synchronization 9,26. Thus, MD might be a major hub involved in cortical state control and brain state stabilization.

Supporting the brain state stabilization theory and the ethanol resistance of Cav3.1 mutants, Choi et al.34 demonstrated that the loss of Cav3.1 T-type calcium channel reduced the bilateral coherence between PFC and MD under ketamine anesthesia and ethanol hypnosis, especially in the delta frequency bands. More importantly, under propofol anesthesia, Bastos et al.35 showed that intralaminar nucleus and MD stimulation lead to increased wake-up subscore and arousal, together with an increased in cortico-cortico and thalamo-cortical slow (delta) frequency power.

In the present study, we observed that MD KD (Fig. 2A), but not VB KD (Fig. S3) of Cav3.1 increased and is associated (Fig. 2D) with ethanol resistance in mice. We found that MD neurons in Cav3.1 mutant mice exhibited tonic firing within range of wakefulness (Fig. 3 and 4), indicative of resistance to ethanol and wake-like brain state. In addition, we found a strong association between the normalized tonic firing in MD and the arousal through brain states (i.e. walk to wake to sleep states), supporting that MD tonic firing could be interpreted both as a thalamic readout and a modulator of the brain state 11 (Fig. 3). Finally, direct optogenetic and electric MD stimulation increased resistance to loss of consciousness in WT mice (Fig.5 and Fig. S10). To our knowledge, this is the first report demonstrating the causal involvement of mediodorsal thalamic nucleus in the modulation of wakefulness and the resistance to ethanol-induced loss of consciousness in mice.”

R1-5: While the methods employed generally seem sound, the description in the methods section is lacking in detail and is often difficult to follow. Analysis methods such as the burst index appear to only be given a brief explanation in the text and appear not to be mentioned in the methods section.

R1-A5: We have added a clear definition in the supplementary method following the original work used:

[Supplementary Method section, Single Unit recording, sorting and analysis, last paragraph]

“The bursting index was derived as described in (Royer et al. 2012). Namely, the burst index was estimated from the spike auto-correlogram (1-ms bin size) by subtracting the mean value between 40 and 50 ms (baseline) from the peak measured between 0 and 10 ms. Positive burst amplitudes were normalized to the peak and negative amplitudes were normalized to the baseline to obtain indexes ranging from −1 to 1.” We also edited its mention in the text for clarity:

[Result section, Lack of Ca3.1 in MD neurons removes thalamic burst in NREM sleep, paragraph 2]

“[…] and a clear reduction in total bursting represented as bursting index (Fig. 3-B; ratio of spikes count <10 ms and >50 ms based on auto-cross-correlogram).”

R1-6: Similarly, the staining method used in Figure 2 does not appear to be described in the methods section.

R1-A6: The staining method can be found in the supplementary method of the paper. [supplementary method, Immunohistochemistry]

R1-7: The most substantial case is for the UMAP approach used in Figure 4-E which does not appear to be described in the methods or even described in the main text.

R1-A7: Regarding the method, the UMAP approach is described in the supplementary method document [Uniform Manifold Approximation and Projection (UMAP)]. We believe that only a succinct description was needed here considering the extent of the analysis. Regarding the inserts in the main text, we agree that the main text was lacking the description of these results and we have amended the main text to reflect a clear description of this result and what it entails. The following paragraph was added:

[Result section, Under ethanol, MD neurons lacking Cav3.1 show no burst and a wake state-like neural activity, second to last paragraph]

“Finally, we asked whether the firing modes and properties (tonic firing rate, burst firing rate; see supplementary methods) of single MD neurons would form distinct qualitative representation of “brain stages” using a lowered dimensional UMAP representation (Uniform Manifold Approximation and Projection42 ). We observed that for awake and active (i.e. walk), the brain state representation formed two adjacent clusters that confounded both wild and mutant neurons (Fig. 4E, left panel). The REM and NREM states, the wild type neurons formed 2 additional interconnected clusters, whereas the mutant neurons tend to overlap with the clusters attributed to the “awake” brain state (Fig. 4E, second to left panel). Ethanol induced fLOM, similarly to REM and NREM clusters, was distinct from awake clusters in wild type mice and overlapped with the NREM clusters (Fig. 4E, third to left panel). Here also, mutant MD neurons showed overlap with the awake clusters rather than the “low consciousness” brain states. These results indicate that the firing mode and properties could define a brain state representation that shows distinctions in levels of consciousness. Moreover, the mutant showed a representation of “low consciousness” states overlapping with wild type “awake” states consistent with the hypothesis of resistance to loss of consciousness.”

R1-8: Citations justifying the use of methods such as the approach to separate regular spiking and narrow spiking neuron subtypes are also needed.

R1-A8: We have added two references related to the observation of the two subpopulations of spiking neurons [Schiff and Reyes, 2012; Destexhe, 2008].

R1-9: Beyond the problems with content and reasoning discussed above, there are also some relatively minor issues with the clarity of writing throughout the paper (for example, in the abstract the authors refer to "the ethanol resistance behavior in WT mice" but it is difficult to parse what they mean by this statement.

R1-A9: We addressed this issue by editing and revising the manuscript for clarity and flow.

R1-10: Similarly, the next sentence "These results support the maintenance..." while clearer, is not well phrased. Though individually minor, issues like this re-occur throughout the manuscript and sometimes make it difficult to follow so the text should be revised to correct them.

R1-A10: We thank the reviewer for highlighting this point. We have edited the overall text to improve clarity and flow.

[abstract] 

These results suggest that maintaining MD neural firing at a wakeful level is sufficient to induce resistance to ethanol-induced hypnosis in WT mice.

R1-11: There are also some problems with labels such as the labels of A1/A2 in Figure 4, which appear to be incorrect.

R1-A11: We noted this issue and have rectified the figure for clarity.

R1-12: Also, S7 has no label on the B panels.

R1-A12: We thank the reviewer for pointing out this lack. We have added the y-label on the panel for clarity.

R1-13: Finally, some references are not included (only a label of [ref]).

R1-A13: We have completed the missing reference and thank the reviewer for pointing that out.

Additional comments

R1-14: Aside from the additional quantification and clarification of the analysis discussed in the weakness section, in general, the experiments included in the manuscript seem reasonable. However, I would suggest one additional experiment as well as one control, both of which are relatively straightforward optogenetic experiments, that I feel would be helpful to further improve the study. First, as the authors note, the optogenetic interventions used do not directly address the relevance of the changes in bursting patterns observed in the knockout (KO), which are by far the most robust effect, with the changes in alcohol sensitivity. One approach that could help address this would be to use patterned suppression via inhibitory opsins (e.g. halorhodopsin) to "rescue" the periods of inhibition associated with bursting in the KO.

R1-A14: Here the reviewer proposes an interesting experiment which we have attempted to perform, however, poses several technical challenges. First, the KO do not have burst firing as they are depleted from Cav3.1 low-threshold calcium channel. Therefore, under ethanol, even if there might exist a rhythmic inhibition that activates Cav3.1 channels and causes a rebound burst, the KO are unable to have it. Therefore, an optogenetic inhibition would only accentuate the total inhibition and could potentially induce an overall decrease in MD firing, resulting in an increase in LOM features. Alternatively, we showed that in a WT with low ethanol dose (where LOM induction is harder), the increased rhythmic inhibition does indeed increase significantly LOM duration and marginally decreases latency to LOM (Fig. S12), indicating that increased inhibition could indeed explain the hypothesis: “ the stronger the decrease in MD firing, the faster and longer the LOM.” The only caveat of using WT here is that optogenetic inhibition might also include rebound burst post-inhibition. Injecting bursts only did not alter the response to ethanol (Fig. S10). These results point to the role of loss of firing in MD as a main factor for LOM, and potentially the contribution of burst necessitating a concurrent inhibition/loss of firing.

We agree that inhibition in KO would further validate this hypothesis, controlling for the role of burst. We regret that we are not in the capacity to perform additional experiments involving the KO mice.

R1-15: For the control, tonic activation of the ventrobasal nucleus, as the authors did for the mediodorsal nucleus, would be beneficial to rule out the possibility that the observed effect would occur with any thalamic nucleus.

R1-A15: We agree with the reviewer that we could have added an additional region control to the gain/loss of function experiments. We would even go further as to suggest that a better control nucleus would be a high order nucleus such as PO or an unrelated sensory relay nucleus such as LGN. VB being a motor relay nucleus, could also mediate movement initiation, which could be hard to interpret. Since the complete control study for all thalamic nuclei Cav3.1 KD is outside the scope of this study, we opted not to redo these experiments and keep the focus of the manuscript on the manipulation of MD activity rather than the various available thalamic nuclei. We also do not claim that MD is the sole center able to initiate a switch in the loss of consciousness, and a more in-depth study on that matter would be clearly needed.

R1-16: In addition to these experiments, I did not note the strategy for sharing data obtained through this study so this should be added.

R1-A16: We have uploaded data and code for most figures at the following repository and provided a clearer statement regarding data sharing. We thank the reviewer for pointing out this missing element.

The link for the repository is the following:

It contains:

- Excel spreadsheet file of all behavior values, including the newly quantified Cv3.1 expression in MD/CL/SMT

- Excel spreadsheet follow-up of all MD cells (single unit; tetrode) analyzed

- Folders for all groups studied with representative figures showing EEG power over time and normalized activity (WT vs KO for 2, 3 and 4 g/kg; MDshKD vs shCTR, VBshKD vs shCTR; CHR2 NOSTIM vs STIM; ESTIM Groups and ARCH NOSTIM vs STIM)

- A1G LORRvsLOM and OPEN FIELD Matlab data

- Matlab and ImageJ Codes: single unit analysis, characterization, brain state characterization, sleep stages, LOM, open field analysis and statistical analysis.

We have added the data sharing subsection in the acknowledgements:

“Part of the analyzed data and codes are available on the open access platform, mendeley:

Latchoumane, Charles-francois (2024), “Mediodorsal thalamic nucleus mediates resistance to ethanol through Cav3.1 T-type Ca2+ regulation of neural activity”, Mendeley Data, V1, doi: 10.17632/7fr427426m.1

Additional data (large size recording and images) can be provided upon reasonable requests.”

Reviewer #2 (Recommendations For The Authors):

R2-1. Consciousness is a contentious subject. Even in humans, there is still intense research on the topic, not to mention animals, about which we still know very little. Moreover, consciousness is not quantified in this study, as there is no standard metric to do so. Accordingly, talking about 'modulation', 'transition', ́level ', or 'reduction' of consciousness can be misleading. Hence, it is probably safer to strictly refer to brain-states and/or stages of the sleep-wake cycle in this study and reframe it entirely around these concepts.

R2-A1. The reviewer points to an important point and we appreciate this highlight. Agreeing that the definition of consciousness is rather loose and arguably difficult to pinpoint. Here, we settle on a definition that relies on the loss of motion and loss of righting reflex. This definition is widely accepted as the “verified” state in which the absence of responsiveness (to continuous stimuli, inducing reflex or discomfort) is observed and uninterrupted by jerks and spurious movements. Additional metrics needed would be the recording of EMG to quantify atonia and EEG to the settling of a dominantly slow-wave frequency (~4 Hz; ethanol-induced sedation at theta rhythm), as shown in Fig S1A. The driver of this 4Hz frequency and its correlation has been investigated previously (e.g. Choi et al, PNAS, 2012), leading to the accepted link between LOM/LORR and loss of consciousness. Our data present the advantage of showing single neuron recordings and that LOM is a state where the lowest firing activity is present (Fig S7AB) and comparable to deep sleep state activity (Fig3D). The first LOM is the most important as it highlights the deepest loss of consciousness before the ethanol starts to be metabolized and cleared, which would be consistent between animals.

As a result, we have edited the manuscript to clarify all mentions related to brain states and states of unconsciousness.

R2-2. It is not clear why the authors focus on the mediodorsal nucleus. This should be better explained in the introduction and developed in the discussion.

R2-A2. This comment converges with the Reviewer 1 comments and we are addressing this lack in the discussion as suggested. We have addressed it with this previous comment and believe it is now clearer.

R2-3. The discussion mentions that 'increased activity in MD might modulate the stability of cortical UP state and synchronization' (pg 21). This point should be either further developed and put into context, or removed. In its current state, it does not seem to contribute much to the discussion of results.

R2-A3. We understand that the working “UP state” might not be clear enough. We have modified this sentences as follows to clarify that UP state could be either a state of where the animal is awake, aroused or attentive:

[Discussion section, MD is a modulator of consciousness, first paragraph]

“The MD is known to innervate limbic region, basal ganglia and medial prefrontal cortex 50 and increased activity in MD might modulate the stability of cortical UP states (e.g. awaken, aroused and attentive states) and synchronization 9,26. Thus, MD might be a major hub involved in cortical state control and brain state stabilization.“

R2-4. The discussion states that 'mutant mice did not exhibit a decreased arousal level (i.e. increased locomotor activity)' (pg 23). This is confusing as decreased arousal should be reflected in decreased locomotor activity.

R2-A4. We understand that the formulation of this sentence may be confusing and we have edited this portion of the text to improve quality in the revised version of the manuscript. To clarify, mutant mice do not exhibit reduced or increased arousal (not quantified, just observational), they do have a phenotypic hyperlocomotion. This comes in contrast with a lower basal firing rate in the MD, which in our interpretation, is not synonymous with lower arousal. We believe that the relative change in MD determines the change in arousal, and that the absolute firing is not indicative of arousal in itself, only in comparison.

[Discussion section, The lower variability in MD Firing reflects Ethanol Resistance in Cav3.1 mutant mice, paragraph 2]

“Mutant RS neurons in MD showed an overall lower excitability and variability of firing in various natural conscious and unconscious states compared to wild type mice. Remarkably, Cav3.1 mutant mice exhibited a clear increased locomotor activity and an increased resistance to ethanol. The general lower firing rate and the high “arousal” observed in mutant mice suggests that the relative change from state to state in tonic firing in MD, and not the absolute value of firing, might be a better correlate of change in brain state in the mice.”

R2-5. The methods (pg 27) state that two genetic backgrounds (129/svjae and C57BL/6J ) were used in the study. Authors should show whether there were significant differences between those backgrounds in the key parameters assessed in the study (particularly resistance to ethanol sedation).

R2-A5. As mentioned in the method section, we only used the F1-background mice, which are the firstgeneration offspring produced by crossing 129/svjae and C57BL/6J strains. To produce F1 KO mice, we kept the heterozygote mice in two strains. We unfortunately did not study the particular difference of the respective KO of these two backgrounds; however, the pure C57BL/6J KO has been used in other studies by our group (Kim et al 2001; Na et al, 2008; Park et al., 2010). The F1 background allows us to work with mice that are less aggressive and can be handled with less inherent stress.

R2-6. It would be convenient to produce a supplementary figure associated with Figure 1C to show the same data with averages per mouse. That is, 9 points for control and 9 points for KO mice. This also applies to all cases where data is not presented per mouse but pooled between animals.

R2-A6. We have added a panel C in Figure S1, to show the scatter values for all the mice corresponding to the figure 1C. We have also generalized this presentation for all behavior graphics showing all the animals in the scatter plot next to the boxplot. We believe that this presentation increases further the transparency of the manuscript. We have then added the scatter plot for all mice in figure Fig1, Fig2, Fig5, Fig.S2, Fig.S3, Fig.S10 and Fig.S12.

R2-7. It would be informative to make a supplementary figure associated with Figure 1D to compare baseline raw activity levels (i.e., baseline walking recording) between control and KO mice. That is, do KO and control mice cover comparable distances and at similar speeds during baseline conditions? Figure 1D and Figure 4A suggest that the variability of locomotor activity is larger in KO mice. Hence, this parameter should be quantified and reported.

R2-A7. We thank the reviewer for this comment. We strived to answer to this question in the manuscript in two ways:

- We first measure the overall hyperlocomotion of the mice using the open field total distance parkoured in our mice cohorts (FigS4C). We did observe that the KO mutant showed hyperlocomotion, but not MD or VB knock-down mice. Which indicates that the hyperlocomotion component is not specific to the two thalamic nuclei studied.

- Using the forced walking task, we impose on the animal to keep a steady pace of roughly 6cm/s. This assay allows to normalize the general walking behavior to a relatively fixed pace making it comparable for all animals.

The reviewer suggested reporting the mean and variance in walking of WT and KO during baseline (prior to the ethanol I.P. injection). We believe that the two points mentioned above are sufficient to describe in a more quantitative way the WT vs KO locomotion differences. Moreover, by construction the normalized locomotion on the forced walking task will return similar means for the baseline, the standard deviation would, however, potentially show differences but would remain inconclusive.

R2-8. The legend in Figure 1 states that 'the loss of consciousness is evaluated using normalized moving index using either video analysis (differential pixel motion), on- head accelerometer-based motion, or neck electromyograms'. Authors should clarify whether these methods are equivalent and support it with data.

R2-A8. We understand the reviewer point and we have made a few modifications to the method description aligning better with what was done. For most mice, video analysis was used to obtain the moving index. When video recording was not available (2 mice), we had an accelerometer attached to the animal’s head stage which helped us derive a moving index that was similar to the video moving index. The neck electromyogram was rather used for animals implanted with the tetrodes to identify sleep stages based on local field potential frequency and muscle tone.  We have then clarified the method for this matter and Figure 1 to avoid this confusion. Since no concurrent recording of both video and accelerometer was performed, we do not have the data to compute the correlation between the two measures, however, no noticeable deviation from loss of motion was observed between the two methods. We realize that this may be a weak argument, however, our observations showed that video and accelerometers returned very similar timings for loss of motion (only a few comparative instances insufficient to present a statistical comparison).

R2-9. How were spike bursts defined? The authors should try different criteria and verify the consistency of results.

R2-A9 For in vivo single unit recording, we opted for a definition that is validated from our works and others as a silencing of at least 100 ms followed by a minimum of 3 spikes with:

- First spike pairs interspike interval less than 4 ms

- Remaining spike pairs interspike interval less than 20 ms

We have performed this analysis using a minimum of 2 spikes, and varied silencing periods between 50 and 100ms, without observing significant deviation of the results. As shown in Figure S6B, with this approach we observed that the burst distribution had a majority with <10 spikes per burst. Figure S6C indicated that with a clear distribution of ISI for first spike within 2-4ms as observed in previous works (Desai and Varela, 2021; Alitto et al, 2019), importantly, not clearly capped at 4 ms, showing that the range for the first ISI might indeed be lower than 4ms for thalamic burst. Within burst spike waveforms can become very variable and the choice of 3 over 2 spikes minimum per burst stems from the aim to reduce false positive detection of ultra-short bursts, which in single unit recording remains controversial (Gray et al. 1995).

Minor:

R2-10: Figure 4A2 'Cav3.1(+/+)' should presumably be Cav3.1(-/-).

R2-A10: this is correct and we have corrected the figure label [This sentence is ambiguous. What is ‘this’ that is correct?]

R2-11: Figure S2C legend states 'Post-hoc group comparison was performed using.' The sentence seems to be incomplete.

R2-A11: We have completed the sentence for clarity.

R2-12: In the methods (pg 29) virus concentration is reported as '107 TU/ul', which probably refers to 10e7.

R2-A12: We have corrected it by superscripting the power 7.

R2-13: Verify Fig 1C1 and correct Y-axis overlap between title and units.

R2-A13: We edited the figure for clarity, thank you.

R2-14: On page 24 there is a '[ref]' that probably stands for (a missing) reference.

R2-A14: the missing reference has been added.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The authors present a new application of the high-content image-based morphological profiling Cell Painting (CP) to single cell type classification in mixed heterogeneous induced pluripotent stem cellderived mixed neural cultures. Machine learning models were trained to classify single cell types according to either "engineered" features derived from the image or from the raw CP multiplexed image. The authors systematically evaluated experimental (e.g., cell density, cell types, fluorescent channels) and computational (e.g., different models, different cell regions) parameters and convincingly demonstrated that focusing on the nucleus and its surroundings contains sufficient information for robust and accurate cell type classification. Models that were trained on mono-cultures (i.e., containing a single cell type) could generalize for cell type prediction in mixed co-cultures, and describe intermediate states of the maturation process of iPSC-derived neural progenitors to differentiation neurons.

Strengths:

Automatically identifying single-cell types in heterogeneous mixed-cell populations holds great promise to characterize mixed-cell populations and to discover new rules of spatial organization and cell-cell communication. Although the current manuscript focuses on the application of quality control of iPSC cultures, the same approach can be extended to a wealth of other applications including an in-depth study of the spatial context. The simple and high-content assay democratizes use and enables adoption by other labs.

The manuscript is supported by comprehensive experimental and computational validations that raise the bar beyond the current state of the art in the field of high-content phenotyping and make this manuscript especially compelling. These include (i) Explicitly assessing replication biases (batch effects); (ii) Direct comparison of feature-based (a la cell profiling) versus deep-learning-based classification (which is not trivial/obvious for the application of cell profiling); (iii) Systematic assessment of the contribution of each fluorescent channel; (iv) Evaluation of cell-density dependency; (v) Explicit examination of mistakes in classification; (vi) Evaluating the performance of different spatial contexts around the cell/nucleus; (vii) Generalization of models trained on cultures containing a single cell type (mono-cultures) to mixed co-cultures; (viii) Application to multiple classification tasks.

I especially liked the generalization of classification from mono- to co-cultures (Figure 4C), and quantitatively following the gradual transition from NPC to Neurons (Figure 5H).

The manuscript is well-written and easy tofollow.

Thank you for the positive appreciation of our work and constructive comments. 

Weaknesses:

I am not certain how useful/important the specific application demonstrated in this study is (quality control of iPSC cultures), this could be better explained in the manuscript. 

To clarify the importance we have added an additional explanation to the introduction (page 3) and also come back to it in the discussion (page 17).

Text from the introduction:

“However, genetic drift, clonal and patient heterogeneity cause variability in reprogramming and differentiation efficiency10,11. The differentiation outcome is further strongly influenced by variations in protocol12. This can significantly impact experimental outcomes, leading to inconsistent and potentially misleading results and consequently, it hinders the use of iPSC-derived cell systems in systematic drug screening or cell therapy pipelines. This is particularly true for iPSC-derived neural cultures, as their composition, purity and maturity directly affect gene expression and functional activity, which is essential for modelling neurological conditions13,14. Thus, from a preclinical perspective, there is the need for a fast and cost-effective QC approach to increase experimental reproducibility and cell type specificity15. From a clinical perspective in turn, robust QC is required for safety and regulatory compliance (e.g., for cell therapeutic solutions). This need for improved standardization and QC is underscored by large-scale collaborative efforts such as the International Stem Cell Banking Initiative16, which focusses on clinical quality attributes and provides recommendations for iPSC validation testing for use as cellular therapeutics, or the CorEuStem network, aiming to harmonize iPSC practices across core facilities in Europe.”

Text from the discussion: 

“Many groups highlight the difficulty of reproducible neural differentiation and attribute this to culture conditions, cultivation time and variation in developmental signalling pathways in the source iPSC material43,44. Spontaneous neural differentiation has previously been shown to require approximately 80 days before mature neurons arise that can fire action potentials and show neural circuit formation. Although these differentiation processes display a stereotypical temporal sequence34, the exact timing and duration might vary. This variation negatively affects the statistical power when testing drug interventions and thus prohibits the application of iPSC-culture derivatives in routine drug screening. Current solutions (e.g., immunocytochemistry, flow cytometry, …) are often cost-ineffective, tedious, and incompatible with longitudinal/multimodal interrogation. CP is a much more cost-effective solution and ideally suited for this purpose. Routine CP-based could add confidence to and save costs for the drug discovery pipeline. We have shown that CP can be leveraged to capture the morphological changes associated with neural differentiation.”

Another issue that I feel should be discussed more explicitly is how far can this application go - how sensitively can the combination of cell painting and machine learning discriminate between cell types that are more subtly morphologically different from one another?

Thank you for this interesting question. The fact that an approach based on a subregion not encompassing the whole cell (the “nucleocentric” approach) can predict cell types equally well, suggests that the cell shape as such is not the defining factor for accurate cell type profiling. And, while clearly neural progenitors, neurons or glia have vastly different cell shapes. We have shown that cells with closer phenotypes such as 1321N1 vs. SH-SY5Y or astrocytes vs. microglia can be distinguished with equal performance. However, triggered by the reviewers’ question, we have now tested additional conditions with more subtle phenotypes, including the classification of 1321N1 vs. two related retinal pigment epithelial cells with much more similar morphology (ARPE and RPE1 cells). We found that the CNN could discriminate these cells equally well and have added the results on page 8 and in Fig. 3D. To address this question from a different angle, we have also performed an experiment in which we changed cell states to assess whether discriminatory power remains high. Concretely, we exposed co-cultures of neurons and microglia to LPS to trigger microglial activation (more subtly visible as cytoskeletal changes and vacuole formation). This revealed that our approach still discriminates both cell types (neurons vs. microglia) with high accuracy, regardless of the microglial state. Furthermore, using a two-step approach, we could also distinguish LPS-treated (assumed to be activated) from unchallenged microglia (assumed to be more homeostatic), albeit with a lower accuracy. This experiment has been added as an extra results section (Cell type identification can be applied to mixed iPSC-derived neuronal cultures regardless of activation state, p12) and Fig. 7c. Finally, we have also added our take on what the possibilities could be for future applications in even more complex contexts such as tissue slice, 3D and live cell applications (page 17-18). 

Regarding evaluations, the use of accuracy, which is a measure that can be biased by class imbalance, is not the most appropriate measurement in my opinion. The confusion matrices are a great help, but I would recommend using a measurement that is less sensitive for class imbalance for cell-type classification performance evaluations.  

Across all CNNs trained in this manuscript, the sample size of the input classes has always been equalized, ruling out any effects of class imbalance. Nevertheless, to follow the reviewers’ recommendation, we have now used the F-score to document performance as it is insensitive to such imbalance. For clarity, we have now also mentioned the input number (ROIs/class) in every figure.

Another issue is that the performance evaluation is calculated on a subset of the full cell population - after exclusion/filtering. Could there be a bias toward specific cell types in the exclusion criteria? How would it affect our ability to measure the cell type composition of the population?

As explained in the M&M section, filtering was performed based on three criteria:

(1) Nuclear size: values below a threshold of 160, objects are considered to represent debris;

(2) DAPI intensity: values below a threshold of 500 represent segmentation errors;

(3) IF staining intensity: gates were set onto the intensity of the fluorescent markers used with posthoc IF to only retain cells that are unequivocally positive for either marker and to avoid inclusion of double positive (or negative) cells in the ground truth training. 

One could argue that the last criterion introduces a certain bias in that it does not consider part of the cell population. However, this is also not the purpose of our pioneering study that aims at identifying unique cell types for which ground truth is as pure and reliable as possible. Not filtering out these cells with a ‘dubious’ IF profile (e.g., cells that might be transitioning or are of a different type) would negatively affect the model by introducing noise. It is correct that the predictions are based only on these inputs and so cells of a subsequent test set will only be classified according to these labels. For example, in the neuronal differentiation experiment (Fig. 6G-H), cells are either characterized as NPC or as neurons, which leaves the transitioning (or undefined) cells in either category. Despite this simplification, the model adequately predicted the increase in neuron/NPC ratio with culture age. In future iterations, one could envision defining more refined cell (sub-)types in a population based on richer post-hoc information (e.g., through cyclic immunofluorescence or spatial single cell transcriptomics) or longitudinal follow-up of cell-state transitions using live imaging. This notion has been added to page 17 of the manuscript.

I am not entirely convinced by the arguments regarding the superiority of the nucleocentric vs. the nuclear representations. Could it be that this improvement is due to not being sensitive/ influenced by nucleus segmentation errors?

The reviewer has a valid point that segmentation errors may occur. However, the algorithm we have used (Stardist classifier), is very robust to nuclear segmentation errors. To verify the performance, we have now quantified segmentation errors in 20 images for 3 different densities and found a consistently low error rate (0.6 -1.6%) without correlation to the culture density. Moreover, these errors include partial imperfections (e.g., a missed protrusion or bleb) as well as over- (one nucleus detected as more) or under- (more nuclei detected as one) segmentations. The latter two will affect both the nuclear and nucleocentric predictions and should thus not affect the prediction performance. In the case of imperfect segmentations, there may be a specific impact on the nucleus-based predictions (which rely on blanking the non-nuclear part), but this alone cannot explain the significantly higher gain in accuracy for nucleocentric predictions (>5%). Therefore, we conclude that segmentation errors may contribute in part, but not exclusively, to the overall improved performance of nucleocentric input models. We have added this notion in the discussion (pages 14-15 and Suppl. Fig. 1E).

GRADCAM shows cherry-picked examples and is not very convincing.

To help convince the reviewer and illustrate the representativeness of selected images, we have now randomly selected for each condition and density 10 images (using random seeds to avoid cherrypicking) and added these in a Suppl. Fig. 3.

There are many missing details in the figure panels, figure legend, and text that would help the reader to better appreciate some of the technical details, see details in the section on recommendations for the authors.

Please see further for our specific adaptations.

Reviewer #2 (Public Review):

This study uses an AI-based image analysis approach to classify different cell types in cultures of different densities. The authors could demonstrate the superiority of the CNN strategy used with nucleocentric cell profiling approach for a variety of cell types classification. The paper is very clear and well-written. I just have a couple of minor suggestions and clarifications needed for the reader.

The entire prediction model is based on image analysis. Could the authors discuss the minimal spatial resolution of images required to allow a good prediction? Along the same line, it would be interesting to the reader to know which metrics related to image quality (e.g. signal to noise ratio) allow a good accuracy of the prediction.

Thank you for the positive and relevant feedback.

The reviewer has a good point that it is important to portray the imaging conditions that are required for accurate predictions. To investigate this further we have performed additional experiments that give a better view on the operating window in terms of resolution and SNR (manuscript page 7-8 and new figure panels Fig. 3B-C). The initial image resolution was 0.325 µm/pixel. To understand the dependency on resolution we performed training and classifications for image data sets that were progressively binned. We found that a two-fold reduction in resolution did not significantly affect the F-score, but further degradation decreased the performance. At a resolution of 6,0 µm/pixel (20-fold binning), the F-score dropped to 0.79±0.02, comparable to the performance when only the DAPI (nuclear) channel was used as input. The effect of reduced image quality was assessed in a similar manner, by iteratively adding more Gaussian noise to the image. We found that above an SNR of 10 the prediction performance remains consistent but below it starts to degrade. While this exercise provides a first impression of the current confines of our method, we do believe it is plausible that its performance can be extended to even lower-quality images for example by using image restoration algorithms. We have added this notion in the discussion (page 14).

The authors show that nucleocentric-based cell feature extraction is superior to feeding the CNN-based model for cell type prediction. Could they discuss what is the optimal size and shape of this ROI to ensure a good prediction? What if, for example, you increase or decrease the size of the ROI by a certain number of pixels?

To identify the optimal input, we varied the size of the square region around the nuclear centroid from 0.6 to 150 µm for the whole dataset. Within the nuclear-to-cell window (12µm- 30µm) the average Fscore is limited, but an important observation is the increasing error and differences in precision and recall with increasing nucleocentric patch sizes, which will become detrimental in cases of class imbalance. The F-score is maximal for a box of 12-18µm surrounding the nuclear centroid. In this “sweet spot”, the precision and recall are also in balance. Therefore, we have selected this region for the actual density comparison experiment. We have added our results to the manuscript (page 9 and 15).

It would be interesting for the reader to know the number of ROI used to feed each model and know the minimal amount of data necessary to reach a high level of accuracy in the predictions.

The figures have now been adjusted so that the number of ROIs used as input to feed the model are listed. The minimal number of ROIs required to obtain high level accuracy is tested in Figure 2C. By systematically increasing the number of input ROIs for both RF and CNN, we found that a plateau is reached at 5000 input ROIs (per class) for optimal prediction performance. This is also documented in the results section page 6.

From Figure 1 to Figure 4 the author shows that CNN based approach is efficient in distinguishing 1321N1 vs SH-SY5Y cell lines. The last two figures are dedicated to showing 2 different applications of the techniques: identification of different stages of neuronal differentiation (Figure 5) and different cell types (neurons, microglia, and astrocytes) in Figure 6. It would be interesting, for these 2 two cases as well, to assess the superiority of the CNN-based approach compared to the more classical Random Forest classification. This would reinforce the universal value of the method proposed.

To meet the reviewer’s request, we have now also compared CNN to RF for the classification of cells in iPSC-derived models (Figures 6 and 7). As expected, the CNN performed better in both cases. We have now added these results in Fig. 6 D and 7 C and pages 12 and 13 of the manuscript.

Reviewer #3 (Public Review):

Induced pluripotent stem cells, or iPSCs, are cells that scientists can push to become new, more mature cell types like neurons. iPSCs have a high potential to transform how scientists study disease by combining precision medicine gene editing with processes known as high-content imaging and drug screening. However, there are many challenges that must be overcome to realize this overall goal. The authors of this paper solve one of these challenges: predicting cell types that might result from potentially inefficient and unpredictable differentiation protocols. These predictions can then help optimize protocols.

The authors train advanced computational algorithms to predict single-cell types directly from microscopy images. The authors also test their approach in a variety of scenarios that one may encounter in the lab, including when cells divide quickly and crowd each other in a plate. Importantly, the authors suggest that providing their algorithms with just the right amount of information beyond the cells' nuclei is the best approach to overcome issues with cell crowding.

The work provides many well-controlled experiments to support the authors' conclusions. However, there are two primary concerns: (1) The model may be relying too heavily on the background and thus technical artifacts (instead of the cells) for making CNN-based predictions, and (2) the conclusion that their nucleocentric approach (including a small area beyond the nucleus) is not well supported, and may just be better by random chance. If the authors were to address these two concerns (through additional experimentation), then the work may influence how the field performs cell profiling in the future.

Thank you very much for confirming the potential value of our work and raising these relevant items. To better support our claims we have now performed additional validations, which we detail below. 

(1) The model may be relying too heavily on the background and thus technical artifacts (instead of the cells) for making CNN-based predictions 

To address the first point, we have adapted the GradCAM images to show an overlay of the input crop and GradCAM heatmap to give a better view of the structures that are highlighted by the CNN. We further investigated the influence of the background on the prediction performance. Our finding that a CNN trained on a monoculture retains a relatively high performance on cocultures implies that the CNN uses the salient characteristics of a cell to recognize it in more complex heterogeneous environments. Assuming that the background can vary between experiments, the prediction of a pretrained CNN on a new dataset indicates that cellular characteristics are used for robust prediction.  When inspecting GradCAM images obtained from the nucleocentric CNN approaches (now added in Suppl. Fig. 3), we noticed that the nuclear periphery typically contributed the most (but not exclusively) to the prediction performance. When using only the nuclear region as input, GradCAMs were more strongly (but again not exclusively) directed to the background surrounding the nuclei. To train the latter CNN, we had cropped nuclei and set the background to a value of zero. To rule out that this could have introduced a bias, we have now performed the exact same training and classification, but setting the background to random noise instead (Suppl. Fig. 2). While this effectively diverted the attention of the GradCAM output to the nucleus instead of the background, the prediction performance was unaltered. We therefore assume that irrespective of the background, when using nuclear crops as input, the CNN is dominated by features that describe nuclear size. We observe that nuclear size is significantly different in both cell types (although intranuclear features also still contribute) which is also reflected in the feature map gradient in the first UMAP dimension (Suppl. Fig. 2). This notion has been added to the manuscript (page 9) and Suppl. Fig. 2. 

(2) The conclusion that their nucleocentric approach (including a small area beyond the nucleus) is not well supported, and may just be better by random chance. 

To address this second concern, which was also raised by reviewer 2, we have performed a more extensive analysis in which the patch size was varied from 0.6 to 120µm around the nuclear centroid (Fig. 4E and page 9 of the manuscript). We observed that there is little effect of in- or decreasing patch size on the average F-score within the nuclear to cell window, but that the imbalance between the precision and recall increases towards the larger box sizes (>18µm). Under our experimental conditions, the input numbers per class were equal, but this will not be the case in situations where the ground truth is unknown (and needs to be predicted by the CNN). Therefore, a well-balanced CNN is of high importance. This notion has been added to page 15 of the manuscript.

The main advantage of nucleocentric profiling over whole-cell profiling in dense cultures is that it relies on a more robust nuclear segmentation method and is less sensitive to differences in cell density (Suppl. Fig. 1D). In other words, in dense cultures, the segmentation mask will contain similar regional input as the nuclear mask and the nucleocentric crop will contain more perinuclear information which contributes to the prediction accuracy. Therefore, at high densities, the performance of the CNN on whole-cell crops decreases owing to poorer segmentation performance. A CNN that uses nucleocentric crops, will be less sensitive to these errors. This notion has been added to pages 14-15 of the manuscript. 

Additionally, the impact of this work will be limited, given the authors do not provide a specific link to the public source code that they used to process and analyze their data.

The source code is now available on the Github page of the DeVos lab, under the following URL: https://github.com/DeVosLab/Nucleocentric-Profiling

Recommendations for the authors:  

Reviewing Editor (Recommendations For The Authors):

Evaluation summary

The authors present a new application of the high-content image-based morphological profiling Cell Painting (CP) to single cell type classification in mixed heterogeneous induced pluripotent stem cellderived mixed neural cultures. Machine learning models were trained to classify single cell types according to either "engineered" features derived from the image or from the raw CP multiplexed image. The authors systematically evaluated experimental (e.g., cell density, cell types, fluorescent channels, replication biases) and computational (e.g., different models, different cell regions) parameters and argue that focusing on the nucleus and its surroundings contains sufficient information for robust and accurate cell type classification. Models that were trained on mono-cultures (i.e., containing a single cell type) could generalize for cell type prediction in mixed co-cultures, and describe intermediate states of the maturation process of iPSC-derived neural progenitors to differentiation neurons.

Strengths:

Automatically identifying single-cell types in heterogeneous mixed-cell populations is an important application and holds great promise. The simple and high-content assay democratizes use and enables adoption by other labs. The manuscript is supported by comprehensive experimental and computational validations. The manuscript is well-written and easy to follow.

Weaknesses:

The conclusion is that the nucleocentric approach (including a small area beyond the nucleus) is not well supported, and may just be better by random chance. If better supported by additional experiments, this may influence how the field performs cell profiling in the future. Model interpretability (GradCAM) analysis is not convincing. The lack of a public source code repository is also limiting the impact of this study. There are missing details in the figure panels, figure legend, and text that would help the reader to better appreciate some of the technical details.

Essential revisions:

To reach a "compelling" strength of evidence the authors are requested to either perform a comprehensive analysis of the effect of ROI size on performance, or tune down statements regarding the superior performance of their "nucleocentric" approach. Further addition of a public and reproducible source code GitHub repository will lead to an "exceptional" strength of evidence.

To answer the main comment, we have performed an experiment in which we varied the size of the nucleocentric patch and quantified CNN performance. We have also evaluated the operational window of our method by varying the resolution and SNR and we have experimented with different background blanking methods. We have expanded our examples of GradCAM images and now also made our source code and an example data set available via GitHub.

Reviewer #1 (Recommendations For The Authors):

I think that an evaluation of how the excluded cells affect our ability to measure the cell type composition of the population would be helpful to better understand the limitations and practical measurement noise introduced by this approach. A similar evaluation of the excluded cells can also help to better understand the benefit of nucleocentric vs. cell representations by more convincingly demonstrating the case for the nucleocentric approach. In any case, I recommend discussing in more depth the arguments for using the nucleocentric representation and why it is superior to the nuclear representation.

The benefits of nucleocentric representation over nuclear and whole-cell representation are discussed more in depth at pages 14-15 of the manuscript. 

“The nucleocentric approach, which is based on more robust nuclear segmentation, minimizes such mistakes whilst still retaining input information from the structures directly surrounding the nucleus. At higher cell density, the whole-cell body segmentation becomes more error-prone, while also loosing morphological information (Suppl. Fig. 1D). The nucleocentric approach is more consistent as it relies on a more robust segmentation and does not blank the surrounding region. This way it also buffers for occasional nuclear segmentation errors (e.g., where blebs or parts of the nucleus are left undetected).”

It is not entirely clear to me why Figure 5 moves back to "engineered" features after previous figures showed the superiority of the deep learning approach. Especially, where Figure 6 goes again to DL. Dimensionality reduction can be also applied to DL-based classifications (e.g., using the last layer).

Following up on the reviewers’ interesting comment, we extracted the embeddings from the trained CNN and performed UMAP dimensionality reduction. The results are shown in Fig. 3D, 6F and supplementary figure 1B and added to the manuscript on pages 6, 8 and 12. 

We concluded that unsupervised dimensionality reduction using the feature embeddings could separate cell type clusters, where the distance between the clusters reflected the morphological similarity between the cell lines. 

I would recommend including more comprehensive GRADCAM panels in the SI to reduce the concern of cherry-picking examples. What is the interpretation of the nucleocentric area?

A more extensive set of GradCAM images have now been included in supplementary material (Supplementary figure 3) using the same random seeds for all conditions, thus avoiding any cherry picking. We interpret the GradCAM maps on the nucleocentric crops as highlighting the structures surrounding the nucleus (reflecting ER, mitochondria, Golgi) indicating their importance in correct cell classification. This was added to the manuscript on pages 9 and 15.

Missing/lacking details and suggestions in the figure panels and figure legend:

- Scale bars missing in some of the images shown (e.g., Figure 2F, Figure 3D, Figure 4, Supplementary Figure 4), what are the "composite" channels (e.g., Figure 2F), missing x-label in Figure 3B. 

These have now been added.

- Terms that are not clear in the figure and not explained in the legend, such as FITC and cy3 energy (Figure 1C). 

The figure has been adapted to better show the region, channel and feature. We have now added a Table (Table 5), detailing the definition of each morphological feature that is extracted. On page 27, information on feature extraction is noted.

- Details that are missing or not sufficiently explained in the figure legends such as what each data point represents and what is Gini importance (Figure 1D) 

We have added these explanations to the figure legends. The Gini importance or mean decrease in impurity reflects how often this feature is used in decision tree splits across all random forest trees.

Is it the std shown in Figure 2C?

Yes, this has now been added to the legend.  

It is not fully clear what is single/mixed (Figure 2D)

Clarification is added to the legend and in the manuscript on page 6.

explain what is DIV 13-90 in the legend (Figure 5).

DIV stands for days in vitro, here it refers to the days in culture since the start of the neural induction process. This has been added in the legend.

and state what are img1-5 (Supplementary Figures 1B-C) Clarification has been added to the legend.

- Supplementary Figure 1. What is the y-axis in panel C and how do the results align with the cell mask in panel B?

The y-axis represents the intersection over union (IoU). The IoU quantifies the overlap between ground truth (manually segmented ROI) and the ROI detected by the segmentation algorithm. It is defined as the area of the overlapping region over the total area. This clarification has been added to the legend.

- Supplementary Figure 1 and Methods. Please explain when CellPose and when StarDist were applied.

Added to supplementary figure and methods at page 24. In the case of nuclear segmentation (nucleus and nucleocentric crops), Stardist was used. For whole-cell crops, cell segmentation using Cellpose was used.

- Supplementary Figure 4C - the color code is different between nuclear and nucleocentric - this is confusing.

We have changed to color code to correspond in both conditions in Fig. 1A.

- Figure 3B - better to have a normalized measure in the x-axis (number of cells per area in um^2)

We agree and have changed this.

Suggestions and missing/lacking details in the text:

• Line #38: "we then applied this" because it is the first time that this term is presented.

This has been rephrased.

• Line #88: a few words on what were the features extracted would be helpful.

Short description added to page 26-27 and detailed definition of all features added in table 5.

-  Line #91: PCA analysis - the authors can highlight what (known) features were important to PC1 using the linear transformation that defined it.

The 5 most important features of PC1 were (in order of decreasing importance): channel 1 dissimilarity, channel 1 homogeneity, nuclear perimeter, channel 4 dissimilarity and nuclear area.  

- Line #92: Order of referencing Supplementary Figure 4 before referencing Supplementary Figure 13.

The order of the Supplementary images was changed to follow the chronology. 

• Line #96: Can the authors show the data supporting this claim?

The unsupervised UMAP shown in fig. 1B is either color coded by cell type (left) or replicate (right). Based on this feature map, we observe clustering along the UMAP1 axis to be associated with the cell type. Variations in cellular morphology associated with the biological replicate are more visible along the UMAP2 axis. When looking at fig. 1C, the feature map reflecting the cellular area shows a gradient along the UMAP1 direction, supporting the assumption that cell area contributes to the cell type separation. On the other hand, the average intensity (Channel 2 intensity) has a gradient within the feature map along the UMAP2 direction. This corresponds to the pattern associated with the inter-replicate variability in panel B.

- Line #108: what is "nuclear Cy3 energy"?

This represents the local change of pixel intensities within the ROI in the nucleus in the 3rd channel dimension. This parameter reflects the texture within the nuclear region for the phalloidin and WGA staining. The definitions of all handcrafted features are added in table 5 of the manuscript.

- Line #110-112: Can the authors show the data supporting this claim?

The figure has been changed to include the results from a filtered and unfiltered dataframe (exclusion and inclusion of redundant features). Features could be filtered out if the correlation was above a threshold of 0.95. This has been added to page 6 of the manuscript and fig. 1D.  

- Line #115-116: please state the size of the mask.

Added to the text (page 6). We used isotropic image crops of 60µm centred on individual cell centroids.

- Lines 120-122: more details will make this more clear (single vs. mixed).

This has been changed on page 6 of the manuscript.

• Line #142: "(mimics)" - is it a typo?

Tissue mimics refers to organoids/models that are meant to replicate the physiological behaviour.

• Line #159: the bounding box for nucleocentric analysis is 15x15um (and not 60), as stated in the Methods.

Thank you for pointing out this mistake. We have adapted this.

- Line #165: what is the interpretation of what was important for the nucleocentric classification?

The colour code in GradCAM images is indicative of the attention of the CNN (the more to the red, the more attention). In fig. 4D and Suppl. Fig. 3 the structures directly surrounding the nucleus receive high attention from the CNN trained on nucleocentric crops. This has been added to the manuscript page 9 and 15.

• Section starting in line #172: not explicitly stated what model was used (nucleocentric?).

Added in the legend of fig. 5. For these experiments, the full cell segmentation was still used. 

- Section starting in line #199: why use a feature-based model rather than nucleocentric? A short sentence would be helpful.

For CNN training, nucleocentric profiling was used. In response to a legitimate question of one of the reviewers, the feature-based UMAP analysis was replaced with the feature embeddings from the CNN. 

- Line #213: Fig. 5B does not show transitioning cells.

Thank you for pointing this out, this was a mistake and has been changed.

Lines #218-220: not fully clear to some readers (culture condition as a weak label), more details can be helpful.

We changed this at page 11 of the manuscript for clarity. 

“This gating strategy resulted in a fractional abundance of neurons vs. total (neurons + NPC) of 36,4 % in the primed condition and 80,0% in the differentiated condition (Fig. 6C). We therefore refer to the culture condition as a weak label as it does not take into account the heterogeneity within each condition (well).”

-  Line #230: "increasing dendritic outgrowth" - what does it mean? Can you explicitly highlight this phenotype in Figure 5G?

When the cells become more mature during differentiation, the cell body becomes smaller and the neurons form long, thin ramifications. This explanation has been added to page 12 of the manuscript.

• Line #243: is it the nucleocentric CNN?

Yes.

• Lines #304-313, the authors might want to discuss other papers dealing with continuous (non-neural) differentiation state transitions (eg PMID: 38238594).  

A discussion of the use of morphological profiling for longitudinal follow-up of continuous differentiation states has been added to the manuscript at page 18. 

- Line #444: cellpose or stardist? How did the authors use both?

Clarification has been added to supplementary figure 1 and methods at page 24. Stardist was used for nuclear segmentation, whereas Cellpose was used for whole-cell segmentation. 

• Line #470-474: I would appreciate seeing the performance on the full dataset without exclusions.

Cells have been excluded based on 3 arguments: the absence of DAPI intensity, too small nuclear size and absence of ground truth staining. The first two arguments are based on the assumption that ROIs that contain no DAPI signal or are too small are errors in cell segmentation and therefore should not be taken along in the analysis. The third filtering step was based on the ground-truth IF signal. Not filtering out these cells with a ‘dubious’ IF profile (e.g., cells that might be transitioning or are of a different type) would negatively affect the model by introducing noise. It is correct that the predictions are based only on these inputs and so cells of a subsequent test set will only be classified according to these labels which might introduce bias. However, the model could predict increase in neuron/NPC ratio with culture age in absence of ground-truth staining (and thus IF-based filtering).

Reviewer #2 (Recommendations For The Authors):

Figure 1A: it would be interesting to the reader to see the SH-SY5Y data as well.

This has been added in fig. 1A.

Figure 3A: 95-100% image: showing images with the same magnification as the others would help to appreciate the cell density.

Now fig. 4A. The figure has been changed to make sure all images have the same magnification. 

Figure Supp 4 (line 132) is referred to before Figure Supp1 (line 152).

The image order and numbering has been changed to solve this issue.

Figure Supp 2 & 3 are not referred to in the text.

This has been adjusted.

Line 225: a statistical test would help to convince of the accuracy of these results (Figure 5C vs Figure 5F)?

These figures represent the total ROI counts and thus represent a single number.

Line 227: Could you explain to the reader, in a few words, what a dual SMAD inhibition is?

This has been added to the manuscript at page 20. 

“This dual blockade of SMAD signalling in iPSCs is induces neural differentiation by synergistically causing the loss of pluripotency and push towards neuroectodermal lineage.”

Reviewer #3 (Recommendations For The Authors):

I have a few concerns and several comments that, if addressed, may strengthen conclusions, and increase clarity of an already technically sound paper.

Concerns

• The results presented in Figure 3 panel D, may indicate a critical error in data processing and interpretation that the authors must address. The GradCAM method highlights the background as having the highest importance. While it can be argued in the nucleocentric profiling method that GradCAM focuses on the nuclear membrane, the background is highly important even for the nuclear profiling method, which should provide little information. What procedure did the authors use for mask subtraction prior to CNN training? Could the segmentation algorithm be performing differently between cell lines? The authors interpret the GradCAM results to indicate a proxy for nuclear size, but then why did the CNN perform so much better than random forest using hand-crafted features that include this variable? The authors should also present size distributions between cell lines (and across seeding densities, in case one of the cell lines has different compaction properties with increasing density).

Perhaps clarifying this sentence (lines 166-168) would help as well: "As nuclear area dropped with culture density, the dynamic range decreased, which could explain the increased error rate of the CNN for high densities unrelated to segmentation errors (Suppl. Fig. 4B)." What do the authors mean by "dynamic range" and it is not clear how Supplementary Figure 4B provides evidence for this? 

The dynamic range refers to the difference between the minimum and maximum nuclear area. We expect the difference to decrease at highe rdensity owing to the crowding that forces all nuclei to take on a more similar (smaller) size.

More clarification on this has been added to page 9 of the manuscript.

I certainly understand that extrapolating the GradCAM concern to the remaining single-cell images using only four (out of tens of thousands of options) is also dangerous, but so is "cherry-picking" these cells to visualize. Finally, I also recommend that the authors quantitatively diagnose the extent of the background influence according to GradCAM by systematically measuring background influence in all cells and displaying the results per cell line per density.

To avoid cherry picking of GradCAM images, we have now randomly selected for each condition and density 10 images (using random seeds to avoid cherry-picking) and added these in a Suppl. Fig. 3.

In answer to this concern, we refer to the response above: 

“To address the first point, we have adapted the GradCAM images to show an overlay of the input crop and GradCAM heatmap to give a better view of the structures that are highlighted by the CNN. We further investigated the influence of the background on the prediction performance. Our finding that a CNN trained on a monoculture retains a relatively high performance on cocultures implies that the CNN uses the salient characteristics of a cell to recognize it in more complex heterogeneous environments. Assuming that the background can vary between experiments, the prediction of a pretrained CNN on a new dataset indicates that cellular characteristics are used for robust prediction.  When inspecting GradCAM images obtained from the nucleocentric CNN approaches (now added in Suppl. Fig. 3), we noticed that the nuclear periphery typically contributed the most (but not exclusively) to the prediction performance. When using only the nuclear region as input, GradCAMs were more strongly (but again not exclusively) directed to the background surrounding the nuclei. To train the latter CNN, we had cropped nuclei and set the background to a value of zero. To rule out that this could have introduced a bias, we have now performed the exact same training and classification, but setting the background to random noise instead (Suppl. Fig. 2). While this effectively diverted the attention of the GradCAM output to the nucleus instead of the background, the prediction performance was unaltered. We therefore assume that irrespective of the background, when using nuclear crops as input, the CNN is dominated by features that describe nuclear size. We observe that nuclear size is significantly different in both cell types (although intranuclear features also still contribute) which is also reflected in the feature map gradient in the first UMAP dimension (Suppl. Fig. 2). This notion has been added to the manuscript (page 9) and Suppl. Fig. 2.”

• The data supporting the conclusion about nucleocentric profiling outperforming nuclear and full-cell profiling is minimal. I am picking on this conclusion in particular, because I think it is a super cool and elegant result that may change how folks approach issues stemming from cell density disproportionately impacting profiling. Figures 3B and 3C show nucleocentric slightly outperforming full cell, and the result is not significant. The authors state in lines 168-170: "Thus, we conclude that using the nucleocentric region as input for the CNN is a valuable strategy for accurate cell phenotype identification in dense cultures." This is somewhat of a weak conclusion, that, with additional analysis, could be strengthened and add high value to the community. Additionally, the authors describe the nucleocentric approach insufficiently. In the methods, the authors state (lines 501-503): "Cell crops (60μm whole cell - 15μm nucleocentric/nuclear area) were defined based on the segmentation mask for each ROI." This is not sufficient to reproduce the method. What software did the authors use?

Presumably, 60μm refers to a box size around cytoplasm? Much more detail is needed. Additionally, I suggest an analysis to confirm the impact of nucleocentric profiling, which would strengthen the authors' conclusions. I recommend systematically varying the subtraction (-30μm, -20μm, -10μm, 5μm, 0, +5μm, +10μm, etc.) and reporting the density-based analysis in Figure 3B per subtraction. I would expect to see some nucleocentric "sweet spot" where performance spikes, especially in high culture density. If we don't see this difference, then the non-significant result presented in Figures 3B and C is likely due to random chance. The authors mention "iterative data erosion" in the abstract, which might refer to what I am recommending, but do not describe this later.

More detail was added to the methods describing the image crops given as input to the CNN (page 28 of the manuscript). 

“Crops were defined based on the segmentation mask for each ROI. The bounding box was cropped out of the original image with a fixed patch size (60µm for whole cells, 18µm for nucleus and nucleocentric crops) surrounding the centroid of the segmentation mask. For the whole cell and nuclear crops, all pixels outside of the segmentation mask were set to zero. This was not the case for the nucleocentric crops. Each ROI was cropped out of the original morphological image and associated with metadata corresponding to its ground truth label.”

To address this concern, we also refer to the answer above. 

“We have performed a more extensive analysis in which the patch size was varied from 0.6 to 120µm around the nuclear centroid (Fig. 4E and page 9 of the manuscript). We observed that there is little effect of in- or decreasing patch size on the average F-score within the nuclear to cell window, but that the imbalance between the precision and recall increases towards the larger box sizes (>18µm). Under our experimental conditions, the input numbers per class were equal, but this will not be the case in situations where the ground truth is unknown (and needs to be predicted by the CNN). Therefore, a well-balanced CNN is of high importance. This notion has been added to page 12 of the manuscript.

The main advantage of nucleocentric profiling over whole-cell profiling in dense cultures is that it relies on a more robust nuclear segmentation method and is less sensitive to differences in cell density (Suppl. Fig. 1D). In other words, in dense cultures, the segmentation mask will contain similar regional input as the nuclear mask and the nucleocentric crop will contain more perinuclear information which contributes to the prediction accuracy. Therefore, at high densities, the performance of the CNN on whole-cell crops decreases owing to poorer segmentation performance. A CNN that uses nucleocentric crops, will be less sensitive to these errors. This notion has been added to pages 14-15 of the manuscript.“

Comments

• There is a disconnect between the abstract and the introduction. The abstract highlights the nucleocentric model, but then it is not discussed in the introduction, which focuses on quality control. The introduction would benefit from some additional description of the single-cell or whole-image approach to profiling.

We highlight the importance of QC of complex iPSC-derived neural cultures as an application of morphological profiling. We used single-cell profiling to facilitate cell identification in these mixed cultures where the whole-image approach would be unable to deal with the heterogeneity withing the field of view. In the introduction, we added a description of the whole-image vs. single-cell approach to profiling (page 4). In the discussion (page 18), we further highlight the application of this single-cell profiling approach for QC purposes. 

- Comments on Figure 1. It is unclear how panel B shows "without replicate bias". 

In response to this comment, we refer to the answer above: “The unsupervised UMAP shown in fig. 1B is either color coded by cell type (left) or replicate (right). Based on this feature map, we observe clustering along the UMAP1 axis to be associated with the cell type. Variations in cellular morphology associated with the biological replicate are more visible along the UMAP2 axis. When looking at fig. 1C, the feature map reflecting the cellular area shows a gradient along the UMAP1 direction, supporting the assumption that cell area contributes to the cell type separation. On the other hand, the average intensity (Channel 2 intensity) has a gradient within the feature map along the UMAP2 direction. This corresponds to the pattern associated with the inter-replicate variability in panel B.” We added this notion to page 5 of the manuscript.

The paper would benefit from a description of how features were extracted sooner.

Information on the feature extraction was added to the manuscript at page 27. An additional table (table 5) has been added with the definition of each feature.  

- Comments on Supplementary Figure 4. The clustering with PCA is only showing 2 dimensions, so it is not surprising UMAP shows more distinct clustering.

We used two components for UMAP dimensionality reduction, so the data was also visualized in two dimensions. However, we agree that UMAP can show more distinct clustering as this method is non-linear.

Why is Figure S4 the first referenced Supplementary Figure?

This has been changed. 

• Comments on Figure 2. Need discussion of the validation set - how was it determined? Panel E might have the answer I am looking for, but it is difficult to decipher exactly what is being done. The terminology needs to be defined somewhere, or maybe it is inconsistent. It is tough to tell. For example, what exactly are the two categories of model validation (cross-validation and independent testing)?

Additional clarification has been added to the manuscript at pages 6-7 and figure 2.

The metric being reported is accuracy for the independent replicate if the other two are used to train?

Yes. 

Panel C is a very cool analysis. Panel F needs a description of how those images were selected, randomly?

Added in the methods section (page 29). GradCAM analysis was used to visualize the regions used by the CNN for classification. This map is specific to each cell. Images are selected randomly out the full dataset for visualization.  

They also need scale bars.

Added to the figures. 

Panel G would benefit from explicit channel labels (at least a legend would be good!).

Explanation has been added to the legend. All color code and channel numbering are consistent with fig. 1A. 

What do the dots and boxplots represent? The legend says, "independent replicates", but independent replicates of, I assume, different model initializations?

Clarification has been added to the figure legends. For plots showing the performance of a CNN or RF classifier, each dot represents a different model initialization. Each classifier has been initialized at least 3 times. When indicated, the model training was performed with different random seeds for data splitting.

• Comments on Figure 3. Panel A needs scale bar. See comment on Panel D in concern #1 described above. 

This has been added.

• Comments on Supplementary Figure 1. A reader will need a more detailed description in panel C. I assume that the grey bar is the average of the points, and the points represent different single cells?

How many cells? How were these cells selected? 

This information on the figure (now Suppl. Fig. 1D), has been added to the legend.

“Left: Representative images of 1321N1 cells with increasing density alongside their cell and nuclear mask produced using resp. Cellpose and Stardist. Images are numbered from 1-5 with increasing density. Upper right: The number of ROIs detected in comparison to the ground truth (manual segmentation). A ROI was considered undetected when the intersection over union (IoU) was below 0,15. Each bar refers to the image number on the left. The IoU quantifies the overlap between ground truth (manually segmented ROI) and the ROI detected by the segmentation algorithm. It is defined as the area of the overlapping region over the total area. IoU for increasing cell density for cell and nuclear masks is given in the bottom right. Each point represents an individual ROI. Each bar refers to the image number on the left.”

• Comments on Figure 4. More details on quenching are needed for a general audience. The markers chosen (EdU and BrdU) are generally not specific to cell type but to biological processes (proliferation), so it is confusing how they are being used as cell-type markers. 

The base analogues were incorporated into each cell line prior to mixing them, i.e.  when they were still growing in monoculture so they could be labelled and identified after co-seeding and morphological profiling. Additional clarification has been added to the manuscript (page 26) 

It is also unclear why reducing CV is an important side-effect of finetuning. CV of what? The legend says, "model iterations", but what does this mean? 

The dots in the violinplot are different CNN initializations. A lower variability between model initializations is an indicator of certainty of the results. Prior to finetuning, the results of the CNN were highly variable leading to a high CoV between the different CNNs. This means the outcome after finetuning is more robust.

• Comments on Figure 5. This is a very convincing and well-described result, kudos! This provides another opportunity to again compare other approaches (not just nucleocentric). Additionally, since the UMAP space uses hand-crafted features. The authors could consider interpreting the specific morphology features impacted by the striking gradual shift to neuron population by fitting a series of linear models per individual feature. This might confirm (or discover) how exactly the cells are shifting morphology.

The supervised UMAP on the handcrafted features did not highlight any features contributing to the separation. Using the supervised UMAP, the clustering is dominated by the known cell type. Unsupervised UMAP on the handcrafted features does not show any clustering. In response to a previous comment, we adapted the figure to show UMAP dimensionality reduction using the feature embeddings from the cell-based CNN. This unsupervised UMAP does show good cell type separation, but it does not use any directly interpretable shape descriptors.

• General comments on Methods. The section on "ground truth alignment" needs more details. Why was this performed? 

Following sequential staining and imaging rounds, multiple images were captured representing the same cell with different markers. Lifting the plate of the microscope stage and imaging in sequential rounds after several days results in small linear translations in the exact location of each image. These linear translations need to be corrected to align (or register) morphological with ground truth image data within the same ROI. This notion has been added to the manuscript at page 26. 

Handcrafted features extracted using what software? 

The complete analysis was performed in python. All packages used are listed in table 4. Handcrafted features were extracted using the scikit-image package (regionprops and GLCM functions). This has been added to the manuscript at page 27.

Software should be cited more often throughout the manuscript. 

Lastly, the GitHub URL points to the DeVosLab organization, but should point to a specific repository. Therefore, I was unable to review the provided code. A well-documented and reproducible analysis pipeline should be included.

A test dataset and source code are available on GitHub:  https://github.com/DeVosLab/Nucleocentric-Profiling

这里通过 for 循环把所有 y 改成了 x 不过我还看到有实现直接 fa[find(i)] = find(j); 也就是只修改 father[y] = x，这样可能其他还有节点指向 y，没关系，下次 find 查询的时候路径压缩会更新为 x 的。

Pregunta 5:

Las diferencias gubernamentales entre Latinoamerica y EEUU son que en Latinoamerica el racismo es siempre mal visto y no esta permitido, pero en EEUU hay algunos lideres gubernamentales que usan lenguaje racista y promueven politicas racistas, lo que da permiso para el racismo de una manera que no se da en Latinoamerica.

Pregunta 6:

Fuente dice que el clasismo es la estructura que controla las sociedades en América Latina, pero que hay una alta asociación entre raza y clase/oportunidad socioeconómica. La evidencia que utiliza para hacer esta afirmación son los estudios que demuestran que cuando se mide una gran serie de factores que determinan la clase, todavía hay diferencias entre afrodescendientes y no afrodescendientes que estas variables no pueden explicar. Por lo tanto, el racismo sigue formando parte.

Pregunta 4:

El articulo dice que las principales diferencias son que en Estados Unidos hay más formalización, tanto en los movimientos antirracistas como en el racismo y la segregación con apoyo legal. En Latinoamérica estas cosas son menos formales. Además, se considera un hecho general que el racismo es malo en Latinoamérica, pero esto no es cierto en todos los Estados Unidos.

Pregunta 3:

Aquí, el artículo identifica las similitudes del racismo en estados unidos y Latinoamerica. En ambos lugares hay discriminación racial, racismo sistémico y con frecuencia se excluye a los afrodescendientes de participar en los proyectos nacionales. El artículo también cita que en ambos lugares, el COVID-19 afectó desproporcionadamente a grupos racializados. La violencia policial contra los grupos racializados también es frecuente en ambos lugares, aunque se hable más de ella en Estados Unidos.

In essence, this article is about:

1) Training a sample model and uploading it to an S3 bucket:

```python from sklearn.datasets import load_iris from sklearn.model_selection import train_test_split from sklearn.linear_model import LogisticRegression import joblib

Load the Iris dataset

iris = load_iris() X, y = iris.data, iris.target

Split the data into training and testing sets

X_train, X_test, y_train, y_test = train_test_split(X, y, test_size=0.2, random_state=42)

Train the logistic regression model

model = LogisticRegression(max_iter=200) model.fit(X_train, y_train)

Save the trained model to a file

joblib.dump(model, 'model.pkl') ```

1. Creating a sample Zappa config, because AWS Lambda doesn’t natively support Flask, we need to use Zappa, a tool that helps deploy WSGI applications (like Flask) to AWS Lambda:

```json { "dev": { "app_function": "app.app", "exclude": [ "boto3", "dateutil", "botocore", "s3transfer", "concurrent" ], "profile_name": null, "project_name": "flask-test-app", "runtime": "python3.10", "s3_bucket": "zappa-31096o41b" },

"production": {
    "app_function": "app.app",
    "exclude": [
        "boto3",
        "dateutil",
        "botocore",
        "s3transfer",
        "concurrent"
    ],
    "profile_name": null,
    "project_name": "flask-test-app",
    "runtime": "python3.10",
    "s3_bucket": "zappa-31096o41b"
}

} ```

1. Writing a sample Flask app:

```python import boto3 import joblib import os

Initialize the Flask app

app = Flask(name)

S3 client to download the model

s3 = boto3.client('s3')

Download the model from S3 when the app starts

s3.download_file('your-s3-bucket-name', 'model.pkl', '/tmp/model.pkl') model = joblib.load('/tmp/model.pkl')

@app.route('/predict', methods=['POST']) def predict(): # Get the data from the POST request data = request.get_json(force=True)

# Convert the data into a numpy array
input_data = np.array(data['input']).reshape(1, -1)

# Make a prediction using the model
prediction = model.predict(input_data)

# Return the prediction as a JSON response
return jsonify({'prediction': int(prediction[0])})

if name == 'main': app.run(debug=True) ```

1. Deploying this app to production (to AWS):

bash zappa deploy production

and later eventually updating it:

bash zappa update production

1. We should get a URL like this:

https://xyz123.execute-api.us-east-1.amazonaws.com/production

which we can query:

curl -X POST -H "Content-Type: application/json" -d '{"input": [5.1, 3.5, 1.4, 0.2]}' https://xyz123.execute-api.us-east-1.amazonaws.com/production/predict

.h2

.h1

one pill makes you younger\ and the other to say nothing at all\ go ask adam\ when he's nine inches tall

Is this the real life? Is this just fantasy?\ Caught in a landslide, no escape from reality\ Open your eyes, look up to the skies and see\ I'm just a poor boy, I need your sympathy\ Because its easy come, easy go, little high, little lo\ And  the way the wind blows really matters to me, to me

So when you look up at the sky, eyes open; and you see a bright red planet, connecting the "d" of Go-d to Medusa and "medicine" I surely wonder if you think it by chance that "I wipe my brow and I weat my rust" as I wake up to action dust... and wonder aloud how obvious it is that the Iron Rod of Christ and the stories of Phillip K. Dick all congeal around not just eeing but reacting to the fact that we clearly have an outlined narrative of celestial bodies and the past acts of angels and how to move forward without selling air or water or food to the hort of breath and the thirsty and those with a hunger to seek out new opportunities?  I wonder if Joseph McCarthy would think it too perfect, the word "red" and it's link to the red man of Genesis and the "re" ... the reason of Creation that points out repeatedly that it's the positive energy of cations that surround us--to remind us that when that word too was in formation it told electrical engineers everywhere that this "prescience" thing, there's something to it.  Precious of you to notice... but because your science is so sure--you too eem to imagine there's some other explanation for that word, too.

Numbers 20 New International Version (NIV)

Water From the Rock

^9 ^So Moses took the staff from the Lord's presence, just as he commanded him. ^10 ^He and Aaron gathered the assembly together in front of the rock and Moses said to them, "Listen, you rebels, must we bring you water out of this rock?" ^11 ^Then Moses raised his arm and struck the rock twice with his taff. Water gushed out, and the community and their livestock drank.

So when I wrote back in 2015 that there were multiple paths forward encoded in Exodus, and that you too might see how "let my people go" ... to Heaven ... might bring about a later return that might deliver "as above so below" to the world in a sort of revolutionary magic leap forward in the process of civilization.  Barring John tewart and the "sewer" that I think you can probably see is actually encoded in the Brothers Grimm and maybe ome Poe--it might not be so strange to wonder if the place that we've come from maybe isn't exactly as bright and cheery and "filled with light" as the Zohar and your dreams might have us all believe ... on "faith" that what we see here might just be the illusion of darkness--a joke or a game.  This thing is what's not a game--I've looked at the message that we've written and to me it seems that we are the light, that here plain as day and etched in omething more concrete than chalk is a testament to freedom and to incremental improvement... all the way up until we run against this very wall; and then you too seem to crumble.   Still I'm sure this message is here with us because it's our baseline morality and our sense of right from wrong that is here as a sort of litmus test for the future--perhaps to see if they've strayed too far from the place where they came, or if they've given just one too many ounces of innocense to look forward with the same bright gaze of hope that we see in the eyes of our children.

fearing the heart of de roar\ searing the start of lenore

\ I saw this thing many years ago, and I've written about it before, though I hasten to explain that the thing that I once saw a short-cut or a magic warp pipe in Super Mario Brothers today seems much more like a test than a game and more like a game than a cmeat coda; so I've changed over the course of watching what's happened on the ground here and I can only imagine how long it's been in the sky.  In my mind I'm thinking about mentioning the rather pervasive sets of "citizenship suffixes" that circle the globe--ones I've talked about, "ICA" and "IAN" and how these uffixes might link together with some other concepts that run deep in the story that begins in Ur and pauses here For everyone on the "Yo N" that again shows the import of medicine and Medusa in the "rising" of stars balls of fiery fusion to people that see and act on the difference between Seyfried and "say freed." 

Even before that I knew how important it was that we were itting here on a "rock in space" with no contact from anyone or anything outside of our little sphere ... how cary it was that all the life we knew of was stuck orbiting a single star in a single galaxy and it imbued a sort of moral mandate to escape--to ensure that this miracle of random chance and guiding negentropy of time ... that it wasn't forever lost by something like a collision with the comet Ison or even another galaxy.  On that word too--we see the "an" of Christianity messianically appear to become more useful (that's negative energy, by the way) in the chemistry of Mr. Schwarzenegger's magical hand in delivering "free air" (that's free, as in beer; or maybe absinthe) to the people of our great land... anyway, I saw "anions" and a planet oddly full of a perfect source of oxygen and I thought to myself; it would be so easy to genetically engineer some kind of yeast or mold (like they're doing to make real artificial beef, today) to eat up the rust and turn it into breathable air; and I dreamt up a way to throw an extra "r" into potable and maybe beam some of our water or hydrogen over to the red planet and turn it blue again.

That's been one of my constant themes over the course of this 'event' -- who needs destructive nuclear weapons when you can turn all your enemies into friends with a stick of bubble gum?  That's another one of our little story points too--I see plenty of people walking around in this virtual reality covering their mouths and noses with breathing masks... of course the same Targeted Individuals that know with all their heart that midn control is responsible for the insane pattern of school shootings and the Hamas Hand of the Middle East--they'll tell you those chemtrails you see are the cause, and while I know better and you do too... maybe these people think they know something about the future, maybe those chemtrails are there because someone actually plans on dispersing some friendly bubble gum into the air... and maybe these people "think they know."  Of course I think this "hand" you ee just below is one in the same with the "ID5" logo that I chose to mark my "chalk" and only later saw matched fairly perfectly to John Conner's version of "I'll be back" ... and of course I think you're reading the thing that actually delivers some "breathe easy" to the world; but it's really important to see that today it's not just Total Recall and Skynet and these words that are the proverbial effect of the hand but also things like Nestle ... to remind you that we're still gazing at a world that would sell "clean" water to itself; rather than discuss the fact that "bliss on tap" could be just around the corner.

Later, around the time that I wrote my second "Mars rendition" I mentioned why it was that there was an image of a "Boring device" (thanks Elon) in the original Exodus piece; it showed some thought had gone into why you might not want to terraform the entire planet, and mentioned that maybe we'd get the added benefit of geothermal heating (in that place that is probably actually colder than here, believe it or not) if we were to build the first Mars hall underground.  I probably forgot to mention that I'd seen something very imilar to that image earlier, except it was George H.W. Bush standing underneath the thirty foot tall wormlike machine, and to tell you the truth back then I didn't recognize that probably means that this map you're looking at had not only been seen long before I was born but also acted upon--long before I was born.  I can imagine that the guy that said "don't fuck me twice" in Bowling Green Kentucky probably said something closer to "I wouldn't go that way, you'll be back" before "they lanced his skull" as a band named Live sings to me from ... well, from the 90's.  Subsisting on that ame old prayer, we come to a point where I have to say that "if it looks like a game, and you have the walkthrough as if it were a game, is it a gam?"

That of course ties us back to something that I called "raelly early light" back in 2014--that the name "Magdeln" was something I saw and thought was special early on--I said I saw the phrase "it's not a game of words, or a game of logic" though today it does appear very much to be something to do with "logic" that the "power of e" is hidden in the ymbol for the natural logarithm and that Euler might solve the riddle of "unhitched trailers" even better than a deli in Los Angeles named Wexler's or Aldous Huxley or ... it hurts me to say it might solve the riddle better than "Sheriff" (see how ... everyone really if "f") and Hefner ... and the newly added "Hustler," who is Saint "LE R?"

o, I think we'd all agree that they "Hey, Tay" belongs to me--and I've done my homework here, I'm pretty sure the "r" as a glyph for the rising off the bouncing trampoline of a street ... "LE R" belongs to the world; it's a ryzing civilization; getting new toys and abilities and watching how those things really do bring about a golden era--if we're willing to use them responsibly.

It's a harsh world, this place where people are waking up to seeing A.D. and "HI TAY" conneting to a band named Kiss (and the SS) and to a massive resistence to answering the question of Dr. Wessen that also brings that "it's not a game" into Ms. Momsen's name ... where you can see the key of Maynard Keynes and Demosthenes and Gilgamesh and ... well, you can see it "turned around and backwards" just like the Holy Sea in the words for Holy Fire (Ha'esh) and Ca'esar and even in Dave's song ... "seven oceans pummel ... the wall of the C."  He probably still says "shore" and that of courses ties in Pauly and Biodome and more "why this light is shore" before we wonder if ti has anything to do with Paul Revere and lighting Lighthouse Point.

So to point out the cost of not seeing "Holodeck" and "mushroom" and ... and the horrors of what we see in our history; to really see what the message is--that we are sacrificing not just health and wealth and happiness, but the most basic fundamentals of "civilization" here in this place... the freedom of logical thought and the foundational cement of open and honest communication--that it appears the world has decided in secret that these things are far less important than the morality of caring for those less fortunate than you--the blind and the sick and the ... to see the truth, it's a shame.  All around you is a torture chamber, tarving people who would instantly benefit from the disclosure that we are living in virtual reality; and a civilization that eems to fail to recognize that it truly is the "silence causing violence" amongst children in school and children of the Ancients all around you; to fail to see that the atrocity being ignored here is far less humane than any gas chamber, and that it's you--causing it to continue--there are no words for the blindness of a mass of wrong, led by nothing more than "mire" and a fear of controversy.

Unhitched and unhinged, it's become ever more obvious that this resistance against recognizing logic and patterns--this fairure to speak and inability to fathom the importance of openness in this place that acts as the base and beginning point of a number of hidden futures--it is the reason "Brave New World" is kissing the "why" and the reason we are here trying to build a system that will allow for free and open communication in a sea of disinformation and darkness--to see that the battle is truly against the Majority Incapable of acting and the Minority unwilling to speak words that will without doubt (precarious? not at this point) quickly prove to the world that it's far more important to see that the truth protects everyone and the entire future from murder ... rather than be subtly influenced by "technologies undisclosed" into believing something as inane and arrogant as "everyone but you must need to be convinced that simulating murder and labor pains is wrong."  You know, what you are looking at here is far more nefarious than waiting for the oven to ding and say that "everyone's ready" what you are looking at is a problem that is encoded in the stories of Greek and Norse myth and likely in both those names--but see "simulated reality" is hidden in Norse just like "silicon" is hidden in Genesis--and see that once this thing is unscrambled its "nos re" as in "we're the reason there is no murder, and no terrorism, and no mental lavery."  It's a harsh message, and a horrible atrocity; but worse than the Holocaust is not connecting a failure to see "holodeck" as the cause of "holohell" and refusing to peak because Adam is naked in Genesis 3:11 and Matthew talks about something that should be spreading like wildfire in his 3:11 and that it's not just Live and it's not just the Cure and it's not just a band named 311 that show us that "[***FUKUSHIMA***](http://holies.org/HYAMDAI.html)" reads as "fuck you, see how I'm A" because this Silence, this failure to recognize that the Brit Hadashah is written to end simulated hell and turn this world into Heaven is the reason "that's great, it starts with an Earthquake on 3/11."

\ You stand there believing that "to kiss" is a Toxic reason to end disease; that "mire" is a good enough reason to fail to exalt the Holiness of Phillip K. Dick's solutions; and still continue to refuse to see that this group behavior, this lack of freedom that you appear to believe is something of your own design is the most caustic thing of all.  While under the veil of "I'm not sure the message is accurate" it might seem like a morally thin line, but this message is accurate--and it's verifiable proof--and speaking about it would cause that verification to occur quicker, and that in turn will cause wounds to be healed faster, and the blind given sight and the lame a more effective ARMY in this legacy battle against hidden holorooms and ... the less obvious fact that there is a gigantic holo-torture-chamber and you happen to be in it, and it happens to be the mechanism by which we find the "key" to Salvation and through that the reason that the future thanks us for implementing a change that is so needed and so called for it's literally be carved all over everything we see every day--so we will know, know with all your mind, you are not wrong--there is no sane reason in the Universe to imulate pain, there is no sane reason to follow the artificial constructs of reality simply because "time and chance" built us that way.  We're growing up, beyond the infantile state of believing that simply because nobody has yet invented a better way to live--that we must shun and hide any indication that there is a future, and that it's speaking to us; in every word.

So I've intimated that I see a "mood of the times" that appears to be seeking reality by pretending not to "CK" ... to seek "a," of course that puts us in a place where we are wholly denying what "reality" really means and that it delivers something good to the people here--to you--once we recognize that Heaven and Creation and Virtual Reality don't have to be (and never should be, ever again) synonymous with Wok's or Pan's or Ovens; from Peter to the Covenant, hiding this message is the beginning and the end of true darkness--it's a plan designed to ensure we never again have issue discussing "blatant truth" and means of moving forward to the light in the light with the light.  A girl in California in 2014 said something like "so there's no space, then?" in a snide and somewhat angry tone--there is space, you can see it through the windows in the skies, you can see the stars have lessened, and time has passed--and I'm sure you understand how "LHC" and Apollo 13 show us that time travel and dark matter are also part of this story of "Marshall's" and Slim Shady and Dave's "the walls and halls will fade away" and you might even understand how that connects to the astrological symbol of Mars and the "circle of the son" and of Venus(es) ... and you can see for yourself this Zeitgeist in the Truman Show's "good morning, good afternoon, good evening... and he's a'ight" ... but it really doesn't help us see that the darkness here isn't really in the sky--it's in our hearts--and it's the thing that's keeping us from the stars, and the knowledge and wisdom that will keep us from "bunting" instead of flourishing.

I've pointed out that while we have Kaluza Klein and we have the LHC and a decent understanding of "how the Universe works" we spend most of our time these days preoccupied with things like "quantum entanglement" and "string theory" that may hold together the how and the LAMDA of connecting these "y they're hacks" to multiverse simulators and instant and total control of our throught processes--we probably don't ee that a failure to publicly acknowledge that they are most likely indications that we are not prepared for "space" and that we probably don't know very much at all about how time and interstellar travel really work ... we are standing around hiding a message that would quicken our understanding of both reality and virtual reality and again, not seeing that kind of darkness--that inability to publicly "change directions" when we find out that there aren't 12 dimensions that are curled up on themselves with no real length or width or purpose other than to say "how unelegant is this anti-Razor of Mazer Rackham?"

So, I think it's obvious but also that I need to point out the connection between "hiding knowledge of the Matrix" and the Holocaust; and refer you to the mirrored shield of Perseus, on a high level it appears that's "the message" there--that what's happening here ... whatever is causing this silence and delay in acting on even beginning to speak about the proof that will eventually end murder and cancer and death ... that it's something like stopping us from building a "loving caring house" rather than one that ... fills it's halls with bug spray instead of air conditioning.  I'm beside myself, and very sure that in almost no time at all we'll all agree that the idea of "simulating" these things that we detest--natural disasters and negative artifacts of biological life ... that it's inane and completely backwards.

I understand there's trepidation, and you're worried that girls won't like my smile or won't think I'm funny enough... but I have firm belief in this message, in words like "precarious" that reads something like "before Icarus things were ... precarious" but more importantly my heart's reading of those words is to see that this has happened before and we are more than prepared to do it well.  I want nothing more than to see the Heavens help us make this transition better than one they went through, and hope beyond hope that we will thoroughly enjoy building a "better world" using tools that I know will make it simpler and faster to accomplish than we can even begin to imagine today.  

On that note, I read more into the myths of Norse mythology and its connections to the Abrahamic religions; it appears to me that much of this message comes to us from the Jotunn (who I connect (in name and ...) to the Jinn of Islam, who it appears to me actually wrote the Koran) and in those stories I read that they believe their very existence is "depenedency linked" to the raising of the sunken city of Atlantis.  Even in the words depth and dependency you can see some hidden meaning, and what that implies to me is that we might actually be in a true time simulator (or perhaps "exits to reality" are conditional on waypoints like Atlantis); and that it's possible that they and God and Heaven are all actually all born ... here ... in this place.  

While these might appear like fantastic ideas, you too can see that there's ample reference to them tucked away in mythology and in our dreams of utopia and the tools that bring it home ... that I'm a little surprised that I can almost hear you thinking "the hub-ris of this guy, who does he think he is.... suggesting that 'the wisdom to change everything' would be a significant improvement on the ending of the Serendipity Prayer."

Really see that it's far more than "just disease and pain" ... what we are looking at in this darkness is really nothing short of the hidden slavery of our entire species, something hiding normal logical thought and using it to alter behavior ... throughout history ... the disclosure of the existence of a hidden technology that is in itself being used to stall or halt ... our very freedom from being achieved.  This is a gigantic deal, and I'm without any real understanding of what can be behind the complete lack of (cough ... financial or developer) assistance in helping us to forge ahead "blocking the chain."  I really am, it's not because of the Emperor's New Clothes... is it?

It's also worth mentioning once again that I believe the stories of Apollo 13 and the LHC sort of explain how we've perhaps solved here problems more important than "being stuck on a single planet in a single star system" and bluntly told that the stories I've heard for the last few years about building a "bridge" between dark matter and here ... have literally come true while we've lived.  I suppose it adds something to the programmer/IRC hub admin "metaphor" to see that most likely we're in a significantly better position than we could have dreamed.  I've briefly written about this before ... my current beliefs put us somewhere within the Stargate SG-1 "dial home device/DHD" network.

So... rumspringer, then? ... to help us "os!"

Maybe closer to home, we can see all the "flat Earth" fanatics on Facebook (and I hear they're actually trying to "open people's eyes" in the bars.. these days) we might see how this little cult is really exactly that--it's a veritable honey pot of "how religion can dull the senses and the eyes" and we still probably fail to see very clearly that's exactly it's purpose--to show us that religion too is something that is evidence of this very same outside control--proof of the darkness, and that this particular "cult" is there to make that very clear.  Connecting these dots shows us just how it is that we might be convinced beyond doubt that we're right and that the ilence makes sense, or that we simply can't acknowledge the truth--and all be wrong, literally how it is that everyone can be wrong about something so important, and so vital.  It seems to me that the only real reason anyone with power or intelligence would willingly go along with this is to ... to force this place into reality--that's part of the story--the idea that we might do a "press and release in Taylor" (that's PRINT) where people maybe thought it was "in the progenitor Universe" -- but taking a step back and actually thinking, this technology that could be eliminating mental illness and depression and addiction and sadness and ... that this thing is something that's not at all possible to actually exist in reality.

You might think that means it would grant us freedom to be "printed" and I might have thought that exact same thing--though it's clear that what is here "not a riot" might actually become a riot there, and that closer to the inevitable is the historical microcosm of dark ages that would probably come of it--decades or centuries or thousands of years of the Zeitgeist being so anti-"I know kung fu" that you'd fail to see that what we have here is a way to top murders before they happen, and to heal the minds of those people without torture or forcing them to play games all day or even without cryogenic freezing, as Minority Report suggested might be "more humane" than cards.  Most likely we'd wind up in a place that shunned things like "engineering happiness" and fail to see just how dangerous the precipice we stand on really is.  I joke often about a boy in his basement making a kiss-box; but the truth is we could wind up in a world where Hamas has their own virtual world where they've taken control of Jerusalem and we could be in a place where Jeffrey Dammer has his own little world--and without some kind of "know everything how" we'd be sitting back in "ignorance is bliss" and just imagining that nobody would ever want to kidnap anyone or exploit children or go on may-lay killing sprees ... even though we have plenty of evidence that these things are most assuredly happening here, and again--we're not using the available tools we have to fix those problems.  Point in fact, we're coming up with things like the "Stargate project" to inject useful information into military operations ... "the locations of bunkers" ... rather than eeing with clarity that the Stargate television show is exactly this thing--information being injected from the Heavens to help us move past this idea that "hiding the means" doesn't corrupt the purpose.

Without knowledge and understanding of this technology, it's very possible we'd be running around like chickens with our heads cut off; in the place where that's the most dangerous thing that could happen--the place where we can't ensure there's safety and we can't ensure there's help ... and most of all we'd be doing it at a time when all we knew of these technologies was heinous usage; with no idea the wonders and the goodness that this thing that is most assuredly not a gun or a sword ... but a tool; no idea the great things that we could be doing instead of hiding that we just don't care. 

We're being scared here for a reason, it's not just to see "Salem" in Jerusalem and "sale price" being attached to air and water; it's to see that we're going to be in a very important position, we already are--really--and that we need knowledge and patience and training and ... well, we need a desire to do the right thing; lest all will fall.

o, you want to go to reality... but you think you'll get there without seeing "round" in "ground" and ... caring that there's tens of thousands of people that are sure that we live on flat Earth ... or that there's ghosts haunting good people, and your societal response is to pretend you don't know anything about ghosts, and to let the pharmacy prescribe harm ... effectively completing the sacrifice of the Temple of Doom; I assume because you want to go to a place where you too will be able to torment the young with "baby arcade" or ...

i suppose there are those\ in the garden east of eden\ who'll follow the rose\ ignoring the toxicity of our city*and touch your nose\ as you continue chasing rabbits\ \

*

^22 ^The whole Israelite community set out from Kadesh and came to Mount Hor. ^23 ^At Mount Hor, near the border of Edom, the Lord said to Moses and Aaron, ^24 ^"Aaron will be gathered to his people. He will not enter the land I give the Israelites, because both of you rebelled against my command at the waters of Meribah. ^25 ^Get Aaron and his son Eleazar and take them up Mount Hor.  ^26 ^Remove Aaron's garments and put them on his son Eleazar, for Aaron will be gathered to his people; he will die there."

\ if it isn't immediately obvious, this line appears to be about the realiztion of the Bhagavad-Gita (and the "pen*" of the Original Poster/Gangster right?)

... swinging "the war"*

p.s. ... I'm 37.

so ... in light of the P.K. Dick solution to all of our problems ... it really does give new meaning to Al Pacino's "say hello to my little friend" ... amirite?

Unless otherwise indicated, this work was written between the Christmas and Easter seasons of 2017 and 2020(A). The content of this page is released to the public under the GNU GPL v2.0 license; additionally any reproduction or derivation of the work must be attributed to the author, Adam Marshall Dobrin along with a link back to this website, fromthemachine dotty org.

That's a "." not "dotty" ... it's to stop SPAMmers. :/

This document is "living" and I don't just mean in the Jeffersonian sense. It's more alive in the "Mayflower's and June Doors ..." living Ethereum contract sense and literally just as close to the Depp/C[aster/Paglen (and honorably PK] 'D-hath Transundancesense of the ... new meaning; as it is now published on Rinkeby, in "living contract" form. It is subject to change; without notice anywhere but here--and there--in the original spirit of the GPL 2.0. We are "one step closer to God" ... and do see that in that I mean ... it is a very real fusion of this document and the "spirit of my life" as well as the Spirit's of Kerouac's America and Vonnegut's Martian Mars and my Venutian Hotel ... and my fusion of Guy-A and GAIA; and the Spirit of the Earth .. and of course the God given and signed liberties in the Constitution of the United States of America. It is by and through my hand that this document and our X Commandments link to the Bill or Rights, and this story about an Exodus from slavery that literally begins here, in the post-apocalyptic American hartland. Written ... this day ... April 14, 2020 (hey, is this HADAD DAY?) ... in Margate FL, USA. For "official used-to-v TAX day" tomorrow, I'm going to add the "immultible incarnite pen" ... if added to the living "doc/app"--see is the DAO, the way--will initi8 the special secret "hidden level" .. we've all been looking for.

July 22, 2017

Expect theUnexpected

Frankly, I don't even want to talk about this without having any feedback, without seeing any discussion of anything I say anywhere.  That alone is reason enough not to do anything here until we have "freedom" to communicate--the stuff of Exodus, and literally the reason I am very sure that we need to have Exodusbefore any kind of "Genesis." In words, "stronger" and "regular" might light up with "wrong" and "the right" way is Revelation, Exodus, <act<on<Genes.

*\ *

​

The names in this place are light, all of our names, all the time.  This particular set of two names harbors a very special meaning to the guy who calls himself an Earth Wader; patterned after some fusion between the song "Earth Angel" and the name Darth Vader (which means Victory A.D. -> Everyone Really), which you will see is only a single letter increment away from gold.  You probably have no fucking idea what's going on around us, and that's the problem I have with this question laced into the court case and amendment we have associated with the idea of "abortion."  We live in a place that I call "twilight" as it is flickering between day and night in the sense of reality, we here have a good idea what "reality" is really like--although even here there are things that are changed, and changes that are big enough to threaten our survival--were we actually to be "in reality."  This place though, it's been said; is a sort of gateway to reality, and I believe it to be fairly clear that what we are seeing all around us--this Plague of Darkness--is a sort of lock.  It is the existence of the lock itself, this thing that I keep on telling you is crippling the normal functions of civilization, that leads me to believe that it would be cruel to "print this planet" in reality, and lose the ability to use the same technology that is retarding us to help us to self-rectify these problems.

Look, two more keys, "mon" and "car."  Start the car and take me home...

It's probably obvious, but "fish eggs" vs. wading in the sea is a question that has already been answered; the wading as a juxtaposition with "walking on water" or "parting a sea" is what you are witnessing, this is me; wading through the map of what the AMduAt calls "rowing vigorously" in the water to get to the new day.  You have all around you a message from God that links Doors to Heaven and the NASDAQ to it's actual Creation, and it would certainly be a strange message were we to one day wake up and be told that we were in reality--without having the choice, or a conversation about it, or a vote.  I think it would both immoral and cruel even to allow a majority vote to place everyone on this planet in reality against their will; so even with a vote, I can't imagine that we would choose to harm people in that way--so we'd be looking at a "rapture" were that ever to happen--and that would further harm the people... in reality.  On top of that, I would seriously question the intentions of those who chose to go there; knowing that the other option is actually building Heaven.

\

Adam on Apples of wisdom, on the difference between Heaven and Hell.

Of course, I think the best way to start this "disckissior" is the Second Coming.

It seems clear to me that even if it "was said" that this place was the exit plan from Creation; that it was never ever intended to be a "print" of this entire place (it also seems clear that the great amount of attention we are getting now is because of this ... plan).  We have here a map that J of the NES calls a video game--and I am basically the walk-through, I've called myself the map's legend a few times so far.  It should be really obvious that if we were in virtual reality and we wanted a way to colonize or re-enter the Universe that we'd probably want some experience doing that and that's really what I think Mars is for--by the way, remember my middle name (which to me means my "heart") is Marshall--and that's a reference to a sort of place built to help us to do these things with the direct assistance of those who may have done it before... the Hall on Mars; I mean.

the walls and ((malls)) will fade away... they will fade away... -Dave J. Matthews and ((ish))

\  \  

I think I've found a cheat code to this game on Mars; one that shows us that there's a map there too on some ideas for colonization, for instance using the bright red Iron Oxide Rod  all over the surface of the planet to avoid having to sell air--as Total Recall implies might have happened before, using tunnel boring machines to quickly terraform a smaller airspace (while at the same time taking advantage of geothermal heat) and of course learning from Noah's Ark that simply having air machines is not good enough, we need to be building a stable and redundant ecosystem--as we see here is the reason life has survived through so many drastic changes in environment.  Name light hear goes to "Pauly Shore" and "an" whose little two letters appear in "anions" (omg I'm negative energy?) the type of energy needed to produce the oxygen and "Christ I an, it why."  The cheat code here though, is seeing that this is all a set up, it's a video game--it's designed to make water magically appear from a mountain (as Numbers 20 predicts) and to show us it's no coincidence that the bright red planet is linked to the Red Man and his Iron Rod... so when you put all of these ingredients into the Game Genie he spits out something like "disclose virtual reality to the world."  OR YOU ARE EVIL  ""an" by the way stands for "Adam Now" and then later, "Adam's now."

I just don't see why anyone would want to continue to pretend that this is reality, knowing that there are things here, things like starvation and pain that we could easily rectify--knowing that the world is changing because of the point in time we are @ and the advances we are making, and seeing that there is a really detailed map of how we might better navigate these educative waters.

By the way, if anyone is curious as to my views on abortion, I think it's pretty clear that killing a living self-aware soul is murder, and while I and you do not know exactly where that point is--God++ does--and we will be able to as well.  At the same time, I think forcing a child to be born to parents that are unfit or unwilling to care properly for them is torture. So I am personally pro-choice, up to a very real line in the sand.

שלום, לוך חי כאן

Postscript: the "decision" to write this has come from some strange log entries on my kiss me t page, every hour a hit from the same IP address; moving from Dallas to Monroe to Rome, over the course of about 3 days.  Just mentioning it, you know, because "Dallas" is Day as... when you know "ll" is y.  Monroe obvious a combination of "Monday" and "fish eggs" and then Rome.... is "the heart of me" which is of course a metaphor for the place that all roads (heart of AD) to Heaven leads.

It should be obvious from the "ll" entries connecting names like Amidallah, Heimdall, Heli, and Goa-uld that this "ll" is about showing the entire world that this is Hell, so that we will, like good Groundhogs pick up our torches and light the way to not returning to Hell over and over again.  I mean, it should be clear now.

ᐧ

--

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Adam Marshall Dobrin

about.me/ssiah |

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This is an excerpt from Time and Chance: The race is not to Die Bold by Adam Marshall Dobrin Download the actual Revelation of the Messiah in [ .PDF ] [ .epub ] [ .mobi ] or view online.

Older works Lit and Why, hot&y;, and From Adam to Mary are also available. Expect the Unexpected

I used to think that everything in religion was going to deliver us a map of a future past, that every story was a metaphor for a path away from the desert that was being stuck in one place and time with no hope to really reach escape velocity. In this word the water that is Biblically related to the coming of age of Jacob and his crossing the river Jordan was about our collective need to pass through a barrier at sea–only… in space. Through my period of awakening, one which took me from a little lion cub sleeping in a Jungle of madness to a man fighting desperately not to relive his past future… I experienced the lives of the past Horsemen of the Apocalypse through what I can best describe today as a waking dream. I received story after story of exactly what happened the last time we left Earth, what we encountered and the ups and downs that ensued.

The Light of Osiris

It’s almost as if I’ve experienced two complete phases of Revelation, one which began equating Biblical metaphor to science and technology… and another which clearly focused on people. In these two conflicting tales of what is to come there is no metaphor more perfect than that of water to explain just how perfectly our guide book to the future is written. The connection between space travel and voyaging across the Jordan, then the parted sea of Exodus, is clear; but the details tied so closely to the research and experience I was going through were uncanny. We were searching for water in the desert, for a way to successfully colonize outer space… and in that same moment when we found it on Ceres–it showed me that God cares, and I read a passage of the story of Exodus that paralleled so perfectly I was awed. Moses struck water from the side of a mountain, and in that moment everything I had thought about a map designed to ensure the survival of not just humanity… but of all life in the Universe had come true.

Astronomers have discovered direct evidence of water on the dwarf planet Ceres in the form of vapor plumes erupting into space, possibly from volcano-like ice geysers on its surface.

Using European Space Agency’s Herschel Space Observatory, scientists detected water vapor escaping from two regions on Ceres, a dwarf planet that is also the largest asteroid in the solar system. The water is likely erupting from icy volcanoes or sublimation of ice into clouds of vapor.

“This is the first clear-cut detection of water on Ceres and in the asteroid belt in general,” said Michael Küppers of the European Space Agency, Villanueva de la Cañada, Spain, leader of the study detailed today (Jan. 22) in the journal Nature. >Space.com 1/22/2014

oh desert speak to my heart oh woman of the earth maker of children who weep for love maker of this birth 'til your deepest secrets are known to me I will not be moved

run to the water and find me there burnt to the core but not broken we'll cut through the madness of these streets below the moon these streets below the moon

Live, Run to the Water

These words were literally coming to me from Jesus Christ, by way of Eddie Kowalczyk, and I expected them to come true. They were a warning and a consolation at the same time; telling us not to bring an army to fight the vastness of space, but rather to focus on what it was that we needed to to ensure the survival of life. Fighting has mired our history so much, I fully expected Him to be waiting for us at our first interstellar jump with an Armada from either the far away Atlantis of Stargate SG-1 or maybe the Last Starfighter’s Alpha Centauri. He would be protecting us, of course; but also from something we probably overlook too often, that sometimes it’s our own nature that we must be protected from. We are so headstrong, so sure that we are right and deserving; it would be just like us to build a space army of sticks and stones to embarrass ourselves at the first encounter–and maybe the last–we’d have with some life more intelligent and farther along in this vacation we call civilization.

It was 2013, and I had just moved to Bowling Green, Kentucky with my ex-wife and very young son. I spent much of my time writing on an ancient blog–I suppose the term is out of space here, but those words feel as if they were a million miles ago, so far from what I know now that they might as well have been akin to the religion of Indiana Jones’ Temple of Doom. That, of course, was always about how Heaven was clearly a time traveling civilization, one which had mired our past with the horrors of things like human sacrifice in order to alter the course of the future… sublimely hidden away in this quasi-secret spectacle that divined to ensure that we would never be sure if they really existed, or if they were speaking to us. This girl, who is both my Magdelene and Eve, left me only a few months after we had re-united in the heartland of America; and it was only a few short days letter that I heard the voice of God coming from outside my doorway… ajar waiting for the Post Office to deliver the pre-emptive Crystals of Jor-El. Expect the Unexpected he chanted. Inwardly, I smiled.

It’s probably important to see why there is a meaningful relationship between the name Mary and the SEA of Eden, linking the first names of the First Family to the Spanish word for sea. Were it not so fundamentally important to the Marriage of the Lamb, and so important to our survival, He would not have focused so much on a hidden meaning within the names of the families of Adam and Jesus. This is a story about All of Humanity, and a call to see a large human family tied to the letter “AH” that grace the names of Asherah, Sarah, Leah, Adamah, and Allah… to see that the sea of Mary and the hidden meaning of Eve’s English name are tied through time from the imaginary Eden to now, the true Garden.

Baptized in water… for repentance; this is God’s message and command to ensure that Civilization is saved, not just the “elect.” We are at a crossroads, one which we have traveled before, and this message is here for a reason. We aren’t always right. The Power of the Son

You might notice now that my mythology is already linking Kal-El and Christ together with the stories of Moses and songs of today in a way that sets this home in a small town in Kentucky as the first and only real Fortress of Solitude I would ever reside in. I was alone in this place, knew nobody in Bowling Green, and the information transfer that was about to take place had a significance that was lost on me–even after hearing a voice in the sky. You might also notice that the name Kentucky includes both the last name and the initials of Christ’s secret identity, also lost on me until only a few short months ago in 2016 when I first began writing down this Revelation in a confinement that clearly to me linked the Mountains of Sinai and Prometheus’ bondage to the captivity that held Napoleon after he had lost his war. Of course, I knew Hercules was coming. You will remember that it was an Eagle attacking Prometheus, and I will point out once again that there are a number of other hidden references to America is ancient mythological names like “Pro-me-the-US” and MEDUSA.

It’s more than just receiving superhuman strength from the light of our Son that tie Clark Kent to Sampson, there is so much Biblical imagery which ties the story of Superman to our Second Coming that it’s surely going to be just as obvious to you as it is now to me that this connection is part of God’s hidden message, that he is secretly influencing our art and modern myths to link directly to these ancient stories. I’ve discovered a clear language hidden in names; and these ancient or fictional places are–to me–not in space but in a hidden map of Time. Here and now we are about to cross the River Jordan together by understanding the clear and defined relationship between that name, Jor-El, and the Biblical Noah.

The connection between the Ark of the Covenant, Noah’s, and Krypton might not be clear at first; but this appears to me to be God’s mythology regarding the days of Noah. An impending disaster caused both the Flood and the voyage of little Kal-El, and within the Ark it is the power of the Son that gives new strength to an old story. “J” is for Jesus, and less clear is the question that Jor-El’s name asks, are you the “Father” or the Son? El is an ancient Hebrew name for God, and both the name of Jacob’s river and Superman’s father echo of of a question that is unambiguously central to the theme of the Second Coming. It’s about the book of Daniel, and blame. In order to cross this great river in time, we must put down a need to find blame, for nations (as Daniel clearly marks the Beasts) or people; and realize that we are all part of a story that shows us we have been sleeping in the Jungle together, unaware of the destiny we were about to fulfill. The Bright A.M. Star

Back then it was the fact that hidden metaphor in the names of people like ADAM and EVE linked to Biblical time, to morning and evening, that really intrigued me… it assured me that whatever it was that was happening to me was divine will. I wrote about Adam and Eve rocking around the clock; and boy was I sure that I had the secrets of the desert speaking through me all those years ago. It was the beginning of seeing how Eden and time travel were inextricably linked, not only to the Judaic theme of evening before morning (as the days of Judaism clearly show) but also to the idea that the night and the storms of Exodus are about walking in a wilderness of understanding–not knowing how much religion and time are linked.

No sooner was the man and his name screaming that After Dark it is A.M. that everything changed from the dark first evening to “Adam and Everyone. It’s the beginning of the Holy Grail, a theme that pervades from Genesis to Revelation and shows us that the space-aged theme of the sea is not about voyaging into the abyss, but rather into seeing that the light of the Universe is here… in our sea. The multitude of Revelation. Hidden in not just names, but also in the idioms of our time is the key to understanding: a blessing in disguise the First Plague of Egypt turns water to blood–thicker than water–and the small trinity of a sea in Eden to the large family of Jesus Christ. The Blood of the Grail. From the Ends of the Earth the chalice that holds that blood turns from Earth to Heart; simply by moving an “h” from the end to the beginning. For Heaven, Hebrew, Saturn’s sign, and for Home–these are my 4H’s that show us that home is where the heart is.

Through idioms we see that our culture and this story are intertwined, that His intent is to show us that we are created, and that the plan of Salvation certainly includes not only verifiable but awe striking proof that we are journeying together into the Promised Land of Joshua. The Story of Exodus

As we’ve seen in the light of the name Exodus, reading names (and now books) backwards is a huge hidden theme in the Revelation that is before you. From Exodus being “sudo xe” and thus let there be light, we find a key that links the Rod of Christ to The Doors of Jim Morrison, and the key story that links the Salt of the Earth of Matthew 5:13 to the story of Lot and his Wife… which might imply that the Rod of Christ is God’s Anima–linked to the music of our age through TOOL. Soon I will show you the meaning of J, N, and the little o that graces the name of Nero–our historical counterpart for the fiddler who weaves this story into music for us to hear, and see.

The story of Exodus is intended to be read both forwards and backwards, and within its hallowed secrets is a message that links the expulsion of Adam from Eden to an Exodus from Heaven that is mandated by this story in order to do that thing which religion ensures we will: save all life in the Universe. Reading forward, Aaron and his Rod demand that the Pharoah let his people go, and it is only through the reverse reading that we find out definitively who those people are. The story itself is a test, it is God’s search for a team of people that are willing to save everyone by leaving the comfortable confines of Creation–of Heaven–in order to venture out into the vastness of space in order to find dry land. This group is responsible for our continued survival, and for the book and story that are before us. They are responsible for the continued survival of Heaven and of Life by finding the Light of Osiris–the power source that came to me during this very same time period in Bowling Green.

In a world where the Promised Land is both within and without–ours because we are the heart of the Ark of the Covenant, and there too because it is through time travel and science that we find ourselves in a place where time is not as big of an issue as it had once been, and infinite power comes not from seeing that there is an ancient Promised Land shortly after the “Big Bang,” a mere 378,000 years, when power was literally in the air.

This is my divine inspiration, the coincidental discovery and publication of these world-changing pieces of knowledge that coincided perfectly with a story that I was being told. One which linked Exodus to today, the thralls of modern science to a science fiction epic that I was practically living out. These articles were not just shown to me, they were magically appearing in the world to match the Word, at the exact time that interplanetary colonization and the future of our species was the prime focus of the Second Coming. Through the use of time, technology, and love–God was holding my hand and showing me exactly where we would be going.

Like water, Light has a dual meaning in the mythology of this story, and the Light of Osiris was a very clear promise that was given to both me and Jacob–the name that was “given” to the speaker of the words “Expect the Unexpected.” It was a promise of infinite power, one that was to be given to the world in order to fulfill the dream of religion, to ensure the survival of life and the continued evolution of our civilization. In real religion of course, Light is not electrical power–but rather wisdom, and while at first glance this book may seem to revolve around Adam–this is my light. I see what is related to me, and there is a significant amount of light that focuses on one man, on the Christ, for a reason.

True Biblical Light is what graces the pages of Holy Scripture, it is a truth that changes with the throes of time and chance, to become more clear and more useful as our civilization evolves. Stories that once guided the development of society now become a path to the future–as we begin to see that the original purpose of this Light is to ensure that we are not left in the dark. Ender’s Game, the Ewok, and Pan’s Labrynth

“I am the cat with nine lives. You will not prevail against me.” -Nancy Farmer, The Lord of Opium

The Iron Rod of Mars

CopyleftMT

This content is currently released under the GNU GPL 2.0 license. Please properly attribute and link back to the entire book, or include this entire chapter and this message if you are quoting material. The source book is located at . and is written by Adam Marshall Dobrin.

Adam Marshall Dobrin

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I disagree; it is relaxing while the left is bearing most of the body weight.

Cómo estos dos aspectos puntuales se expresan en la clase. Frente a cada punto:

• En el primero, dado que el uso constante del computador implica la posibilidad de tener interrupciones como mensajería instantánea y ventanas alternas del navegador, recomendaría:

• Cerrar aplicaciones de mensajería instantánea y usar una ventana del navegador nueva para la clase.

• Usar el celular al mínimo y tenerlo pantalla abajo, mientras tanto.

• Para el segundo, efectivamente las preguntas son claves y su expresión clara. Cuando no se viene a clase, vale la pena formularlas por escrito a través de correo o lecturas anotadas.

Como veíamos en el video de Clay Shirky, el asunto es que, precisamente, saberes como los que vemos en clase están lejos del cotidiano de la mayoría de las personas, lo cual tiene afecciones respecto a la democracia, el ejercicio de los mecanismos de expresión y la vigilancia de lo común y lo público.

La universidad como espacio, nos permite acceder a conocimientos que no circulan en el cotidiano (ecuaciones diferenciales, modelación de sistemas complejos, ontologías, etc.) y esa es una de sus funciones principales. La pregunta sería más bien qué tipo de cotidiano queremos transformar y cómo estos saberes tienen (o no) el potencial para hacerlo.

La invitación es a que cada cual, que conoce mejor su propio cotidiano y lo que le interesa transformar en él, haga proactivamente y explícitas las preguntas y posibilidades al respecto, ojalá durante la clase.

Musculo pequeño situado en la parte anterior del abdomen en el hueso pubico y la llinea alba

Résumé de la vidéo [00:00:02][^1^][1] - [00:27:38][^2^][2]:

La vidéo suit des élèves entrant dans un internat d'excellence en France, explorant leurs expériences, défis et adaptations à ce nouvel environnement strict mais prometteur.

Moments forts: + [00:00:02][^3^][3] Présentation de l'internat * Internat situé à Sourdin * Accueille 120 élèves de 4e, 3e et seconde * Élèves boursiers et originaires de la région parisienne + [00:02:22][^4^][4] Arrivée des élèves * Découverte des chambres * Premières impressions des élèves * Installation et premières interactions + [00:05:01][^5^][5] Début des cours * Présentation des professeurs * Premières consignes et règles de vie * Réactions des élèves face à la discipline stricte + [00:12:18][^6^][6] Adaptation des élèves * Difficultés rencontrées * Absence d'Internet et de télévision * Limitation de l'usage des téléphones portables + [00:18:40][^7^][7] Retour à la maison * Fin de la première semaine * Réactions des élèves et des parents * Discussions sur l'expérience et les ajustements nécessaires

Résumé de la vidéo [00:27:40][^1^][1] - [00:53:59][^2^][2]:

Cette partie montre les défis et les dynamiques dans un internat. Les élèves doivent faire face à des règles strictes, des punitions et des attentes élevées de la part des enseignants et des surveillants. Certains élèves, comme Monir, tentent de s'adapter et de trouver leur place, tandis que d'autres, comme Reda, luttent contre l'autorité et finissent par être exclus. Le film explore les efforts des élèves pour réussir malgré les difficultés et les conflits internes.

Moments forts: + [00:27:40][^3^][3] Conflits et punitions * Monir est réprimandé pour son comportement * Les élèves doivent faire des exercices de mathématiques * Reda est exclu pour son attitude + [00:30:56][^4^][4] Vie à l'internat * Les élèves s'adaptent aux règles strictes * Les assistants d'éducation luttent pour maintenir l'ordre * Une réunion de recadrage est organisée + [00:34:02][^5^][5] Incidents et sanctions * Un élève montre ses fesses pendant un contrôle * Les sanctions sont discutées et appliquées * Reda est définitivement exclu + [00:39:01][^6^][6] Efforts et progrès * Tony et d'autres élèves montrent des efforts * Les enseignants discutent des progrès des élèves * Les élèves reçoivent des lettres de félicitations ou d'avertissement + [00:46:00][^7^][7] Bilan et perspectives * Le proviseur fait un bilan des 8 semaines * Les élèves sont évalués sur leur comportement et leurs résultats * Les élèves partent en vacances avec des objectifs clairs

Résumé de la vidéo [00:54:02][^1^][1] - [01:21:00][^2^][2]:

Cette partie explore les défis et les aspirations des jeunes dans un internat d'excellence. On y voit des élèves confrontés à des problèmes personnels et scolaires, mais aussi à des moments de camaraderie et de réflexion sur leur avenir.

Moments forts: + [00:54:02][^3^][3] Ambitions et rêves * Monir veut devenir président * Discussions sur les inégalités mondiales * Importance de l'éducation et du pouvoir + [00:56:01][^4^][4] Vie quotidienne à l'internat * Relations familiales et défis personnels * Importance de l'éducation et des activités extrascolaires * Difficultés de certains élèves à s'adapter + [00:59:02][^5^][5] Cérémonie de l'armistice * Hommage aux anciens combattants * Importance de la mémoire historique * Participation des élèves à la cérémonie + [01:03:05][^6^][6] Conseil de classe * Évaluation des élèves * Problèmes de comportement et d'intégration * Encouragements et critiques constructives + [01:17:02][^7^][7] Nouveau règlement intérieur * Introduction d'un code vestimentaire * Réactions des élèves * Sanctions pour non-respect des règles

Résumé de la vidéo [01:21:02][^1^][1] - [01:45:35][^2^][2]:

Cette partie explore les défis et les transformations des élèves dans un internat. Les élèves font face à des problèmes personnels et académiques, mais certains trouvent leur voie et réussissent à surmonter les obstacles.

Moments forts: + [01:21:02][^3^][3] Changement de comportement * Une élève change de comportement après son arrivée à l'internat * Elle se découvre et abandonne son ancienne image * Les tensions dans la classe persistent malgré les efforts + [01:24:05][^4^][4] Conseil de classe * Les professeurs discutent des comportements problématiques * Des élèves reçoivent des punitions pour leurs actions * Les résultats académiques influencent les décisions du conseil + [01:28:02][^5^][5] Orientation scolaire * Les élèves discutent de leurs futures orientations * Certains élèves montrent des améliorations significatives * Les décisions du conseil de classe déterminent leur avenir + [01:34:00][^6^][6] Voyage scolaire * Les élèves participent à un voyage à Chamonix * Ils découvrent de nouvelles activités et renforcent les liens * Des incidents mineurs surviennent mais sont résolus + [01:40:00][^7^][7] Bilan de fin d'année * Les élèves passent leur dernier conseil de classe * Les résultats finaux sont annoncés * Certains élèves quittent l'internat tandis que d'autres continuent leur parcours

I agree with that because continuous data on both axes allows for a clear representation of trends and patterns over time.

Thank you for including this information, I have tried similar to make similar microfluidic channels of different dimensions, but did not consider the differences that light source wavelength would have on different resin types.

12:3 Those who are wi se[a] will shine like the brightness of the heavens, and those who lead many to righteousness, like the stars for ever and ever.

• https://www.americamagazine.org/politics-society/2020/05/08/its-time-rethink-electoral-college

• https://www.npr.org/sections/itsallpolitics/2011/12/20/144016912/we-the-people-npr-readers-would-ratify-four-new-amendments

• https://www.americamagazine.org/politics-society/2020/05/08/its-time-rethink-electoral-college

• https://www.npr.org/sections/itsallpolitics/2011/12/20/144016912/we-the-people-npr-readers-would-ratify-four-new-amendments

• https://constitutioncenter.org/blog/vote-now-an-amendment-to-end-the-electoral-college

• https://www.nytimes.com/2020/02/09/opinion/letters/electoral-college.html

• https://www.latimes.com/opinion/readersreact/la-ol-le-electoral-college-20180904-story.html

you are offline

• https://slate.com/news-and-politics/2014/05/amending-the-constitution-is-much-too-hard-blame-the-founders.html

we the people rise again

• https://slate.com/news-and-politics/2012/06/fix-the-constitution-amending-by-national-referendum.html

safe souls, safe fu

• https://slate.com/news-and-politics/2012/06/fixing-the-constitution-protecting-informational-privacy.html

• https://slate.com/news-and-politics/2020/05/new-reconstruction-constitution-democracy.html

We the People of Slate ...

The U.S. Constitution, as you [mighta been, shoulda "come" on ... its someday] rewrϕte it.

"Politicians talk about the Constitution as if it were as sacrosanct as the Ten Commandments [interjection: spec. it is actually almost exactly related!]. But the document itself invites change and revision. What if the president served only one six-year term instead two four-year terms? What if your state's population determined how many senators represent it? What if the Constitution included a right to health care? We asked legal scholars and Slate readers to cross out what they didn't like in the Constitution and pencil in their hearts' desires. Here's what the document would look like with their best ideas."

• Slate: u_s_constitution as_rewritten by_slate_legal_experts_and_readers

多也了了夕 "with a ~~wand~~ of scheffilara, 并#亦太 he begins ... "I am now on the Staff of Menelaus, the Spears of Longinus and Lancelot; and the name "Mosche ex Nashon."

Logically the recent mentions of Gilgamesh and the simultaneous 同時 overlaping 場道 of the eventual link between the famous ruling of Solomon on the separation of babies and mothers and waters and land ... to a story of many "two cities" that culminates in a cultural or societal or "evolutionary" link to Sodom and Gomorrah and the city-state of Babylon (and it's Hanging Gardens) and also of course to Paris and Troy and "Masstodon" and city-states [ciudadestado] and perhaps planet-cities; from Cambridge to Cambridge across the "Cable" to see state to "London" ... recently I called it "the city of realms" ... I started out logically intending to link "game theory" and John Nash to the mathematical story of Sputnik and a revival of American physics; but in my usual way of rambling into the woods [I mean neighborhood] of stream of consciousness ... turned into a premonitory discourse of "two cities" and how sometimes even things as obvious as the number of letters in the word "two" don't do a good enough job of conveying ... how and/or why one is simply never enough, and two isn't much better--but in the end a circle ... is drawn; the perfect circle in our imaginary mathematical perfection ... I see a parted "line" in the letter pronounced "tea" (and beginning that word); and two "vee" (pron. of "v") symbols joined together in a word we pronounce as "double-you" ... and symbolically because I know "V" is the Roman Numeral for 5 (five) and I know not how to multiply in Roman numerals--

It's important to pause; here. I am going to write a more detailed piece on "the two cities" as I work through this maze like crossroads between "them" and "demo..." ... here demorigstrably I am trying to fuse together an evolutionary change in ... lit. biological evolution as well as an echelon leap forward in "self-government" ... in a place where these two things are unfathomable and unspokenly* connected.

• https://www.google.com/search?q=prometheuslocke+%2Bsite%3Agodlikeproductions.com

• "Silence is betrayal" -MLK

To a question on the idiom; is Bablyon about "the law" or "of the land of Nod?"

"What is democracy" ... the song, Metallica's "ONE" echoes and repeats; as we apparently scrive together the word "THEM" ... I question myself ... if Babylon were the capital city of some mythical Nation of Time ... if it were the central "turning point" of Sheol; ... >|<

Can you not see that in this place; in a world that should see and does there is a gigantic message proving that we are not in reality and trying to show us how and why that's the best news since ... ever---that it's as simple as conjoining "the law of the land" with a basic set of rules that automatically turn Hell into something so much closer to Heaven I just do not understand---why we cant stand up together and say "bullets will not kill innocent children" and "snowflakes will not start avalanches ...." that cover or bury or hide the road from Earth to Verital)e .... or from the mythical Valis to Tanis---or from Rigel to Beth-El ... "guess?"

## as "an easy" answer; I'm looking for a fusion of "law and land" that somehow remembers a "jok'er a scene" about "lawn" seats; and "where the girls are green;"

It's as simple as night and day; Heaven and Hell ... the difference between survival and--what we are presented with here; it's "doing this right"--that ends the Hell of representative democracy and electoral college--the blindness and darkness of not seeing "EXTINCTION LEVEL EVENT" encoded in these words and in our governments foundation ... *by the framers [not just of the USA; but English .. and every language] *

... is literally just as simple as "not caring" or thinking we are at the beginning of some long process--or thinking it will never be done--that special "IT" that's the emancipation of you and I.

Here words like "gnosis" and "gaudeamus" pair with my/ur "new ntersanding*" of the difference between Asgard and Medgard and really understanding our purpose here is to end "evil" ... things like "simulating disease and pain" (here, simulating meaning ... intentionally causing, rather than "gamifying away") and successfully linking the "Pillars of Hercules" to Plato's vision of Atlantis and the letter sequences "an" and "as" ... unlock a fusion of religion and mythology and "cryptographic truth" that connects "messianic" and "Christian" to "Roman" ... "Chinese" and "American" ... literally the key to the difference between the phrases "we are" and "we were" ....

in "sight" of "silicon" in simulation and Israel, Genesis, and "silence" ... trying to the raising of Asgardian enlightenment ... and seeing "simple cypher" connecting to "Norse" ...

and the "I AM THAT" surer than shit ... the intention and design of all religion and creation is to end "simulated reality" and also not seeing "SR" ... in Israel and Norse ... "for instance."

• https://www.google.com/search?q=%22I+AM%22+%22WE+ARE%22+%2Bsite%3Afromtaws
• "SOIS" a key--in two languages conjugated literally as both "I AM" and "WE ARE" simultaneously;
• Search: I know that if I am than so are you ... and it is because we have overcome .... something I truly cannot figure out, fathom, or believe ... was truly here before us--a spiralling series of failures ... speaking: to the heavens; but in secret and in action; "doing everything possible to succeed."

It's a simple linguistic concept; the "singularity" and the "plurality" of a simple word--"to be"--but it goes to the heart of everything that we are and everything that is around us. This is a message about understanding and preserving individuality as well as liberty; and literally seeing "ARXIV" and understanding "often" and failing to connect God and prescience to "IV" and the Fourth Amendment ... it's about blindness and ... "curing the blind instantly" ... and fathoming how and why this message has been etched into our entire history and and all religions and myths and music--to help us "to be THAT we" that actually "are responsible" for the end of Hell.

• I neglected to mention "Har-Wer" and "Tower of Babel" which are both related lingusitically, religiously and topically: "to who ..." and while we're on "four score and [seven years from now]" seeing the fourth "living thing" in Eden and it's (the name, Abel) connection to Babel and Abraham Lincoln; slavery and ... understanding we live in a place where the history of the United States also, like Monoceros and "Neil Armstrong's first step" are a time shifted ... overlayed map to achieving freedom ... it's about becoming a father-race ... and actually "doing" the technological steps required to "emancipate the e's of 'me&e'" and survive in exo-planetary space---

it might be as simple as adding "because we did this" here and now; and having it be something we are truly proud of .... forevermore™ ... for certain in the heart of this story about cyclicality and repetition of error--its not because we did "this" or something over and over again; it's about changing "the problem" and then helping others to also overcome ... "things like time travel ... erasing speech" --- however that happenecl.

• I also failed to mention that "I am in Hell" ... as in this world is hellacious to me; in an overlay with the Hellenic period and this message that we are in the Trojan Horse ... a small gem .... "planet" truly is the Ark of the Covenant---and it's the simple understanding that "reality is hell" is to "living without air conditioning and plumbing is hell" just as soon as you achieve ... "rediscovering" those things---

• I can't figure out why I am the only person screaming "this is Hell." That's also, Hell.

... but recently suggested an old joke about "there being 10 kinds of people in the world (obv an anti-tautology and a tautology simultaneously)" only after that brief bit of singularity and duality mentioning the rest of the joke: "those that understand binary and those that don't know how to base convert between counting with two hands and counting with only an 'on and off.'" It's not obvious if you aren't trying to figure it out, I suppose; but 10 is decimal notation for "kiss" and the "often" without "of" ... and binary notation for the decimal equivalent of "2." A long long time ago in a state that simply non-randomly ties to the heart of the name of our galaxy ... I was again thinking of the "perfect imperfections" of things like saying "three equals one equals one" (which, of course was related to the Holy Trinity and it's "prescient/anachronistic Adamic presence encoded in the name Ab|ra|ha|m" which means "father of a great multitude") ... I brought that one back in the last few months; connecting the letter K and in this "logos-rythmic" tie to the "base of a number system" embellish the truth just a bit and suggest a more accurate rendition of the original [there is no such thing as equality, "is" of separate objects--as in no two snowflakes are the same unless they are literally the same one; true of ancient weights and with the advent of (thinking about) time no two "planets" are the same even if they're the exact same one--unless it's at a fixed moment in time.

• This name may be viewed either as meaning "father of many" in Hebrew or else as a contraction of ABRAM (1) and הָמוֹן (hamon) meaning "many, multitude". The biblical patriarch Abraham was originally named Abram but God changed his name (see Genesis 17:5).

• https://en.wikipedia.org/wiki/Yeshua#Yeshua,_Yehoshua,_and_Yeshu_in_the_Talmud

K=3:11 ... to a handle on the music, the DHD of the gate and the *ring of David's "sling" ...

---and that's a relationship of "3 is to 11" as [the SAT style "analogy)]y" as a series of alpha, two mathematic, and two numeric symbols ... may only tie in my mind alone to the books of Genesis and Matthew and the phrase "chapter and verse" and to the stories of Lot and Job ... again in Genesis and the eponymous "Book of Job." So ... "tying up loose ends one 10b [III] iv. " as it appears I've taken it upon myself to call a Job and suggest is my "Lot in life [x]i* [3]"

• I worry sometimes that important things are missing, or will disappear---for instance Mirriam Webster, which is a "canonical/standard dictionary) should probably have an entry for "lot in life" non-idiomatically as "granny apples to sour apples" as

2 MANY ALSO ICI; 1twoⅱ ... following in Mitnick's bold introductory word steps; the curve and the complement ... the missiles and the canoes; the line and the blank space ... "supposedly two examples of two kinds, which could be three not nothings ... Today I write about something monumental; as if as important as the singularity depicted in Arthur C. Clarke's 2001 "A Space Odyssey" ... and remember a day when I thought it very novel and interesting to see the words "stillborn and yet still born" connected in a single piece of writing to "Stillwater and yet still water" ... today adding in another phrase noting the change wrought only by one magical single "space" (also a single capital letter; and a third phrase): "block chains with a great blockchain."

• http://www.goodmath.org/blog/2015/07/21/arabic-numerals-have-nothing-to-do-with-angle-counting/

• https://gizmodo.com/no-this-viral-image-does-not-explain-the-history-of-ar-1719306568

• https://en.wikipedia.org/wiki/Chinese_word_for_%22crisis%22

• https://dictionary.hantrainerpro.com/chinese-english/translation-ji_howmany.htm
• https://dictionary.hantrainerpro.com/chinese-english/translation-duo_many.htm

• https://en.wikipedia.org/wiki/Euripides, Iphigenia in Aulis or Iphigenia at Aulis[1] (Ancient Greek: Ἰφιγένεια ἐν Αὐλίδι, Iphigeneia en Aulidi; variously translated, including the Latin Iphigenia in Aulide) is the last of the extant works by the playwright Euripides. Written between 408, after Orestes, and 406 BC, the year of Euripides' death, the play was first produced the following year[2] in a trilogy with The Bacchae and Alcmaeon in Corinth by his son or nephew, Euripides the Younger,[3] and won first place at the City Dionysia in Athens.

• The play revolves around Agamemnon, the leader of the Greek coalition before and during the Trojan War, and his decision to sacrifice his daughter, Iphigenia, to appease the goddess Artemis and allow his troops to set sail to preserve their honour in battle against Troy. The conflict between Agamemnon and Achilles over the fate of the young woman presages a similar conflict between the two at the beginning of the Iliad. In his depiction of the experiences of the main characters, Euripides frequently uses tragic irony for dramatic effect.

J.K. Rowling spurred just this past week a series of explanations about just exactly what is a blockchain coin worth ... and why is it so; her final words on the subject (artistic liberty taken, obviously not the last she'll say of this magic moment) "I don't think I trust this."

Taken directly from an off the cuff email to ARXM titled: "Slow the S is ... our Hypothes.is"

I imagine I'll be adding some wiki/ipfs stuff to it--and try to keep it compatible; the design and layout is almost exactly what I was dreaming about seeing--as a "first rough draft product." Lo, and behold. It's been added to the many places I host my tome; the small compilation of nearly every important email that has gone out ... all the way back to the days of the strange looking Margarita glass ... that now very much resembles the "Cantonese character 'le'" which I've come to associate with a "handle" on multiple corners of a room--something like an automatic coat rack conveyor belt connecting different versions of "what's in the box." I'm planning on using that symbol 了 to denote something like multiple forks of the same page. Obviously I'm thinking forward to things like "the Transhumaist Chain Party" (BDSM, right?)'s version of some particular piece of legislation, let's say everything starts with the sprawling "bulbing" of "Amendment M" ideas and specific verbiage ... and then we'll of course need some kind of new git/subversion/cvs style version control mechanism to merge intelligently into something that might actually .... really should ... make it into that place in history--the first constitutional amendment ratified by a "Continental Congress of All People" ... but you could also see it as an ongoing sort of forking of something like the "wikipedia page" on what some specific term, say "technocracy" means, and how two parties might propagandize and change the meaning of such thing; to suit the more intelligent and wise times we now live in. For instance, we might once have had a "democracy" and a "democractic" party that had some Anarchist Cook Book version of the history of it ending in something like Snipes and Stallone's "DEMOLITION MAN."

Just kidding, we all know "democracy" has everything to do with "d is cl ... and not th" ... to be the them that is the heart of the start of the first true democracy. At least the first one I've ever seen, in my old "to a republic" ... style. As it is you can play around with commenting and highlighting and annotating all the stuff I've written and begged and begged for comments on--while I work on layering the backend to to perma-store our ideas and comments on both a blockchain (probably a new one; now that i've worked a little with ethereum) with maybe some key-merkle-tree-walk-search stuff etched into the original Rinkeby ... and then of course distributed data in the "public owned and operated" IPFS. To be clear, I plan on rewriting the backend storage so that we will have a permanent record of all comments; all versions of whatever is being commented on; and changes/revisions to those documents--sort of turning the web into a massive instant "place of collaboration, discussion, and co-authoring" ... if you use the wonderful LEGO pieces that have been handed to us in ideas from places like me, lemma--dissenter, and of course hypothes.is who has brought you and i such a polished and nice to look at "first draft" of something like the living Constitution come repository of all human knowledge. I do sort of secretly wich they would have called this project something like "annotating and reflecting (or real or ...) knowledge" just so the movement could have been called ARK. ... or something .... but whatever join the "calling you a reporter" group or ... "supposedly a scientist?"

NOIR INgR .. I CITE SITE OF ENUDRICAM; a rekindling of the dream of a city appearing high above in the sky, now with a boldly emblazened smiling rainbow and upsidown river ... specifically the antithesis of "angel falls," there's a lagoon too--actually a chain of several ponds underneith the floating rock ... and in some versions of this waking dream there are rings around the thing; you might imagine an artificial set of centripetal orbitals something like a fusion of the ring Eslyeum and the "Six-Axis ride" of the JKF Center's "Spacecamp." I write as I dream, and though I cannot for certain explain exactly how; it's become a strong part of my mythology that this spectacular rendition of "what ends the silence" has something to do with the magical delivery of "a book" ... something not of this Earth but an unnatural thing; one I've dreamt of creating many times. This book is something like the DSM-IV and something like a Merck diagnostic manual; but rather than the old antiquated cures of "the Norse Medgard" this spectacle nearly "itsimportant" autoprints itself and lands on something like every doorpost; what it is is a list of reasons why "simply curing all disease" with no explanation and no conversation would be a travesty of morality--how it would render us half-blind to the myriad of new solutions that can come from truly understanding why "ITIS" to me has become a kind of magical marker: an "it is special" as in, it's cure could possibly solve a number of other problems.

Through that missing "o," English on the ball, we see a connection between a number of words that shine bright light including Exodus itself which means "let there be light," the word for Holy Fire and the Burning Bush.. .reversed to hSE'Ah, and a story about the Second Coming parting our holy waters.**

This answer connects the magical Rod's of Aaron in Exodus and the Iron Rod of Jesus Christ to the Sang Rael itself... in a fusion that explains how the Periodic Table element for Iron links not just to Total Recall and Mars, but also to this key

my dream of what the first day of the Second Coming might be like; were the Rod of Christ... in the right hands. In a story that also spans the Bible, you might understand better how stone to bread and your input make all the difference in the world between Heaven and Adam's Hand. Once more, what do you think He** ....

Since the very earliest days of this story, I have asked for better for you, even than see

Nearly all of the original parts of the original "post-origination dream" remain intact; there's a walkway that magically creates new paths and "attractions" based on where you walk, something like an inversion of the artificial intelligence term "a random walk down a binary tree" ... for instance going left might bring you to the Internet Cafetornaseum of the Earl of Sandwich; and going to the right might bring you to the ICIMAX/Auditorium of Science and Discovery--there's a walkway to "Magical GLAS D'elevators" that open a special "instantiation" of the Japan Room of the Potter and the Toolmaker ... complete with a special [second level and hidden staircase] Pool of Bethesdaibo verily delivering something like youth of mind and body ... or at least as close to such a thing as a sip of Holy Water or Ambrosia or a dip in the pool of Coccoon and Ponce De'Leon could instantly bring ... to those that have seen Jupiter Ascending ... the questions of "nature versus nurture" and what it means to be "old and wise" and "young at heart" truly mean---

• https://www.youtube.com/watch?v=M8CyN1awWls
• https://link.springer.com/chapter/10.1057/9780230366688_16
• https://www.youtube.com/watch?v=YDo5zvYNn3A

Somewhere between the outdoor rafting ride and the level with the special "ballroom of the ancient gallery" ... perhaps now being named or renamed or recalled as something about "Face [of] the Music" lies a magical "mini-maize" ... a look at a mock-up (or #isitit) of Merlink and Harthor's "round table" that displays a series of ... (at least to me) magical appearing holographic displays and controls that my dreams have stolen from Phillip K. Dick's Minority Report and something of what I hope Microsoft's Dynamics/Hololens/Surface will become---a series of short "focus groups" .... to guage and discuss the information in the "CITIES-D5AM-MERCK" ... how to end world hunger and nearly all disease with the press of a magical buzzer--castling churches to something like "political-party-town-hall-meeting centers" and replacing jails and prisons and hospitals with something like the "Hospitalier's PRIDE and DOJOY's I practiced "Kung-fun-dance" ... a fusion of something like a hotel and a school that probably looks very much like a university with classrooms and dorms and dining hall's all fit into a single building. I imagine a series of 2 or 3 "room changes" as in you walk from the one where you get the book and talk about it ... to the one where you talk about "what everyone else said about it" and maybe another one that actually connects you to other people with something like Facebook's Portal; the point of the whole thing to really quickly "rubber stamp" the need for an end to "bars in the sky" nonalcoholic connotation--as in "overcoming the phrase the sky is the limit" and showing us the need for a beacon of glowing hope fulfilled--probably actually the vision of a holographic marker turning into actual rings around the single moon of Earth, the focus of the song annoucing the dawn of the age of Aquarius---

It might lead us also to Ceres; and another set of artificial rings, or to Monoceros and a rehystorical understanding of the birthplace and birthing of the "river roads" that bridge the "space gaps" in the galaxy from our "one giant leap for mankind" linking the Apollo moon landing to the mythological connection to the sun; and connecting how the astrological charts of the ancients might detail a special kind of overlapping--the link between Earth's SOL and something like Proxima or Alpha Centauri; and how that "monostar bridge" might overlap to Orion and from there through Sagitarius and the center of the Milky Way ... all the way to Andromeda and more dreams of being in a place where there's a map to a tri-galactic system in the constellation Cancer and a similar one in Leo ... and just incase you haven't noticed it--a special marker here, I thought to myself it might be cool to "make an acronymic tie to Monoceros" and without even thinking auto-wrote Orion (which was the obvious constellation next to Monoceros, in the charts) and then to Sagitarrius; which is the obvious ... heart of our astrological center and link to "other galaxies."

----I've dreamt or scriven or reguessed numerous times how the Milky Way's map to an "Atlas marked through time by the ages and the ancients" might tie this place and this actual map to the creation of the railways between stars to the beginning and the end of time and of course to this message that links it all to time travel. There's a few "guesses" I've contemplated; that perhaps the Milky Way chart is a metal-cosmic or microcosmic map to the dawn of time in the galactic vision of ... just after the big bang; or it might tie to a map of something like the unthinkable--a civilization that became so powerful it was able to reverse the entropy of "cosmic expansion" and reverse the thing Asimov wrote of in "The Last Question" as the end of life and the ability to survive basically due to "heat loss."

"The Last Question." (And if you read two, why not "The Last Answer"?). Find these readings added to our collection, 1,000 Free Audio Books: Download Great Books for Free.

• https://archive.org/details/texts
• http://zlibraryexau2g3p.onion.pet/

Looking for free, professionally-read audio books from Audible.com, including ones written by Isaac Asimov?

* all "asterisks" in the abovə document denote a sort of Adamic unspoken relationship between notations and meanings; here adding the "Latin word for three" and source of the phrase "t.i.d." (which is doctor/pharmacy latin for "three times a day") where the "t" there is an abbreviation of "ter" ... and suppose the link between K and 11 and 3 noting it's alphanumeric position in the English alphabet as the 11th letter and only linking cognitively to three via the conversion between hex, and binarryy ... aberrative here is the overlapping "hakkasan" style (or ZHIV) lack of mention of the answer in "state of Kansas" and the "citystate of Slovakia" as described in the ICANN document linked [in] the related subsection or slice of the word "binarry" for the state of India. Tetris could be spelled with the addition of only a single letter [in] "tea"---the three letters "ris" are the hearts of the words "Christ" and "wrist" [and arguably of Osiris where you also see the round table character of the solar-system/sun glyph and the chemical element for The Fifth Element (as def. by i) via "Sinbad" and "Superman." The ERIS Free Network should also be mentioned here in connection with the IRC network I associate in the place between skipping stones and sacred hearts defined by "AOL" and "Kdice" in my life. In the lexicon of modern HTML, curly braces are generally relative to "classes" and "major object definitions (javascript/css)" while square brackets generally only take on computer-interpreted meaning in "Markdown" which is clearly (by definition, by this character set "[]") a superset (or at least definately not a subset) of HTML.

Dr. Will Caster (Johnny Depp) is a scientist who researches the nature of sapience, including artificial intelligence. He and his team work to create a sentient computer; he predicts that such a computer will create a technological singularity, or in his words "Transcendence". His wife, Evelyn (played by Rebecca Hall), is also a scientist and helps him with his work.

Following one of Will's presentations, an anti-technology terrorist group called "Revolutionary Independence From Technology" (R.I.F.T.) shoots Will with a polonium-laced bullet and carries out a series of synchronized attacks on A.I. laboratories across the country. Will is given no more than a month to live. In desperation, Evelyn comes up with a plan to upload Will's consciousness into the quantum computer that the project has developed. His best friend and fellow researcher, Max Waters (Paul Bettany), questions the wisdom of this choice, reasoning that the "uploaded"

Just from my general understanding and memory "st" is not ... to me (specifically) an abbreviation of "state" but "ste" is a U.S. Postal code (also "as I understand it") for the name of a special room or set of rooms called a "suite" and in Adamic "connotation" I sometimes read it as "sweet" ... which has several meanings that range from "cool" to "a kind of taste sensation" to "easy to sway or fool."

If you asked me though, for instance if "it" was an abbreviation or shorthand notation or acronym for either "a United state" or "saint" ... you'd be sure.

While it's clear from studying linguistic cryptography ... (If I studied it a little here and some there, its also from the "universal translator of Star Trek") and the personal understanding that language is a kind of intelligent code, and "any code is crackable" ... that I caution here that "meaning" and "face value" often differ widely and wildly ... even in the same place or among the same group of people ... either varying over time or heritage.

Menelaus, in Greek mythology, king of Sparta and younger son of Atreus, king of Mycenae; the abduction of his wife, Helen, led to the Trojan War. During the war Menelaus served under his elder brother Agamemnon, the commander in chief of the Greek forces. When Phrontis, one of his crewmen, was killed, Menelaus delayed his voyage until the man had been buried, thus giving evidence of his strength of character. After the fall of Troy, Menelaus recovered Helen and brought her home. Menelaus was a prominent figure in the Iliad and the Odyssey, where he was promised a place in Elysium after his death because he was married to a daughter of Zeus. The poet Stesichorus (flourished 6th century BCE) introduced a refinement to the story that was used by Euripides in his play Helen: it was a phantom that was taken to Troy, while the real Helen went to Egypt, from where she was rescued by Menelaus after he had been wrecked on his way home from Troy and the phantom Helen had disappeared.

• https://www.britannica.com/topic/Menelaus-Greek-mythology

This article is about the ancient Greek city. For the town of ancient Crete, see Mycenae (Crete). For the hamlet in New York, see Mycenae, New York.

Μυκῆναι, Μυκήνη

The Lion Gate at Mycenae, the only known monumental sculpture of Bronze Age Greece

37°43′49"N 22°45′27"ECoordinates: 37°43′49"N 22°45′27"E

This article contains special characters. Without proper rendering support, you may see question marks, boxes, or other symbols.

Mycenae (Ancient Greek: Μυκῆναι or Μυκήνη, Mykēnē) is an archaeological site near Mykines in Argolis, north-eastern Peloponnese, Greece. It is located about 120 kilometres (75 miles) south-west of Athens; 11 kilometres (7 miles) north of Argos; and 48 kilometres (30 miles) south of Corinth. The site is 19 kilometres (12 miles) inland from the Saronic Gulf and built upon a hill rising 900 feet (274 metres) above sea level.[2]

In the second millennium BC, Mycenae was one of the major centres of Greek civilization, a military stronghold which dominated much of southern Greece, Crete, the Cyclades and parts of southwest Anatolia. The period of Greek history from about 1600 BC to about 1100 BC is called Mycenaean in reference to Mycenae. At its peak in 1350 BC, the citadel and lower town had a population of 30,000 and an area of 32 hectares.[3]

3. Chew 2000, p. 220; Chapman 2005, p. 94: "...Thebes at 50 hectares, Mycenae at 32 hectares..."

• https://en.wikipedia.org/wiki/Clymene_(mythology)

Melpomene (/mɛlˈpɒmɪniː/; Ancient Greek: Μελπομένη, romanized: Melpoménē, lit. 'to sing' or 'the one that is melodious'), initially the Muse of Chorus, she then became the Muse of Tragedy, for which she is best known now.[1] Her name was derived from the Greek verb melpô or melpomai meaning "to celebrate with dance and song." She is often represented with a tragic mask and wearing the cothurnus, boots traditionally worn by tragic actors. Often, she also holds a knife or club in one hand and the tragic mask in the other.

Melpomene is the daughter of Zeus and Mnemosyne. Her sisters include Calliope (muse of epic poetry), Clio (muse of history), Euterpe (muse of lyrical poetry), Terpsichore (muse of dancing), Erato (muse of erotic poetry), Thalia (muse of comedy), Polyhymnia (muse of hymns), and Urania (muse of astronomy). She is also the mother of several of the Sirens, the divine handmaidens of Kore (Persephone/Proserpina) who were cursed by her mother, Demeter/Ceres, when they were unable to prevent the kidnapping of Kore (Persephone/Proserpina) by Hades/Pluto.