Reviewer #1 (Public Review):

Summary:

In this manuscript, Ketaren, Mast, Fridy et al. assessed the ability of a previously generated llama nanobody library (Mast, Fridy et al. 2021) to bind and neutralize SARS-CoV-2 delta and omicron variants. The authors identified multiple nanobodies that retain neutralizing and/or binding capacity against delta, BA.1 and BA.4/5. Nanobody epitope mapping on spike proteins using structural modeling revealed possible mechanisms of immune evasion by viral variants as well as mechanisms of cross-variant neutralization by nanobodies. The authors additionally identified two nanobody pairs involving non-neutralizing nanobodies that exhibited synergy in neutralization against the delta variant. These results enabled the refinement of target epitopes of the nanobody repertoire and the discovery of several pan-variant nanobodies for further preclinical development.

Strengths:

Overall, this study is well executed and provides a valuable framework for assessing the impact of emerging SARS-CoV-2 variants on nanobodies using a combination of in vitro biochemical and cellular assays as well as computational approaches. There are interesting insights generated from the epitope mapping analyses, which offer possible explanations for how delta and omicron variants escape nanobody responses, as well as how some nanobodies exhibit cross-variant neutralization capacity. These analyses laid out a clear path forward for optimizing these promising next-gen therapeutics, particularly in the face of rapidly emerging SARS-CoV-2 variants. This work will be of interest to researchers in the fields of antibody/nanobody engineering, SARS-CoV-2 therapeutics, and host-virus interaction.

Weaknesses:

A main weakness of the study is that the efficacy statement is not thoroughly supported. While the authors comprehensively characterized the neutralizing ability of nanobodies in vitro, there is no animal data involving mice or hamsters to demonstrate the real protective efficacy in vivo. Yet, in the title and throughout the manuscript, the authors repeatedly used phrases like "retains efficacy" or "remains efficacious" to describe the nanobodies' neutralization or binding capacities. This claim is not well supported by the data and underestimates the impact of variants on the nanobodies, especially the omicron sublineages. For example, the authors showed that S1-RBD-15 had a ~100-fold reduction in neutralization titer against Omicron, with an IC50 at around 1 uM. This is much higher than the IC50 value of a typical anti-ancestral RBD nanobody reported in the previous study (Mast, Fridy et al. 2021). In fact, the authors themselves ascribe nanobodies with an IC50 above 1 uM as weak neutralizers. And there were many in the range of 0.1-1 uM. Furthermore, many nanobodies selected for affinity measurement against BA.4/5 had no detectable binding. Without providing in vivo protection data or including monoclonal antibodies that are known to be efficacious against variants in the in vitro assays as a benchmark, it is difficult to evaluate the efficacy just with the IC50 values.

Comments post revision:

The authors are to be commended for their comprehensive response to the referees' comments. In the revised manuscript, the authors made extensive changes throughout the texts and added new figures that greatly improved their clarity. While the manuscript is still limited in solely relying on in vitro data for efficacy assessment, it nicely demonstrates how the combination of experimental and computational techniques could lead to the discovery of broadly neutralizing nanobody candidates for further lead optimization.

Reviewer #1 (Public Review):

Summary:

The Roco proteins are a family of GTPases characterized by the conserved presence of an ROC-COR tandem domain. How GTP binding alters the structure and activity of Roco proteins remains unclear. In this study, Galicia C et al. took advantage of conformation-specific nanobodies to trap CtRoco, a bacterial Roco, in an active monomeric state and determined its high-resolution structure by cryo-EM. This study, in combination with the previous inactive dimeric CtRoco, revealed the molecular basis of CtRoco activation through GTP-binding and dimer-to-monomer transition.

Strengths:

The reviewer is impressed by the authors' deep understanding of the CtRoco protein. Capturing Roco proteins in a GTP-bound state is a major breakthrough in the mechanistic understanding of the activation mechanism of Roco proteins and shows similarity with the activation mechanism of LRRK2, a key molecule in Parkinson's disease. Furthermore, the methodology the authors used in this manuscript - using conformation-specific nanobodies to trap the active conformation, which is otherwise flexible and resistant to single-particle average - is highly valuable and inspiring.

Reviewer #1 (Public Review):

In the presented manuscript, the authors investigate how neural networks can learn to replay presented sequences of activity. Their focus lies on the stochastic replay according to learned transition probabilities. They show that based on error-based excitatory and balance-based inhibitory plasticity networks can self-organize towards this goal. Finally, they demonstrate that these learning rules can recover experimental observations from song-bird song learning experiments.

Overall, the study appears well-executed and coherent, and the presentation is very clear and helpful. However, it remains somewhat vague regarding the novelty. The authors could elaborate on the experimental and theoretical impact of the study, and also discuss how their results relate to those of Kappel et al, and others (e.g., Kappel et al (doi.org/10.1371/journal.pcbi.1003511)). Overall, the work could benefit if there was either (A) a formal analysis or derivation of the plasticity rules involved and a formal justification of the usefulness of the resulting (learned) neural dynamics; and/or (B) a clear connection of the employed plasticity rules to biological plasticity and clear testable experimental predictions. Thus, overall, this is a good work with some room for improvement.

Reviewer #1 (Public Review):

Summary:

The aim of the present work is to evaluate the role of BMP9 and BMP10 in liver by depleting Bmp9 and Bmp10 from the main liver cell types (endothelial cells (EC), hepatic stellate cells (HSC), Kupffer cells (KC) and hepatocytes (H)) using cell-specific cre recombinases. They show that HSCs are the main source of BMP9 and BMP10 in the liver. Using transgenic ALK1 reporter mice, they show that ALK1, the high affinity type 1 receptor for BMP9 and BMP10, is expressed on KC and EC. They have also performed bulk RNAseq analyses on whole liver, and cell-sorted EC and KC, and showed that loss of Bmp9 and Bmp10 decreased KC signature and that KC are replaced by monocyte-derived macrophages. EC derived from these Bmp9fl/flBmp10fl/flLratCre mice also lost their identity and transdifferentiated into continuous ECs. Liver iron metabolism and metabolic zonation were also affected in these mice. In conclusion, this work supports that BMP9 and BMP10 produced by HSC play a central role in mediating liver cell-cell crosstalk and liver homeostasis.

Strengths:

This work further supports the role of BMP9 and BMP10 in liver homeostasis. Using a specific HSC-Cre recombinase, the authors show for the first time that it is the BMP9 and BMP10 produced by HSC that play a central role in mediating liver cell-cell crosstalk to maintain a healthy liver. Although the overall message of the key role of BMP9 in liver homeostasis has been described by several groups, the role of hepatic BMP10 has not been studied before. Thus, one of the novelties of this work is to have used liver cell specific Cre recombinase to delete hepatic Bmp9 and Bmp10. The second novelty is the demonstration of the role of BMP9 and BMP10 in KC Differentiation/homeostasis which has already been slightly addressed by this group by knocking out ALK1, the high affinity receptor of BMP9 and BMP10 (Zhao et al. JCI, 2022).

Weaknesses:

This work remains rather descriptive and the molecular mechanisms are barely touched upon and could have been more explored.<br /> Some references should be added; In particular, a work that has already demonstrated, using a different approach (in situ hybridization RNAscope), that in the liver BMP9 and BMP10 are expressed by HSC (Tillet et al., J Biol Chem 2018). Another publication (Bouvard et al., Cardiovasc Res, 2021) has previously showed that deletion of Bmp9 and Bmp10 leads to liver fibrosis and could have thus been cited. There is also a reference that is not correctly cited. Ref 26 (Herrera et al., 2014) does not say that "BMP10 is mostly expressed in the heart, followed by the liver" or that "BMP9 and BMP10 also bind to ALK2" as cited in the manuscript.<br /> The gating strategies for cell sorting which is used for bulk RNAseq and FACS analyses should be better described in order to better follow the manuscript. This point is particularly important for KC gating as the authors show that Tim4 is very strongly decreased in Bmp9fl/flBmp10fl/flLratCre (Fig 2c), yet, it seems that this marker is used for gating macrophages (Suppl fig4). Same question with F4/80 which is strongly decreased in Bmp9fl/flBmp10fl/flLratCre (Fig 2d) and also used for gating. It is important to show the gating strategy for both Control and Bmp9fl/flBmp10fl/flLratCre mice.<br /> The authors should explain how they selected the genes shown on each heatmaps and add references that can justify the choice of the genes.<br /> Quantifications of Immunostaining and FACS data should be added as well as statistical analyses.

Reviewer #1 (Public Review):

This study holds significant importance as it assessed antibody levels arising from both COVID-19 vaccination and natural infection in a representative population-based sample. The analysis was conducted with thoughtfulness and rigor. The sampling methodology ensured the representation of the broader Canadian population, including minorities and indigenous communities. Findings suggest, that despite a substantial number of individuals having been previously infected, especially following the first omicron wave, repeat booster vaccination is essential to ensure that individuals develop an optimal antibody response against new exposures to infection, given the waning of antibodies over time. The study findings carry global significance as it informs decisions about the relevance of booster vaccination for reducing infection incidence amid the ongoing challenge of vaccine hesitancy and the continual emergence of new variants.

Among the weaknesses of the study, from my perspective, is the lack of explicit clarification that one objective of achieving repeat booster vaccination is to impart a robust level of protection against acquiring infections. Previous studies have demonstrated that the effectiveness of even only primary-series vaccination against COVID-19 severe disease was high, with slow waning over time. However, even when effectiveness against severity is high, infections may still present a risk for progression to severe COVID-19 among older individuals and those with comorbidities. Another limitation is that the study did not investigate whether there were variations in spike levels based on the last vaccine type administered. Furthermore, it is important to comment on the generalizability of the findings considering that individuals who participated in the research may have been different from those who did not participate and therefore residual confounding cannot be eliminated.

Reviewer #1 (Public Review):

Authors investigated the role of OBOX4 in the zygotic genome activation (ZGA) in mice. Obox4 genes form an array of duplicated genes they were identified as a candidate ZGA factor based on expression patterns during early development. The role of OBOX4 was subsequently studied in embryonic stem cells and early embryos. It was found that transcriptional activation mediated by OBOX4 has similar features as that of DUX, which was previously identified as a zygotic transcription factor involved in ZGA and a major activator of the zygotic expression program. It was, however, unexpected that Dux knock-out did not impair embryonic development. The work by Guo et al. provides several lines of evidence that OBOX4-mediated activation of gene expression considerably overlaps with that of DUX and this redundancy might explain the loss of early developmental phenotype in Dux mutants. Consistent with this model, double mutants of Obox4 and Dux show impaired development. Given the difficulties with investigating details of the genetic model in double mutants at the preimplantation embryo stage, authors not only crossed genetic mutants, but also used (1) nuclear transfer of mutated nuclei of ESCs, which could be characterized on their own in separate experiments, and (2) antisense oligonucleotides (ASO) microinjection, which included a rescue control demonstrating that reintroducing OBOX4 is sufficient to rescue the phenotype caused by blocking both, Dux and Obox4.

This work is important for the field because it reveals functional redundancy and plasticity of the zygotic genome activation in mammals, where the mouse model stands as a remarkable example of genome activation, which massively integrated long terminal repeat (LTR)-derived enhancers from retrotransposons and now two of the key activating zygotic factors appear to be encoded by tandemly duplicated clusters of different phylogenetic age. Identification of OBOX4 as a second factor partially redundant with DUX now allows us to decipher what constitutes the essential part of the ZGA program.

Reviewer #1 (Public Review):

Summary:

This study examines how blood vessels exposed to the cytokine VEGF respond to vascular leakage when the VEGF receptor NRP1 is targeted. This study compares results in in two different body sites of the dermis and in a different organ, the trachea. The authors refer to the two different sites of the dermis as two different organs, but the dermis is one organ. The authors report that vascular leakage is differentially affected by NRP1 targeting in the ear skin compared to the trachea and back skin. They attribute these differences to NRP1 presence in cells other than the vascular endothelium, especially in the ear skin, where they observe higher perivascular NRP1 staining.

The manuscript states that the aim was to uncover the role of NRP1 in VEGF-mediated vascular permeability. This was misleading, because a lot is already known on NRP1 in this pathway, as is evidenced by a large number of publications the authors themselves quote (and sometimes misquote). The main information they wish to add is the possibility that NRP1 may also play a role in other cells to regulate permeability, as they previously suggested for blood vessel growth. Several technical issues and experimental limitations call into question whether the above conclusion can be reached with the data provided.

Strengths:

It is an interesting concept that NRP1 regulates vascular permeability by acting in perivascular cells.

Weaknesses:

(A) Technical limitations due to assay type:

A direct comparison of the skin in two body sites is not warranted given that the authors used different methods to study the two sites. Below is a list of differences reported in their methods section:

(A1) Different tracers were used to visualize VEGF165-induced leakage in different sites.<br /> Ear skin assay: 2 kDa FITC and two different dextrans, 10 kDa TRITC dextran, and another dextran whose molecular weight is not specified. It is not explained why 3 different tracers were used. Figures 1 and 2 report data with 2 kDa TRITC dextran.<br /> Back skin assay: They describe the Miles assay using Evans Blue, which binds to albumin, making it a 67 kDa tracer. However, Figure 1 suggests that 2 kDa dextran was used, and perhaps Evans Blue was only used for the supplemental data. This is relevant because current knowledge suggests that small dyes use the junctional pathway, whereas larger proteins such as albumin can use vesicular transport. The former is thought to be a fast pathway (hence, the authors measured dye extravasation 3 min after VEGF165 injection). The latter pathway is a slower one (hence, measured 30 min after VEGF165 injection in the Miles assay).

Quantification: For ear skin, the number of leakage sites and lag period is quantified, as well as leakage over time. For back skin, the amount of extravasated dye is quantified at a fixed time point. Such different measurements do not allow for direct comparison.

(A2) Mice were prepared in different ways for the different body sites studied:<br /> Ear skin assay: general anesthesia with ketamine-xylazine.<br /> Back skin assay: No anesthesia is described for the back skin Miles assay. This would be a concern because intradermal injections are considered to be painful. For back skin histology, they do report to have used isoflurane anesthesia before perfusion fixation. However, it is not advisable to use used isoflurane anesthesia for perfusion fixation if this has been done via the conventional cardiac route, because opening the chest cavity to access the heart for perfusion causes lung collapse, meaning that the mice cannot breathe the anaesthetic, and there is a risk of them regaining consciousness. The authors should clarify what exactly they have done, for ethical reasons and also because the type of anesthesia can affect vascular studies, for example, see PMID 36418078.

(A3) Differential histamine use:<br /> Back skin assay: uses anti-histamine, as is advised with intradermal injections to minimize vascular leakage due to histamine release after local trauma.<br /> Ear skin assay: no anti-histamine was used, so histamine-induced background leakage might have been present, independently of VEGF165. The authors suggest that the ear skin injection does not cause trauma, but it is unclear how this is possible, given that skin needs to be disrupted for the needle to enter the tissue.

(A4) Different VEGF165 concentration used:<br /> The ear skin assay uses 10 ng VEGF per injection, and the back skin assay 80 ng.

Given all these differences in experimental protocols, as well as different knockdown efficiency (see below), the results for the different sites are not directly comparable. Hence it cannot presently be concluded that the role of NRP1 in both sites is different, and further work is required to make a firm conclusion. In addition, the conflicts between the reported methods and figures need to be resolved.

(B) It is unclear whether appropriate controls were used:

(B1) What genotype and treatment are the control mice for NRP1 targeting? The ideal control would be wild-type mice with the same CreER, injected with tamoxifen according to the same timeline, to account for vehicle, tamoxifen, and tamoxifen-induced CreER toxicity (https://doi.org/10.1038/s44161-022-00125-6). This could be a littermate mouse or, alternatively, a separate experiment should be shown comparing wild-type mice carrying the same CreER as used for the ablation studies and injected with tamoxifen, versus wild-type mice injected with tamoxifen, to demonstrate that the induction regime does not in itself cause phenotypes.

(B2) Has a PBS injection been performed to compare baseline leakage between genotypes, independently of VEGF165 injections? This is an essential control.

(B3) The experimental protocol assays 4 days after 5 consecutive tamoxifen injections, which does not allow much time for drug washout. Moreover, this is a lot of tamoxifen (80 mg x 5 = 400 mg tamoxifen per kg). Due to the possibility that tamoxifen-induced effects might still be present and cause sex-differential effects, the corresponding sex for each individual data point should be indicated in all graphs.

(B4) i.p. peanut oil is used in undefined volumes; this vehicle was shown to cause inflammation if administered i.p. (PMID 33139505). Therefore, inflammation might be present, which might affect different body sites differently.

(C) Validation of NRP1 targeting:<br /> The authors have not performed an NRP1 knockout in the endothelium, as they repeatedly claim. In the lung, there is a good knockdown of around 75%; this may or may not be due to complete EC knockdown with preservation of NRP1 in other cell types. In the trachea, ear skin, and back skin, knockdown was not quantified, although qualitative comparisons by NRP1 immunostaining in Supplementary Figure 1 suggest that the back skin targeting worked better than the ear skin targeting, which would confound results, but in any case, it was neither a knockdown nor knockout. The staining for global targeting looks fainter than for the other genotypes, and the single-channel images seem to have different intensities than the overlays in Supplementary Figure 1 A.

(D) Systemic permeability studies:<br /> Organs have very different baseline permeability, due to the properties of the vascular barrier, i.e. tight barriers in the brain and retina and permeable endothelium in the liver and kidney. In this assay, VEGF is not delivered from the tissue side, as would be typical during inflammation but is delivered through the circulation, which has been shown to differentially affect the VEGF response, at least in some tissues (PMID 25175707). Nevertheless, this is a helpful readout, especially given that PBS controls appear not to have been performed above to establish baseline leakage between genotypes and tissues.

Figure Supplement 3 shows that VEGF induces vascular leakage in all body sites examined, independently of the size of the tracer used, and agreeing with current literature. An additional set of panels should be included with data shown without calculating the fold change relative to the control, set to 1, to account for the endothelium in different organs having different baseline vascular permeability. How do the authors explain that VEGF has the same effect in the ear and back skin in this assay, when NRP1 is present, given that they claim a role for perivascular NRP1 in the ear, but not back skin, for reducing VEGF/VEGFR2 signalling?

(E) Comparing results obtained with different tools:

- The endothelial NRP1 knockdown yielded different results for ear and back skin.<br /> - Anti-NRP1 yielded similar results for ear and back skin.<br /> - The global NRP1 ko yielded similar results for ear and back skin.<br /> Because anti-NRP1 and the global NRP1 knockdown gives similar results for all tissues, the authors deduce that the NRP1 acts in cell types other than endothelial cells to regulate permeability. This is an interesting idea, based on the lab's prior work in angiogenesis. In their trans-interaction scenario, NRP1 would have the same role in ECs in all sites, but non-endothelial NRP1 can override the function of the endothelial NRP1 function depending on its expression levels.

Confidence in this conclusion would require additional experiments:<br /> - Show that the endothelial knockdown works equally well in different body sites, via NRP1 staining and/or by checking recombination efficiency with a reporter.<br /> - Using an analogous assay to measure permeability in different body sites.<br /> - Perform a non-endothelial knockdown, i.e. in pericytes, which is hypothesized to be the source of NRP1 that affects vascular leakage signalling in endothelial cells in trans.

(F) Abstract, introduction, and references:<br /> The authors suggest controversy with regard to NRP1's roles in permeability. However, NRP1's function in VEGF signalling has been defined as being an accessory to VEGFR2, with a role in promoting SFK activation. This function relies on the NRP1 cytoplasmic domain, which mediates VEGFR2 trafficking and signalling; the relevant literature for the NRP1 cytoplasmic domain is mentioned for arteriogenesis (PMID 23639442), but not permeability (PMID 28289053). Another paper is mentioned which describes a VEGFR2-independent pathway for a CendR ligand, but this prior study did NOT make the claim that VEGF signalling is NRP1-independent or promotes it (PMID 27117252). In the eye, NRP1 has been implicated in both SEMA3A and VEGF165-induced permeability, which was also corroborated by the Miles assay in two prior studies (PMID 18180379, PMID 28289053). The last sentence in the abstract is incorrect, because differences in ear versus back skin do not constitute organotypic difference (as the organ is the dermis), and the potential role of perivascular cells is only inferred from the global endothelial NRP1 knockdown, which gives the same result as reported for the endothelial NRP1 knockdown in the literature.

(1) Lines 5/.53: The references for VEGF-NRP1 signalling in age-related macular degeneration are not helpful: Raimondi investigated VEGF-independent NRP1 pathways in angiogenesis, Fernandez-Robredo investigated NRP1 pathways in angiogenesis and showed that fewer vessels correlated with less leakage but did not test VEGF signaling specifically. A more suitable reference would have been PMID 28289053.

(2) Lines 63/64 and repeated in 84-89: The references quoted all showed that NRP1 inhibition reduces vascular permeability, and therefore do not provide evidence for the idea that NRP1 inhibition promotes permeability, as the authors report here for the ear skin; the only study supporting them is one using arterial endothelial cells, which are not permeability-relevant.

(3) Lines 106/107: The references used to underpin organ-specific barrier properties are correct, but as stated above, the dermis is the dermis, and therefore, these references would not be useful to provide support for the idea that the ear and back skin behave differently after NRP1 knockdown.

(G) Additional comments on the figures:<br /> Figure 4: The authors show that VEGFR2 is essential for permeability, and VEGF164 effects are VEGFR2 dependent - this is well established for VEGF164 in the Miles assay, including the accessory role of NRP1 (e.g. PMID 28289053). As the proposed trans function of NRP1 cannot make a difference in VEGFR2 signaling when VEGFR2 is not there, this experiment is only confirmatory of prior VEGFR2 knowledge.

Reviewer #1 (Public Review):

Summary:

In this article, Kremser et al set off to explore how local interactions between cells can drive pattern formation by focusing on the French flag problem whereby an initially homogeneous system breaks axial symmetry to form three distinct regions of different cell fates. The authors use a cellular automata model together with evolution searches on possible rules that determine cell state and tissue level patterning. It is assumed that three cell states are possible and that at each time iteration each cell updates its fate according to the current state of itself and its neighbours. The authors use a computational procedure based on evolution algorithms to identify "fit" update rules that can successfully drive patterning into three distinct domains and go on to provide insights with regards to the function of these rules as well as their properties such as robustness and patterning dynamics. The article is generally well-written, the results seem solid, and the analysis and methods are thorough and generally well-explained. A main concern is the lack of connection between the biology that motivated the analysis and the results, this could be improved in the discussion by making the methods somewhat more concise to allow space to make links back to potential biological mechanisms when the results are presented. We raise some general points and some more specific questions and suggestions for clarification below that we hope will help improve the MS and make it more accessible to a wider audience.

General points:

• Although the authors motivate their work on the premise that biological patterns at the tissue level often are driven by local cell-cell interactions, by the end of the analysis any possible connection to the underlying biology is lost. For example, it would have been useful to discuss how the rules that evolved to dominate the patterning process in the results section could be implemented by cells. Is there a connection that could be made back to Notch signalling and its multiple ligands or to morphogens that diffuse only locally? Would the large number of rules possible in the cellular automata context reflect transcriptional feedback? This is an important point to bring the work "home". At the moment, it feels like a nice computational analysis of cellular automata but the links to the systems that motivate the work are lost in the process.

• When growth is considered (p.14-15) a discussion of timescales seems pertinent. Often patterning takes place at a timescale faster than cell division so the system could be allowed to reach a steady state before a new division event takes place. What are the time scales of updating the phenotype compared with the time scales of division in the model and in relevant biological systems? How would different limiting cases impact conclusions, e.g. new cells added and pattern allowed to reach steady state before more growth versus cells added while patterning dynamics are still updating?

• An interesting question is whether certain elements of rules (out of the 27 possible elements for the system with 3 states) are more or less likely to appear together in an evolved final rule. This may give a mechanistic understanding of what combinations of elements are likely to drive the optimal pattern and which combinations are avoided altogether.

Reviewer #2 (Public Review):

In this study, Torcq and colleagues make carefull observations of the cellular morphology of haemogenic endothelium undergoing endothelial to haematopoietic transition (EHT) to become stem cells, using the zebrafish model. To achieve this, the used an extensive array of transgenic lines driving fluorescent markers, markers of apico-basal polarity (podocalixin-FP fusions) or tight junction markers (jamb-FP fusions). The use of the runx truncation to block native Runx1 only in endothelial cells is an elegant tool to achieve something akin to tissue-specific deletion of Runx1. Overall, the imaging data is of excellent quality. They demonstrate that differences in apico-basal polarity are strongly associated with different cellular morphologies of cells undergoing EHT from HE (EHT pol- and EHT pol+) which raises the exciting possibility that these morphological differences reflect heterogeneity of HE (and potentially HSCs, but this is not addressed in this manuscript) at a very early stage. They then overexpress a truncated form of Runx1 (just the runt domain) to block Runx1 function and show that more HE cells abort EHT and remain associated with the embryonic dorsal aorta. The revised version identifies pard3ab as differentially distributed in dtRunx mutants and correlates that distribution with a potential regulatory role on cell polarity. No direct evidence for their role in EHT is presented.

The manuscript has now been streamlined and reference to figures made much clearer. It provides for a clearer reading, and clearly a well thought out discussion of HE, polarity and the regulation of the EHT process. The evidence for the different cellular morphologies of cells undergoing EHT is strong, and the main claim that tuning apico-basal polarity and junctional recycling underlie morphological complexity of EHT (rather than of HSCs) is well supported by the data.

Reviewer #1 (Public Review):

Summary

The authors investigated the antigenic diversity of recent (2009-2017) A/H3N2 influenza neuraminidases (NAs), the second major antigenic protein after haemagglutinin. They used 27 viruses and 43 ferret sera and performed NA inhibition. This work was supported by a subset of mouse sera. Clustering analysis determined 4 antigenic clusters, mostly in concordance with the genetic groupings. Association analysis was used to estimate important amino acid positions, which were shown to be more likely close to the catalytic site. Antigenic distances were calculated and a random forest model used to determine potential important sites.

This revision has addressed many of my concerns of inconsistencies in the methods, results and presentation. There are still some remaining weaknesses in the computational work.

Strengths

(1) The data cover recent NA evolution and a substantial number (43) of ferret (and mouse) sera were generated and titrated against 27 viruses. This is laborious experimental work and is the largest publicly available neuraminidase inhibition dataset that I am aware of. As such, it will prove a useful resource for the influenza community.

(2) A variety of computational methods were used to analyse the data, which give a rounded picture of the antigenic and genetic relationships and link between sequence, structure and phenotype.

(3) Issues raised in the previous review have been thoroughly addressed.

Weaknesses:

Some concerns regarding the robustness of the machine learning model and potential overfitting remain.

Reviewer #2 (Public Review):

Summary:

The manuscript by Kandoi et al. describes a new 3D retinal organoid model of a mono-allelic copy number variant of the rhodopsin gene that in a patient led to autosomal dominant retinitis pigmentosa. The evidence provided here is relatively strong that the rod photoreceptor phenotype observed in an adult patient with RP in vivo is similar to that phenotype observed in human stem cell-derived retinal organoids. Increases in RHO expression were detected by qPCR, RNA-seq, and IHC support this phenotype. Importantly, the amelioration of photoreceptor rhodopsin mislocalization and related defects using the small molecule drug photoregulin demonstrates an important potential clinical application.

Strengths:<br /> - Retinal organoids derived from patient with adRP.<br /> - RHO mislocalization could explain the phenotype in patients.

Weaknesses:

- Organoids at 300 days do not show PR loss.

Additional minor weaknesses

- Bulk RNAseq methods require greater detail, particularly with respect to how total or mRNA was purified, how was it quantified for concentration and integrity (i.e. Nanodrop, Tape station, Bioanalyzer), what reagents were used for library preparation and how many reads were analyzed per sample.

- Fig. 4. The levels of RHO visualized in tissue sections (panels A-C) does not seem to match the general levels shown for the western blots (panel D) which appear to be far higher in RM western blot samples than in the IHC images. Please clarify why there is such a difference.

- Line 186: by what criteria are the authors able to state that " there were no clear visible anatomical changes in apical-basal retinal cell type distribution (data not shown)". Was this based on histological staining with antibodies, nuclear counter-staining or some other evaluation?

Reviewer #1 (Public Review):

Valk and Engert et al. examined the potential relations between three different mental training modules, hippocampal structure and functional connectivity, and cortisol levels (stress) over a 9-month period. They found that among the three types of mental training: Presence (attention and introspective awareness), Affect (socio-emotional - compassion and prosocial motivation), and Perspective (socio-cognitive - metacognition and perspective taking) modules; Affect training most robustly related to changes in hippocampal structure and function - specifically, CA1-3 subfields of the hippocampus. Moreover, change in intrinsic functional connectivity related to changes in diurnal cortisol release and long-term cortisol exposure. These changes are proposed to result from a combination of factors, which is supported by multivariate analyses showing changes across subfields and training content relate to cortisol changes.

The authors demonstrate that mindfulness training programs are a potential avenue for stress interventions that impact hippocampal structure and cortisol, providing a promising approach to improve health. The data contribute to the literature on plasticity of hippocampal subfields during adulthood, the impact of mental training interventions on the brain, and the link between CA1-3 and both short- and long-term stress changes.

The authors thoughtfully approached the study of hippocampal subfields, utilizing a method designed for T1w images that outperformed Freesurfer 5.3 and that produced comparable results to an earlier version of ASHS. The authors note the limitations of their approaches and provide detailed information on the data used and analyses conducted. The results provide a strong basis from which future studies can expand using computational approaches or more fine-grained investigations of the impact of mindfulness training on cortisol levels and the hippocampus.

Reviewer #1 (Public Review):

The manuscript considers a hierarchical network of neurons, of the type that can be found in sensory cortex, and assumes that they aim to constantly predict sensory inputs that may change in time. The paper describes the dynamics of neurons and rules of synaptic plasticity that minimize the integral of prediction errors over time.

The manuscript describes and analyses the model in great detail, and presents multiple and diverse simulations illustrating the model's functioning. However, the manuscript could be made more accessible and easier to read. The paper may help to understand the organization of cortical neurons, their properties, as well as the function of its particular components (such as apical dendrites).

Reviewer #1 (Public Review):

The authors start from the premise that neural circuits exhibit "representational drift" -- i.e., slow and spontaneous changes in neural tuning despite constant network performance. While the extent to which biological systems exhibit drift is an active area of study and debate (as the authors acknowledge), there is enough interest in this topic to justify the development of theoretical models of drift.

The contribution of this paper is to claim that drift can reflect a mixture of "directed random motion" as well as "steady state null drift." Thus far, most work within the computational neuroscience literature has focused on the latter. That is, drift is often viewed to be a harmless byproduct of continual learning under noise. In this view, drift does not affect the performance of the circuit nor does it change the nature of the network's solution or representation of the environment. The authors aim to challenge the latter viewpoint by showing that the statistics of neural representations can change (e.g. increase in sparsity) during early stages of drift. Further, they interpret this directed form of drift as "implicit regularization" on the network.

The evidence presented in favor of these claims is concise, but on balance I find their evidence persuasive, at least in artificial network models. This paper includes a brief analysis of four independent experiments in Figure 3, which corroborates the main claims of the paper. Future work should dig deeper into the experimental data to provide a finer grained characterization. For example, in addition to quantifying the overall number of active units, it would be interesting to track changes in the signal-to-noise ratio of each place field, the widths of the place fields, et cetera.

To establish the possibility of implicit regularization in artificial networks, the authors cite convincing work from the machine learning community (Blanc et al. 2020, Li et al., 2021). Here the authors make an important contribution by translating these findings into more biologically plausible models and showing that their core assumptions remain plausible. The authors also develop helpful intuition in Figure 5 by showing a minimal model that captures the essence of their result.

Reviewer #1 (Public Review):

In this manuscript, Nagel et al. sought to characterize the composition of urinary compounds, some of which are putative chemosignals. They used urines from adult males and females in three different strains, including one wild-derived strain. By performing mass spectrometry of two classes of compounds: volatile organic compounds and proteins, they found that urines from inbred strains are qualitatively similar to those of a wild strain. This finding is significant because there is a high degree of diversity in different inbred strains and wild mice, with respect to the polymorphisms of chemosensory receptor genes and expression of vomeronasal ligands previously identified. Notably, their study did not characterize steroids, which represent a major class of urinary chemosignals activating vomeronasal neurons. Therefore, important future studies should address the strain dependence of steroid composition in urines.

In the second part of this work, the authors used calcium imaging to monitor the pattern of vomeronasal neuron responses to these urines. By performing pairwise comparisons, the authors found a large degree of strain-specific response and a relatively minor response to sex-specific urinary stimuli. This is a finding generally in agreement with previous calcium imaging work by Ron Yu and colleagues in 2008. The authors extend the previous work by using urines from wild mice. They further report that the concentration diversity of urinary compounds in different urine batches is largely uncorrelated with the activity profiles of these urines. In addition, the authors found that the patterns of vomeronasal neuron response to urinary cues are not identical when measured using different recipient strains.

The pitfalls of this study are the omission of steroids for the mass spectrometry experiments and the indirect (correlational) nature of their mass spectrometry data and activity data. Whether the urinary compounds identified in this study activate vomeronasal neurons were not tested.

Nevertheless, the major contribution of this work is the identification of specific molecules in mouse urines. This work is likely to be of significant interest to researchers in chemosensory signaling in mammals and could provide a systematic avenue to exhaustively identify additional pheromones in mice.

Reviewer #1 (Public Review):

Summary:

This important study investigated the role of the PHOX2B transcription factor in neurons in the key brainstem chemosensory structure, the retrotrapezoid nucleus (RTN), for maintaining proper CO2 chemoreflex responses of breathing in the adult rat in vivo. PHOX2B has an important transcriptional role in neuronal survival and/or function, and mutations of PHOX2B severely impair the development and function of the autonomic nervous system and RTN, resulting in the developmental genetic disease congenital central hypoventilation syndrome (CCHS) in neonates, where the RTN may not form and is functionally impaired. The function of the wild-type PHOX2B protein in adult RTN neurons that continue to express PHOX2B is unknown. By utilizing a viral PHOX2B-shRNA approach for the knockdown of PHOX2B specifically in RTN neurons, the authors' solid results show impaired ventilatory responses to elevated inspired CO2, measured by whole-body plethysmography in freely behaving adult rats, that develop progressively over a four-week period in vivo, indicating effects on RTN neuron transcriptional activity and associated blunting of the CO2 ventilatory response. The RTN neuronal mRNA expression data presented suggests the impaired hypercapnic ventilatory response is possibly due to the decreased expression of key proton sensors in the RTN. This study will be of interest to neuroscientists studying respiratory neurobiology as well as the neurodevelopmental control of motor behavior.

Strengths:

(1) The authors used a shRNA viral approach to progressively knock down the PHOX2B protein, specifically in RTN neurons, to determine whether PHOX2B is necessary for the survival and/or chemosensory function of adult RTN neurons in vivo.

(2) To determine the extent of PHOX2B knockdown in RTN neurons, the authors combined RNAScope® and immunohistochemistry assays to quantify the subpopulation of RTN neurons expressing PHOX2B and Neuromedin B (Nmb), which has been proposed to be key chemosensory neurons in the RTN.

(3) The authors demonstrate that knockdown efficiency is time-dependent, with a progressive decrease in the number of Nmb-expressing RTN neurons that co-express PHOX2B over a four-week period.

(4) Their results convincingly show hypoventilation, particularly in 7.2% CO2 only, for PHOX2B-shRNA RTN-injected rats after four weeks compared to naïve and non-PHOX2B-shRNA targeted (NT-shRNA) RTN-injected rats, suggesting a specific impairment of chemosensitive properties in RTN neurons with PHOX2B knockdown.

(5) Analysis of the association between PHOX2B knockdown in RTN neurons and the<br /> attenuation of the hypercapnic ventilatory response (HCVR), by evaluating the correlation between the number of Nmb+/PHOX2B+ or Nmb+/PHOX2B- cells in the RTN and the resulting HCVR, showed a significant correlation between HCVR and number of Nmb+/PHOX2B+ and Nmb+/PHOX2B- cells, suggesting that the number of PHOX2B-expressing cells in the RTN is a predictor of the chemoreflex response and the reduction of PHOX2B protein impairs the CO2-chemoreflex.

(6) The data presented indicate that PHOX2B knockdown reduces the HCVR and the expression of Gpr4 and Task2 mRNAs. This suggests that PHOX2B knockdown affects RTN neurons' transcriptional activity and decreases the CO2 response, possibly by reducing the expression of key proton sensors in the RTN.

(7) This study's results show that independent of its role during development, PHOX2B is still required to maintain proper CO2 chemoreflex responses in the adult brain, and its reduction in CCHS may contribute to the respiratory impairment in this disorder.

Weaknesses:

(1) The authors found a significant decrease in the total number of Nmb+ RTN neurons (i.e., Nmb+/PHOX2B+ plus Nmb+/ PHOX2B-) in NT-shRNA rats at two weeks post viral injection, and also at the four-week period where the impairment of the chemosensory function of the RTN became significant, suggesting some inherent cell death possibly due to off-target toxic effects associated with shRNA procedures.

(2) The tissue sampling procedures for quantifying numbers of cells expressing proteins/mRNAs throughout the extended RTN region bilaterally have not been completely validated to accurately represent the full expression patterns in the RTN under the experimental conditions.

(3) The inferences about RTN neuronal expression of NMB, GPR4, or TASK2 are based on changes in mRNA levels, so it remains speculation that the observed reduction in Gpr4 and Task2 mRNA translates to a reduction in the protein levels and associated reduction of RTN neuronal chemosensitive properties.

Reviewer #1 (Public Review):

Summary:

The aim of the study described in this paper was to test whether visual stimuli that pulse synchronously with the systole phase of the cardiac cycle are suppressed compared with stimuli that pulse in the diastole phase. To this end, the authors employed a binocular rivalry task and used the duration of the perceived image as the metric of interest. The authors predicted that if there was global suppression of the visual stimulus during systole then the durations of the stimulus that were pulsing synchronously with systole should be of shorter duration than those pulsing in diastole. However, the results observed were the opposite of those predicted. The authors speculate on what this facilitation effect might mean for the baroreceptor suppression hypothesis.

Strengths:

This is an interesting and timely study that uses a clever paradigm to test the baroreceptor suppression hypothesis in vision. This is a refreshingly focussed paper with interesting and seemingly counterintuitive results.

Weaknesses:

The paper could benefit from a clearer explanation of the predicted results. For those not experts in binocular rivalry, it would be useful to explain the predicted results. Does pulsing stimuli in this way change durations in such a task? If there is global suppression of visual stimuli why would this lead to shorter/longer durations in the systole compared to the diastole conditions? In addition, the duration lengths in both conditions seem to be longer than one cardiac cycle. If the cardiac cycle modulates duration it would be interesting to discuss why this occurs on some cycles but not on others. If there is a facilitation effect why does it only occur on some cycles?

Reviewer #1 (Public Review):

Summary:

Pham and colleagues provide an illuminating investigation of aquaporin-4 water flux in the brain utilizing ex vivo and in vivo techniques. The authors first show in acute brain slices, and in vivo with fiber photometry, SRB-loaded astrocytes swell after inhibition of AQP4 with TGN-020, indicative of tonic water efflux from astrocytes in physiological conditions. Excitingly, they find that TGN-020 increases the ADC in DW-MRI in a region-specific manner, potentially due to AQP4 density. The resolution of the DW-MRI cannot distinguish between intracellular or extracellular compartments, but the data point to an overall accumulation of water in the brain with AQP4 inhibition. These results provide further clarity on water movement through AQP4 in health and disease.

Overall, the data support the main conclusions of the article, with some room for more detailed treatment of the data to extend the findings.

Strengths:

The authors have a thorough investigation of AQP4 inhibition in acute brain slices. The demonstration of tonic water efflux through AQP4 at baseline is novel and important in and of itself. Their further testing of TGN-020 in hyper- and hypo-osmotic solutions shows the expected reduction of swelling/shrinking with AQP4 blockade.

Their experiment with cortical spreading depression further highlights the importance of water efflux from astrocytes via AQP4 and transient water fluxes as a result of osmotic gradients. Inhibition of AQP4 increases the speed of tissue swelling, pointing to a role in the efflux of water from the brain.

The use of DW-MRI provides a non-invasive measure of water flux after TGN-020 treatment.

Weaknesses:

The authors specifically use GCaMP6 and light sheet microscopy to image their brain sections in order to identify astrocytic microdomains. However, their presentation of the data neglects a more detailed treatment of the calcium signaling. It would be quite interesting to see whether these calcium events are differentially affected by AQP4 inhibition based on their cellular localization (ie. processes vs. soma vs. vascular end feet which all have different AQP4 expressions).

The authors show the inhibition of AQP4 with TGN-020 shortens the onset time of the swelling associated with cortical spreading depression in brain slices. However, they do not show quantification for many of the other features of CSD swelling, (ie. the duration of swelling, speed of swelling, recovery from swelling).

Significance:

AQP4 is a bidirectional water channel that is constitutively open, thus water flux through it is always regulated by local osmotic gradients. Still, characterizing this water flux has been challenging, as the AQP4 channel is incredibly water-selective. The authors here present important data showing that the application of TGN-020 alone causes astrocytic swelling, indicating that there is constant efflux of water from astrocytes via AQP4 in basal conditions. This has been suggested before, as the authors rightfully highlight in their discussion, but the evidence had previously come from electron microscopy data from genetic knockout mice.

AQP4 expression has been linked with the glymphatic circulation of cerebrospinal fluid through perivascular spaces since its rediscovery in 2012 [1]. Further studies of aging[2], genetic models[3], and physiological circadian variation[4] have revealed it is not simply AQP4 expression but AQP4 polarization to astrocytic vascular endfeet that is imperative for facilitating glymphatic flow. Still, a lingering question in the field is how AQP4 facilitates fluid circulation. This study represents an important step in our understanding of AQP4's function, as the basal efflux of water via AQP4 might promote clearance of interstitial fluid to allow an influx of cerebrospinal fluid into the brain. Beyond glymphatic fluid circulation, clearly, AQP4-dependent volume changes will differentially alter astrocytic calcium signaling and, in turn, neuronal activity.

(1) Iliff, J.J., et al., A Paravascular Pathway Facilitates CSF Flow Through the Brain Parenchyma and the Clearance of Interstitial Solutes, Including Amyloid β. Sci Transl Med, 2012. 4(147): p. 147ra111.<br /> (2) Kress, B.T., et al., Impairment of paravascular clearance pathways in the aging brain. Ann Neurol, 2014. 76(6): p. 845-61.<br /> (3) Mestre, H., et al., Aquaporin-4-dependent Glymphatic Solute Transport in the Rodent Brain. eLife, 2018. 7.<br /> (4) Hablitz, L., et al., Circadian control of brain glymphatic and lymphatic fluid flow. Nature Communications, 2020. 11(1).

Reviewer #1 (Public Review):

Yun et al. examined the molecular and neuronal underpinnings of changes in Drosophila female reproductive behaviors in response to social cues. Specifically, the authors measure the ejaculate-holding period, which is the amount of time females retain male ejaculate after mating (typically 90 min in flies). They find that female fruit flies, Drosophila melanogaster, display shorter holding periods in the presence of a native male or male-associated cues, including 2-Methyltetracosane (2MC) and 7-Tricosene (7-T). They further show that 2MC functions through Or47b olfactory receptor neurons (ORNs) and the Or47b channel, while 7-T functions through ppk23 expressing neurons. Interestingly, their data also indicates that two other olfactory ligands for Or47b (methyl laurate and palmitoleic acid) do not have the same effects on the ejaculate-holding period. By performing a series of behavioral and imaging experiments, the authors reveal that an increase in cAMP activity in pC1 neurons is required for this shortening of the ejaculate-holding period and may be involved in the likelihood of remating. This work lays the foundation for future studies on sexual plasticity in female Drosophila.

The conclusions of this paper are mostly supported by the data, but aspects of the lines used for individual pC1 subtypes and visual contributions as well as the statistical analysis need to be clarified.

(1) The pC1 subtypes (a - e) are delineated based on their morphology and connectivity. While the morphology of these neurons is distinct, they do share a resemblance that can be difficult to discern depending on the imaging performed. Additionally, genetic lines attempting to label individual neurons can easily be contaminated by low-level expression in off-target neurons in the brain or ventral nerve cord (VNC), which could contribute to behavioral changes following optogenetic manipulations. In Figures 5C - D, the authors generated and used new lines for labeling pC1a and pC1b+c. The line for pC1b+c was imaged as part of another recent study (https://doi.org/10.1073/pnas.2310841121). However, similar additional images of the pC1a line (i.e. 40x magnification and VNC expression) would be helpful in order to validate its specificity.

(2) The author's experiments examining olfactory and gustatory contributions to the holding period were well controlled and described. However, the experiments in Figure 1D examining visual contributions were not sufficiently convincing as the line used (w1118) has previously been shown to be visually impaired (Wehner et al., 1969; Kalmus 1948). Using another wild-type line would have improved the authors' claims.

(3) When comparisons between more than 2 groups are shown as in Figures 1E, 3D, and 5E, the comparisons being made were not clear. Adding in the results of a nonparametric multiple comparisons test would help for the interpretation of these results.

Reviewer #1 (Public Review):

Summary:

Olfactory sensory neurons (OSNs) in the olfactory epithelium detect myriads of environmental odors that signal essential cues for survival. OSNs are born throughout life and thus represent one of the few neurons that undergo life-long neurogenesis. Until recently, it was assumed that OSN neurogenesis is strictly stochastic with respect to subtype (i.e. the receptor the OSN chooses to express).

However, a recent study showed that olfactory deprivation via naris occlusion selectively reduced birthrates of only a fraction of OSN subtypes and indicated that these subtypes appear to have a special capacity to undergo changes in birthrates in accordance with the level of olfactory stimulation. These previous findings raised the interesting question of what type of stimulation influences neurogenesis, since naris occlusion does not only reduce the exposure to potentially thousands of odors but also to more generalized mechanical stimuli via preventing airflow.

In this study, the authors set out to identify the stimuli that are required to promote the neurogenesis of specific OSN subtypes. Specifically, they aim to test the hypothesis that discrete odorants selectively stimulate the same OSN subtypes whose birthrates are affected. This would imply a highly specific mechanism in which exposure to certain odors can "amplify" OSN subtypes responsive to those odors suggesting that OE neurogenesis serves, in part, an adaptive function.

To address this question, the authors focused on a family of OSN subtypes that had previously been identified to respond to musk-related odors and that exhibit higher transcript levels in the olfactory epithelium of mice exposed to males compared to mice isolated from males. First, the authors confirm via a previously established cell birth dating assay in unilateral naris occluded mice that this increase in transcript levels actually reflects a stimulus-dependent birthrate acceleration of this OSN subtype family. In a series of experiments using the same assay, they show that one specific subtype of this OSN family exhibits increased birthrates in response to juvenile male exposure while a different subtype shows increased birthrates to adult mouse exposure. In the core experiment of the study, they finally exposed naris occluded mice to a discrete odor (muscone) to test if this odor specifically accelerates the birth rates of OSN types that are responsive to this odor. This experiment reveals a complex relationship between birth rate acceleration and odor concentrations showing that some muscone concentrations affect birth rates of some members of this family and do not affect two unrelated OSN subtypes.

Strengths:

The scientific question is valid and opens an interesting direction. The previously established cell birth dating assay in naris occluded mice is well performed and accompanied by several control experiments addressing potential other interpretations of the data.

Weaknesses:

(1) The main research question of this study was to test if discrete odors specifically accelerate the birth rate of OSN subtypes they stimulate, i.e. does muscone only accelerate the birth rate of OSNs that express muscone-responsive ORs, or vice versa is the birthrate of muscone-responsive OSNs only accelerated by odors they respond to?

This question is only addressed in Figure 5 of the manuscript and the results only partially support the above claim. The authors test one specific odor (muscone) and find that this odor (only at certain concentrations) accelerates the birth rate of some musk-responsive OSN subtypes, but not two other unrelated control OSN subtypes. This does not at all show that musk-responsive OSN subtypes are only affected by odors that stimulate them and that muscone only affects the birthrate of musk-responsive OSNs, since first, only the odor muscone was tested and second, only two other OSN subtypes were tested as controls, that, importantly, are shown to be generally stimulus-independent OSN subtypes (see Figure 2 and S2).

As a minimum the authors should have a) tested if additional odors that do not activate the three musk-responsive subtypes affect their birthrate b) choose 2-3 additional control subtypes that are known to be stimulus-dependent (from their own 2020 study) and test if muscone affects their birthrates.

(2) The finding that Olfr1440 expressing OSNs do not show any increase in UNO effect size under any muscone concentration (Figure 5D, no significance in line graph for UNO effect sizes, middle) seems to contradict the main claim of this study that certain odors specifically increase birthrates of OSN subtypes they stimulate. It was shown in several studies that olfr1440 is seemingly the most sensitive OR for muscone, yet, in this study, muscone does not further increase birthrates of OSNs expressing olfr1440. The effect size on birthrate under muscone exposure is the same as without muscone exposure (0%).

In contrast, the supposedly second most sensitive muscone-responsive OR olfr235 shows a significant increase in UNO effect size between no muscone exposure (0%) and 0.1% as well as 1% muscone.

(3) The authors introduce their choice to study this particular family of OSN subtypes with first, the previous finding that transcripts for one of these musk-responsive subtypes (olfr235) are downregulated in mice that are deprived of male odors. Second, musk-related odors are found in the urine of different species. This gives the misleading impression that it is known that musk-related odors are indeed excreted into male mouse urine at certain concentrations. This should be stated more clearly in the introduction (or cited, if indeed data exist that show musk-related odors in male mouse urine) because this would be a very important point from an ethological and mechanistic point of view.

In addition, this would also be important information to assess if the chosen muscone concentrations fall at all into the natural range.

Related: If these are male-specific cues, it is interesting that changes in OR transcripts (Figure 1) can already be seen at the age of P28 where other male-specific cues are just starting to get expressed. This should be discussed.

(4) Figure 5: Under muscone exposure the number of newborn neurons on the closed sides fluctuates considerably. This doesn't seem to be the case in other experiments and raises some concerns about how reliable the naris occlusion works for strong exposure to monomolecular odors or what other potential mechanisms are at play.

(5) In contrast to all other musk-responsive OSN types, the number of newborn OSNs expressing olfr1437 increases on the closed side of the OE relative to the open in UNO-treated male mice (Figure 1). This seems to contradict the presented theory and also does not align with the bulk RNAseq data (Figure S1).

(6) The authors hypothesize in relation to the accelerated birthrate of musk-responsive OSN subtypes that "the acceleration of the birthrates of specific OSN subtypes could selectively enhance sensitivity to odors detected by those subtypes by increasing their representation within the OE". However, for two other OSN subtypes that detect male-specific odors, they hypothesize the opposite "By contrast, Olfr912 (Or8b48) and Olfr1295 (Or4k45), which detect the male-specific non-musk odors 2-sec-butyl-4,5-dihydrothiazole (SBT) and (methylthio)methanethiol (MTMT), respectively, exhibited lower representation and/or transcript levels in mice exposed to male odors, possibly reflecting reduced survival due to overstimulation."

Without any further explanation, it is hard to comprehend why exposure to male-derived odors should, on one hand, accelerate birthrates in some OSN subtypes to potentially increase sensitivity to male odors, but on the other hand, lower transcript levels and does not accelerate birth rates of other OSN subtypes due to overstimulation.

Reviewer #1 (Public Review):

Summary:

In this study, the author aimed to develop a method for estimating neuronal-type connectivity from transcriptomic gene expression data. They sought to develop an interpretable model that could be used to characterize the underlying genetic mechanisms of circuit assembly and connectivity in various neuronal systems.

Strengths:

Many of the proposed suggestions were addressed by the author from the initial review. In general the claims made by the author are more strongly supported by the data and better situated in the literature. A major improvement includes the application of the model to the C. elegans gap junction neuronal system. Despite several key differences in the dataset as compared to the mouse retina data, the proposed model performs comparably to the SCM model currently considered state of the art in the literature (the author should remain cautious about claiming better performance given extremely marginal differences). In section 7.2, the author clearly outlines additional advantages of the proposed model including superior time and space complexity. The overall model performance remains modest, but it learns the same rules as the SCM model as well as other candidate patterns.

As in the initial submission, the bilinear model recapitulates key connectivity motifs for the mouse dataset. The algorithm is shown to converge across several runs affirming its stability/replicability. The model is also extended to predict connectivity on unknown RGC-BC cell type pairs. Without ground truth, the author posits how it should perform based on known functional properties of the RGC type. The hypotheses are confirmed for 8/10 neuronal types with unknown connectivity. The author more clearly describes how this model can be used experimentally for hypothesis testing and presents a more comprehensive future roadmap regarding validation, avenues for improving the model, and incorporation of growing datasets.

Weaknesses:

While the C Elegans dataset is useful because it enables benchmarking to existing models, the dataset is quite different. The gene expression dimensionality is 18 genes as opposed to over 3000 genes in the mouse dataset. It is a strength that the model still works as intended, but a weakness that the bilinear model could not be tested on a similar mouse dataset. This distinction matters because it remains an open question if the PCA methodology would hold up in a dataset with varied distributions of gene expression. Variations of the PCA methodology could be evaluated further with the present dataset to make the generalizability of the model more convincing.

The Gene Ontology analysis requires more methodological explanation. The author claims, "(the linear nature of the model) enables the direct interpretation of gene expressions by examining their associated weights in the model. These weights signify the importance of each gene in determining the connectivity motifs between the BC and RGC types." If I am correctly understanding the methods, the model weights in each dimension are indexing the importance of a gene expression feature as opposed to the importance of a single gene alone, "the gene expression of the BCs in X and the RGCs in Y were featurized by their respective PCs, resulting in matrices of dimensions 22453 × 11323 and 3779 × 3142, respectively." It would be helpful to explain how gene weights are extracted from a gene expression feature once highlighted.

There could be a more rigorous analysis of the predictive capacity of the model even with the current data. The model recapitulates connectivity patterns from the full dataset and a prediction is demonstrated for unknown data. The model is thus championed as a useful tool for predicting how genetic modifications will influence connectivity, but this is not empirically evaluated.

Appraisal of whether the author achieved their aims, and whether results support their conclusions:

In line with the aims of the paper, the author proposed an interpretable bilinear model to learn a shared latent feature space derived from gene expression profiles to predict synaptic connectivity between various neuron types. The model was shown to generalize to two distinct neuronal systems with varying levels of genomic and cellular resolution. While the performance remains modest, the model performs comparably to the existing state of the art despite improved computational complexity.

Discussion of likely impact of the work on the field, and utility of methods and data to the community:

The author has elaborated substantially on the impact of this work, particularly how it could be leveraged in experimental settings. The clear methodology could be implemented by other researchers to test the model on new datasets and for benchmarking novel methods.

Reviewer #1 (Public Review):

The work by Debashish U. Menon, Noel Murcia, and Terry Magnuson brings important knowledge about histone H3.3 dynamics involved in meiotic sex chromosome inactivation (MSCI). MSCI is unique to gametes and failure during this process can lead to infertility. Classically, MSCI has been studied in the context of DNA Damage repair pathways and little is known about the epigenetic mechanisms behind maintenance of the sex body as a silencing platform during meiosis. One of the major strengths of this work is the evidence provided on the role of ARID1A, a BAF subunit, in MSCI through the regulation of H3.3 occupancy in specific genic regions.

Using RNA seq and CUT&RUN and ATAC-seq, the authors show that ARID1A regulates chromatin accessibility of the sex chromosomes and XY gene expression. Loss of ARID1A increases promoter accessibility of XY linked genes with concomitant influx of RNA pol II to the sex body and up regulation of XY-linked genes. This work suggests that ARID1A regulates chromatin composition of the sex body since in the absence of ARID1A, spermatocytes show less enrichment of H3.3 in the sex chromosomes and stable levels of the canonical histones H3.1/3.2. By overlapping CUT&RUN and ATAC-seq data, authors show that changes in chromatin accessibility in the absence of ARID1A are given by redistribution of occupancy of H3.3. Gained open chromatin in mutants corresponds to up regulation of H3.3 occupancy at transcription start sites of genes mediated by ARID1A.

Interestingly, ARID1A loss caused increased promoter occupancy by H3.3 in regions usually occupied by PRDM9. PRDM9 catalyzes histone H3 lysine 4 trimethylation during meiotic prophase I, and positions double strand break (DSB) hotspots. Lack of ARID1A causes reduction in occupancy of DMC1, a recombinase involved in DSB repair, in non-homologous sex regions. These data suggest that ARID1A might indirectly influence DNA DSB repair on the sex chromosomes by regulating the localization of H3.3. This is very interesting given the recently suggested role for ARID1A in genome instability in cancer cells. It raises the question of whether this role is also involved in meiotic DSB repair in autosomes and/or how this mechanism differs in sex chromosomes compared to autosomes.

The fact that there are Arid1a transcripts that escape the Cre system in the Arid1a KO mouse model might difficult the interpretation of the data. The phenotype of the Arid1a knockout is probably masked by the fact that many of the sequencing techniques used here are done on a heterogeneous population of knockout and wild type spermatocytes. In relation to this, I think that the use of the term "pachytene arrest" might be overstated, since this is not the phenotype truly observed. Nonetheless, the authors provide evidence showing that the spermatids observed in cKO testes that progress in spermatogenesis are the ones expressing Arid1a. This work presents enough evidence to include the BAF complex as part of the MSCI process, which increases our knowledge on specific regulation of the sex chromatin during meiosis.

Reviewer #1 (Public Review):

Summary:

Asymptomatic malaria infections are frequent during the dry season and have been associated with lower cytoadherence of P. falciparum parasites and lower expression of variant surface antigens. The mechanisms underlying parasite adaptation during the low transmission season remain poorly understood. The authors previously established that members of the non-coding RNA RUF6 gene family, transcribed by RNA pol III, are required for expression of the main variant surface antigens in P. falciparum, PfEMP1, which drive parasite cytoadherence and pathogenicity. In this study, the authors investigated the contribution of RNA pol III transcription in the regulation of PfEMP1 expression in different clinical states, either symptomatic malaria cases during the wet season or asymptomatic infections during the dry season.

By reanalyzing RNAseq data from a previous study in Mali, complemented with RT-qPCR on new samples collected in The Gambia, the authors first report the down-regulation of RNA pol III genes (tRNAs, RUF6) in P. falciparum isolates collected from asymptomatic individuals during the dry season, as compared to isolates from symptomatic (wet season) individuals. They also confirm the down-regulation of var (DBLalpha) gene expression in asymptomatic infection as compared to symptomatic malaria. Plasma analysis in the two groups in the Gambian study reveals higher Magnesium levels in dry season as compared to wet season samples, pointing at a possible role of external factors. The authors tested the effect of MgCl2 supplementation on cultured parasites, as well as three other stimuli (temperature, low glucose, Ile deprivation), and show that Ile deprivation and MgCl2 both induce down-regulation of RNA pol III transcription but not pol I or pol II (except the active var gene). Using RNAseq, they show that MgCl2 supplementation predominantly inhibits RNA pol III-transcribed genes, including the entire RUF6 family. Conditional depletion of Maf1 leads to the up-regulation of RNA pol III gene transcription, confirming that Maf1 is a RNA pol III inhibitor in P. falciparum, as described in other organisms. Quantitative mass spectrometry shows that Maf1 interacts with RNA pol III complex in the nucleus, and with distinct proteins including two phosphatases in the cytoplasm. Using the Maf1 cKD parasites, the authors document that down-regulation of RNA pol III by MgCl2 is dependent on Maf1. Finally, they show that MgCl2 results in decreased cytoadherence of infected erythrocytes, associated with reduced PfEMP1 expression.

Strengths:

-The work is very well performed and presented.<br /> -The study uncovers a novel regulatory mechanism relying on RNA pol III-dependent regulation of variant surface antigens in response to external signals, which could contribute to parasite adaptation during the low transmission season.<br /> -Potential regulators of Maf1 were identified by mass spectrometry, including phosphatases, paving the way for future mechanistic studies.

Weaknesses:

-The signaling pathway upstream of Maf1 remains unknown. In eukaryotes, Maf1 is a negative regulator of RNA pol III and is regulated by external signals via the TORC pathway. Since TORC components are absent in the apicomplexan lineage, one central question that remains open is how Maf1 is regulated in P. falciparum. Magnesium is probably not the sole stimulus involved, as suggested by the observation that Ile deprivation also down-regulates RNA pol III activity.<br /> -The study does not address why MgCl2 levels vary depending on the clinical state. It is unclear whether plasma magnesium is increased during asymptomatic malaria or decreased during symptomatic infection, as the study does not include control groups with non-infected individuals. Along the same line, MgCl2 supplementation in parasite cultures was done at 3mM, which is higher than the highest concentrations observed in clinical samples.<br /> -Although the study provides biochemical evidence of Maf1 accumulation in the parasite nuclear fraction upon magnesium addition, this is not fully supported by the immunofluorescence experiments.

Reviewer #1 (Public Review):

Bian et al showed that biomarker-informed PhenoAgeAccel was consistently related to an increased risk of site-specific cancer and overall cancer within and across genetic risk groups. The results showed that PhenoAgeAccel and genetic liability of a bunch of cancers serve as productive tools to facilitate the identification of cancer-susceptible individuals under an additive model. People with a high genetic risk for cancer may benefit from PhenoAgeAccel-imformed interventions.

As the authors pointed out, the large sample size, the prospective design UK Biobank study, and the effective application of PhenoAgeAccel in predicting the risk of overall cancer are the major strengths of the study. Meanwhile, the CPRS seems to be a solid and comprehensive score based on incidence-weighted site-specific polygenic risk scores across 20 well-powered GWAS for cancers.

Reviewer #1 (Public Review):

Summary:

This manuscript is an extension of previous studies by this group looking at the new drug spectinamide 1599. The authors directly compare therapy with BPaL (bedaquiline, pretomanid, linezolid) to a therapy that substitutes spectinamide for linezolid (BPaS). The Spectinamide is given by aerosol exposure and the BPaS therapy is shown to be as effective as BPaL without adverse effects. The work is rigorously performed and analyses of the immune responses are consistent with curative therapy.

Strengths:

(1) This group uses 2 different mouse models to show the effectiveness of the BPaS treatment.

(2) Impressively the group demonstrates immunological correlates associated with Mtb cure with the BPaS therapy.

(3) Linezolid is known to inhibit ribsomes and mitochondria whereas spectinaminde does not. The authors clearly demonstrate the lack of adverse effects of BPaS compared to BPaL.

Weaknesses:

(1) Although this is not a weakness of this paper, a sentence describing how the spectinamide would be administered by aerosolization in humans would be welcomed.

Reviewer #1 (Public Review):

Summary:

In this work, Huang et al used SMRT sequencing to identify methylated nucleotides (6mA, 4mC, and 5mC) in Pseudomonas syringae genome. They show that the most abundant modification is 6mA and they identify the enzymes required for this modification as when they mutate HsdMSR they observe a decrease of 6mA. Interestingly, the mutant also displays phenotypes of change in pathogenicity, biofilm formation, and translation activity due to a change in gene expression likely linked to the loss of 6mA.

Overall, the paper represents an interesting set of new data that can bring forward the field of DNA modification in bacteria.

Major Concerns:

• Most of the authors' data concern Psph pathovar. I am not sure that the authors' conclusions are supported by the two other pathovars they used in the initial 2 figures. If the authors want to broaden their conclusions to Pseudomonas synringae and not restrict it to Psph, the authors should have stronger methylation data using replicates. Additionally, they should discuss why Pss is so different than Pst and Psph. Could they do a blot to confirm it is really the case and not a sequencing artefact? Is the change of methylation during bacterial growth conserved between the pathovar? The authors should obtain mutants in the other pathovar to see if they have the same phenotype. The authors have a nice set of data concerning Psph but the broadening of the results to other pathovar requires further investigation.

• The authors should include proper statistical analysis of their data. A lot of terms are descriptive but not supported by a deeper analysis to sustain the conclusions. For example, in Figure 4E, we do not know if the overlap is significant or not. Are DEGs more overlapping to 6mA sites than non-DEGs? Here is a non-exhaustive list of terms that need to be supported by statistics: different level (L145), greater conservation (L162), significant conservation (L165), considerable similarity (L175), credible motifs (L189), Less strong (L277) and several "lower" and "higher" throughout the text.

• The authors performed SMRT sequencing of the delta hsdMSR showing a reduction of 6mA. Could they include a description of their results similar to Figures 1-2. How reduced is the 6mA level? Is it everywhere in the genome? Does it affect other methylation marks? This analysis would strengthen their conclusions.

• In Figure 6E to conclude that methylation is required on both strands, the authors are missing the control CAGCN6CGC construct otherwise the effect could be linked to the A on the complementary strand.

Reviewer #1 (Public Review):

Summary:

The manuscript presents a compelling model to explain the impact of mosaicism in preimplantation genetic testing for aneuploidies.

Strengths:

A new view of mosaicism is presented with a computational model, that brings new insights into an "old" debate in our field. It is a very well-written manuscript.

Weaknesses:

Although the manuscript is very well written, this is in a way that assumes that the reader has existing knowledge about specific terms and topics. This was apparent through a lack of definitions and minimal background/context to the aims and conclusions for some of the author's findings.

There is a need for some examples to connect real evidence and scenarios from clinical reports with the model.

Reviewer #1 (Public Review):

Summary:

This study by Park and colleagues uses longitudinal saliva viral load data from two cohorts (one in the US and one in Japan from a clinical trial) in the pre-vaccine era to subset viral shedding kinetics and then use machine learning to attempt to identify clinical correlates of different shedding patterns. The stratification method identifies three separate shedding patterns discriminated by peak viral load, shedding duration, and clearance slope. The authors also assess micro-RNAs as potential biomarkers of severity but do not identify any clear relationships with viral kinetics.

Strengths:

The cohorts are well developed, the mathematical model appears to capture shedding kinetics fairly well, the clustering seems generally appropriate, and the machine learning analysis is a sensible, albeit exploratory approach. The micro-RNA analysis is interesting and novel.

Weaknesses:

The conclusions of the paper are somewhat supported by the data but there are certain limitations that are notable and make the study's findings of only limited relevance to current COVID-19 epidemiology and clinical conditions.

(1) The study only included previously uninfected, unvaccinated individuals without the omicron variant. It has been well documented that vaccination and prior infection both predict shorter duration shedding. Therefore, the study results are no longer relevant to current COVID-19 conditions. This is not at all the authors' fault but rather a difficult reality of much retrospective COVID research.

(2) The target cell model, which appears to fit the data fairly well, has clear mechanistic limitations. Specifically, if such a high proportion of cells were to get infected, then the disease would be extremely severe in all cases. The authors could specify that this model was selected for ease of use and to allow clustering, rather than to provide mechanistic insight. It would be useful to list the AIC scores of this model when compared to the model by Ke.

(3) Line 104: I don't follow why including both datasets would allow one model to work better than the other. This requires more explanation. I am also not convinced that non-linear mixed effects approaches can really be used to infer early model kinetics in individuals from one cohort by using late viral load kinetics in another (and vice versa). The approach seems better for making population-level estimates when there is such a high amount of missing data.

(4) Along these lines, the three clusters appear to show uniform expansion slopes whereas the NBA cohort, a much larger cohort that captured early and late viral loads in most individuals, shows substantial variability in viral expansion slopes. In Figure 2D: the upslope seems extraordinarily rapid relative to other cohorts. I calculate a viral doubling time of roughly 1.5 hours. It would be helpful to understand how reliable of an estimate this is and also how much variability was observed among individuals.

(5) A key issue is that a lack of heterogeneity in the cohort may be driving a lack of differences between the groups. Table 1 shows that Sp02 values and lab values that all look normal. All infections were mild. This may make identifying biomarkers quite challenging.

(6) Figure 3A: many of the clinical variables such as basophil count, Cl, and protein have very low pre-test probability of correlating with virologic outcome.

(7) A key omission appears to be micoRNA from pre and early-infection time points. It would be helpful to understand whether microRNA levels at least differed between the two collection timepoints and whether certain microRNAs are dynamic during infection.

(8) The discussion could use a more thorough description of how viral kinetics differ in saliva versus nasal swabs and how this work complements other modeling studies in the field.

(9) The most predictive potential variables of shedding heterogeneity which pertain to the innate and adaptive immune responses (virus-specific antibody and T cell levels) are not measured or modeled.

(10) I am curious whether the models infer different peak viral loads, duration, expansion, and clearance slopes between the 2 cohorts based on fitting to different infection stage data.

Reviewer #1 (Public Review):

Summary:

The author presents the discovery and characterization of CAPSL as a potential gene linked to Familial Exudative Vitreoretinopathy (FEVR), identifying one nonsense and one missense mutation within CAPSL in two distinct patient families afflicted by FEVR. Cell transfection assays suggest that the missense mutation adversely affects protein levels when overexpressed in cell cultures. Furthermore, conditionally knocking out CAPSL in vascular endothelial cells leads to compromised vascular development. The suppression of CAPSL in human retinal microvascular endothelial cells results in hindered tube formation, a decrease in cell proliferation, and disrupted cell polarity. Additionally, transcriptomic and proteomic profiling of these cells indicates alterations in the MYC pathway.

Strengths:

The study is nicely designed with a combination of in vivo and in vitro approaches, and the experimental results are good quality.

Weaknesses:

My reservations lie with the main assertion that CAPSL is associated with FEVR, as the genetic evidence from human studies appears relatively weak. Further careful examination of human genetics evidence in both patient cohorts and the general population will help to clarify. In light of human genetics, more caution needs to be exercised when interpreting results from mice and cell models and how is it related to the human patient phenotype.

Reviewer #2 (Public Review):

Summary:

An article with lots of interesting ideas and questions regarding the evolution of timing of dormancy, emphasizing mammalian hibernation but also including ectotherms. The authors compare selective forces of constraints due to energy availability versus predator avoidance and requirements and consequences of reproduction in a review of between and within species (sex) differences in the seasonal timing of entry and exit from dormancy.

Strengths:

The multispecies approach including endotherms and ectotherms is ambitious. This review is rich with ideas if not in convincing conclusions. Limitations are discussed yet are impactful, namely that differences among and within species are contrast only for ecological hibernation (the duration of remaining sequestered) and not for "heterothermic hibernation" the period between first and last torpor. Differences between the two can have significant energetic consequences, especially for mammals returning to euthermic levels of body temperature whilst remaining in their cold burrows before emerging, eg. reproductively developing males in spring.

Weaknesses:

The differences between physiological requirements for gameatogenesis between sexes that affect the timing of heterothermy and need for euthermy during mammalian hibernator are significant issues that underlie, but are under discussed, in this contrast of selective pressures that determine seasonal timing of dormancy. Some additional discussion of the effects of rapid rapid climate change on between and within species phenologies of dormancy would have been interesting.

Reviewer #2 (Public Review):

Summary:

This work by Cloarec-Ung et al. sets out to uncover strategies that would allow for the efficient and precision editing of primitive human hematopoietic stem and progenitor cells (HSPCs). Such effective editing of HSPCs via homology directed repair has implications for the development of tractable gene therapy approaches for monogenic hematopoietic disorders as well as precise engineering of these cells for clinical regenerative and/or cell therapy strategies. In the setting of experimental hematology, precision introduction of disease relevant mutations would also open the door to more robust disease modeling approaches. It has been recognized that to encourage HDR, NHEJ as the dominant mode of repair in quiescent HSPCs must be inhibited. Testing editing of human cord blood HSPCs the authors first incorporate a prestimulation phase then identify optimal RNP amounts and donor types/amounts using standard editing culture conditions identifying optimal concentrations of AAV and short single-stranded oligonucleotide donors (ssODNs) that yield minimal impacts to cell viability while still enabling heightened integration efficiency. They then demonstrate the superiority of AZD7648, an inhibitor of NHEJ-promoting DNA-PK, in allowing for much increased HDR with toxicities imparted by this compound reduced substantially by siRNAs against p53 (mean targeting efficiencies at 57 and 80% for two different loci). Although AAV offered the highest HDR frequencies, differing from ssODN by a factor by ~2-fold, the authors show that spacer breaking sequence mutations introduced into the ssODN to better mimic the disruption of the spacer sequence provided by the synthetic intron in the AAV backbone yielded ssODN HDR frequencies equal to that attained by AAV. By examining editing efficiency across specific immunophenotypically identified subpopulations they further suggest that editing efficiency with their improved strategy is consistent across stem and early progenitors and use colony assays to quantify an approximate 4-fold drop in total colony numbers but no skewing in the potentiality of progenitors in the edited HSPC pool. Finally, the authors provide a strategy using mutation-introducing AAV mixed with different ratios of silent ssODN repair templates to enable tuning of zygosity in edited CD34+ cells.

Strengths:

The methods are clearly described and the experiments for the most part also appropriately powered. In addition to using state-of-the-art approaches, the authors also provided useful insights into optimizing the practicalities of the experimental procedures that will aid bench scientists in effectively carrying out these editing approaches, for example avoiding longer handling times inherent when scaling up to editing over multiple conditions.

The sum of the adjustments to the editing procedure have yielded important advances towards minimizing editing toxicity while maximizing editing efficiency in HSPCs. In particular, the significant increase in HDR facilitated by the authors' described application of AZD7648 and the preservation of a pool of targeted progenitors is encouraging that functionally valuable cell types can be effectively edited.

The discovery of the effectiveness of spacer breaking changes in ssODNs allowing for substantially increased targeting efficiency is a promising advance towards democratizing these editing strategies given the ease of designing and synthesizing ssODNs relative to the production of viral donors.

The ability to zygosity tune was convincingly presented and provides a valuable strategy to modify this HDR procedure towards more accurate disease modelling.

Weaknesses:

Despite providing convincing evidence that functional progenitors can be successfully edited by their procedure, as the authors acknowledge it remains to be verified to what degree the survival/self-renewal capacity and in vivo regenerative potential of the more primitive fractions is maintained with their strategy. That said the inclusion of LTC-IC assays that verify the lack of effect on these quite primitive cells is encouraging that functionality of stem cells will be similarly spared.

Reviewer #1 (Public Review):

Summary: The type I ABC importer OpuA from Lactococcus lactis is the best studied transporter involved in osmoprotection. In contrast to most ABC import systems, the substrate binding protein is fused via a short linker to the transmembrane domain of the transporter. Consequently, this moiety is called the substrate binding domain (SBD). OpuA has been studied in the past in great detail and we have a very detailed knowledge about function, mechanisms of activation and deactivation as well as structure.

Strengths: Application of smFRET to unravel transient interactions of the SBDs. The method is applied at a superb quality and the data evaluation is excellent.

Weaknesses: The proposed model is not directly supported by experimental data. Rather alternative models are excluded as they do not fit to the obtained data. However, this is now clearly stated in the manuscript

Reviewer #1 (Public Review):

Building on previous work from the Tansey lab, here Howard et al. characterize transcriptional and translational changes upon WIN site inhibition of WDR5 in MLL-rearranged cancer cells. They first analyze whether C16, a newer generation compound, has the same cellular effects as C6, an early generation compound. Both compounds reduce the expression of WDR5-bound RPGs in addition to the unbound RPG RPL22L1. They then investigate differential translation by ribo-seq and observe that WIN site inhibition reduces the translational RPGs and other proteins related to biomass accumulation (spliceosome, proteasome, mitochondrial ribosome). Interestingly, this reduction adds to the transcriptional changes and is not limited to RPGs whose promoters are bound by WDR5. Quantitative proteomics at two time points confirmed the downregulation of RPGs. Interestingly, the overall effects are modest, but RPL22LA is strongly affected. Unexpectedly, most differentially abundant proteins seem to be upregulated 24 h after C6 (see below). A genetic screen showed that loss of p53 rescues the effect of C6 and C16 and helped the authors to identify pathways that can be targeted by compounds together with WIN site inhibitors in a synergistic way. Finally, the authors elucidated the underlying mechanisms and analyzed the functional relevance of the RPL22, RPL22L1, p53 and MDM4 axis.

Comments on revised version:

The authors have answered my points satisfactorily and the manuscript has become clearer and more meaningful as a result. In particular, the measurement of global translation rate is important and validates the upregulation of a number of proteins following WDR5 inhibitor treatment.

Reviewer #1 (Public Review):

Summary:

The authors use truncations, fragments, and HCN2/4 chimeras to narrow down the interaction and regulatory domains for LRMP inhibition of cAMP-dependent shifts in the voltage dependence of activation of HCN4 channels. They identify the N-terminal domain of HCN4 as a binding domain for LRMP, and highlight two residues in the C-linker as critical for the regulatory effect. Notably, whereas HCN2 is normally insensitive to LRMP, putting the N-terminus and 5 additional C-linker and S5 residues from HCN4 into HCN2 confers LRMP regulation in HCN2.

Strengths:

The work is excellent, the paper well written, and the data convincingly support the conclusions which shed new light on the interaction and mechanism for LRMP regulation of HCN4, as well as identifying critical differences that explain why LRMP does not regulate other isoforms such as HCN2.

Reviewer #1 (Public Review):

Summary:

In this study, Pan DY et al. discovered that the clearance of senescent osteoclasts can lead to a reduction in sensory nerve innervation. This reduction is achieved through the attenuation of Netrin-1 and NGF levels, as well as the regulation of H-type vessels, resulting in a decrease in pain-related behavior. The experiments are well-designed. The results are clearly presented, and the legends are also clear and informative. Their findings represent a potential treatment for spine pain utilizing senolytic drugs.

Strengths:

Rigorous data, well-designed experiments as well as significant innovation make this manuscript stand out.

Weaknesses:

All my concerns have been well addressed, no further comments.

Reviewer #1 (Public Review):

Here, using an organoid system, Wong et al generated a new model of hereditary diffuse leukoencephalopathy with axonal spheroids, with which they investigated how CSF1R-mutaions affect the phenotypes of microglia/macrophages, and revealed metabolic changes in microglia/macrophages associated with a proinflammatory phenotype.

In general, this paper is interesting and well-written, and tackles important issues to be addressed.

This study suffers from several major concerns and limitations that dampen the value of the study. As the authors also mentioned, models that perfectly recapitulate the complexity of the HDLS brain the models would be required to better understand the molecular mechanisms of the disease. In this regard, it is unclear how nicely the organoid system in this study can recapitulate the condition in patients with HDLS (e.g. reduced microglia density, downregulated expression of P2YR12, pathological alterations). In addition, the authors used two different models with distinct mutations that could produce different readouts in CSF1R-mediated cellular responses.

Although the reviewer does understand the importance of providing several options/tools to study rare diseases like HDLS and the difficulty of generating stable organoids with less variation, it is unclear if the different outcomes between HD1 and HD2 are generated through different mutations or simply due to different differentiation efficiency from iMacs (e.g. Figure 2B), which needs to be confirmed. Lastly, there is an over-interpretation regarding the results in Figure 6A. There is no difference between isoHD1 iMac control and HD1 Mut iMac.

Reviewer #2 (Public Review):

Summary:

Fertilization is a crucial event in sexual reproduction, but the molecular mechanisms underlying egg-sperm fusion remain elusive. Elofsson A et al. used AlphaFold to explore possible synapse-like assemblies between sperm and egg membrane proteins during fertilization. Using a systematic search of protein-protein interactions, the authors proposed a pentameric complex of three sperm (IZUMO1, SPACA6, and TMEM81) and two egg (JUNO and CD9) proteins, providing a new structural model to be used in future structure-function studies.

Strengths:

(1) The study uses the AlphaFold algorithm to predict higher-order assemblies. This approach could offer insights into a highly transient protein complex, which are challenging to detect experimentally.<br /> (2) The article predicts a pentameric complex between proteins involved in fertilization, shedding light on the architectural aspects of the egg-sperm fusion synapse.

Weaknesses:

The proposed model, which is a prediction from a modeling algorithm, lacks experimental validation of the identity of the components and the predicted contacts.

It is noteworthy that in an independent study, Deneke et al. provides experimental evidence of the interaction between IZUMO1/SPACA6/TMEM81 in zebrafish. This is an important element that supports the findings presented in this manuscript

Regarding the authors response on the question of a global search:<br /> I understand that a global search might be difficult to interpret because a large number of putative false positives. But it is this type of information that is needed to assess the validity of the model and the scoring power in the absence of any experimental validation. At minimum, the search should include a negative control set of proteins known to be unrelated to sperm fertilization or homologous egg-sperm fusion complexes from incompatible species to account for species-specific interactions.

I acknowledge that experimentally validating highly transient complexes presents technical hurdles. However, a high-confidence structural model could enable the design of point mutations specifically disrupting the predicted interactions. Subsequent rescue experiments could then validate the directionality of these interactions. Ultimately, such experiments are crucial for robust model validation.

Reviewer #1 (Public Review):

Summary:

In this study, the authors generated a novel transgenic mouse line OpalinP2A-Flpo-T2A-tTA2 to specifically label mature oligodendrocytes, and at the same time their embryonic origins by crossing with a progenitor cre mouse line. With this clever approach, they found that LGE/CGE-derived OLs make minimum contributions to the neocortex, whereas MGE/POA-derived OLs make a small but lasting contribution to the cortex. These findings are contradictory to the current belief that LGE/CGE-derived OPCs make a sustained contribution to cortical OLs, whereas MGE/POA-derived OPCs are completely eliminated. Thus, this study provides a revised and more comprehensive view on the embryonic origins of cortical oligodendrocytes. To specifically label mature oligodendrocytes, and at the same time their embryonic origins by crossing with a progenitor cre mouse line. With this clever approach, they found that LGE/CGE-derived OLs make minimum contributions to the neocortex, whereas MGE/POA-derived OLs make a small-but-lasting contribution to to cortex. These findings are contradictory to the current belief that LGE/CGE-derived OPCs make a sustained contribution to cortical OLs, whereas MGE/POA-derived OPCs are completely eliminated. Thus, this study has provided a revised and updated view on the embryonic origins of cortical oligodendrocytes.

Strengths:

The authors have generated a novel transgenic mouse line to specifically label mature differentiated oligodendrocytes, which is very useful for tracing the final destiny of mature myelinating oligodendrocytes. Also, the authors carefully compared the distribution of three progenitor cre mouse lines and suggested that Gsh-cre also labeled dorsal OLs, contrary to the previous suggestion that it only marks LGE-derived OPCs. In addition, the author also analyzed the relative contributions of OLs derived from three distinct progenitor domains in other forebrain regions (e.g. Pir, ac). Finally, the new transgenic mouse lines and established multiple combinatorial genetic models will facilitate future investigations of the developmental origins of distinct OL populations and their functional and molecular heterogeneity.

Weaknesses:

Since OpalinP2A-Flpo-T2A-tTA2 only labels mature oligodendrocytes but not OPCs, the authors can not suggest that the lack of LGE/CGE-derived-OLs in the neocortex is less likely caused by competitive postnatal elimination, but more likely due to limited production and/or allocation (line 118-9). It remains possible that LGE/CGE-derived OPCs migrate into the cortex but are later eliminated.

Reviewer #1 (Public Review):

This is an interesting, informative, and well-designed study that combines theoretical and experimental methodologies to tackle the phenomenon of higher-resolution structures/substructures in model biomolecular condensates. However, there is significant room for improvement in the presentation and interpretation of the results. As it stands, the precise definition of "frustration," which is a main theme of this manuscript (as emphasized in the title), is not sufficiently well articulated. This situation should be rectified to avoid "frustration" becoming a "catch-all" term without a clear perimeter of applicability rather than a precise, informative description of the physical state of affairs. There are also a few other concerns, e.g., regarding interpretation of correlation of phase-separation critical temperature and transfer free energy of amino acid residues as well as the difference between critical temperature and onset temperature, and the way the simulated configurations are similar to that of gyroids. Accordingly, the manuscript should be revised to address the following:

(1) It is accurately pointed out on p.4 that elastin-like polypeptides (ELPs) undergo heat-induced phase separation and therefore exhibit lower critical solution temperatures (LCSTs). But it is not entirely clear how this feature is reproduced by the authors' simulation. A relationship between simulated surface tension and "transition temperature" is provided in Fig.1C; but is the "transition temperature" (authors cited ref.41 by Urry) the same as critical temperature? Apparently, Urry's Tt is "critical onset temperature", the temperature when phase separation happens at a given polymer concentration. This is different from the (global) critical temperature LCST - though the two may be correlated-or not-depending on the shape of the phase boundary. Moreover, is the MOFF coarse-grained forcefield (first step in the multi-scale simulation), by itself, capable of reproducing heat-induced phase separation in a way similar to the forcefield of Dignon et al., ACS Cent Sci 5, 821-230 (2019)? Or, is this temperature-dependent effect appearing only subsequently, after the implementation of the MARTINI and/or all-atom steps? Clarification is needed. To afford a more informative context for the authors' introductory discussion, the aforementioned Dignon et al. work and the review by Cinar et al. [Chem Eur J 25, 13049-13069 (2019)], both touching upon the physical underpinning of the LCST feature of elastin, should also be cited along with refs.41-43.

(2) "Frustration" and "frustrated" are used prominently in the manuscript to characterize certain observed molecular configurations (11 times total, in both the title and in the abstract). Apparently, it is the most significant conceptual pronouncement of this work, hence its precise meaning is of central importance to the authors' thesis. Whereas one should recognize that the theoretical and experimental observations are striking without invocation of the "frustration" terminology, usage of the term can be useful if it offers a unifying conceptual framework. However, as it stands, a clear definition of the term "frustration" is lacking, leaving readers to wonder what molecular configurations are considered "frustrated" and what are not (i.e.,is the claim of observation of frustration falsifiable?). For instance, "frustrated microphase separation" appears in both the title and abstract. A logical question one may ask is: "Are all microphase separations frustrated"? If the answer is in the affirmative, does invocation of the term "frustration" add anything to our physical insight? If the answer is not in the affirmative, then how does one distinguish between microphase separations that are frustrated from those that are not frustrated? Presumably all simulated and experimental molecular configurations in the present study are those of lowest free energy for the given temperature. In other words, they are what they are. In the discussion about frustrated phase separation on p.13, for example, the authors appear to refer to the fact that chain connectivity is preventing hydrophobic residues to come together in a way to achieve the most favorable interactions as if there were no chain connectivity (one may imagine in that case all the hydrophobic residues will form a large cluster without microphase separation). Is this what the authors mean by "frustration"? If that's true, isn't that merely stating the obvious, at least for the observed microphase separation? In general, does "frustration" always mean deviation of actual, physical molecular configurations from certain imagined/hypothetical/reference molecular configurations, and therefore dependent upon the choice of the imagined reference configuration? If this is how the authors apply the term "frustration" in the present work, what is the zero-frustration reference state/configuration for microphase separation? And, similarly, what is the zero-frustration reference state/configuration when frustrated EPS-water interactions are discussed (~p.14-p.15, Fig.5)? How do non-frustrated water-protein interactions look like? Is the classic clathrate-like organization of water hydrogen bonds around small nonpolar solute "frustrated"?

(3) In the discussion about the correlation of various transfer free energy scales for amino acids and Urry's critical onset temperature (ref.41) on p.11 and Fig.4, is there any theoretical relationship to be expected between the interactions among amino acids of ELPs and their critical onset temperatures? While a certain correlation may be intuitively expected if the free energy scale "is working", is there any theoretical insight into the mathematical form of this relationship? A clarifying discussion is needed because it bears logically on whether the observed correlation or lack thereof for different transfer energy scales is a good indication of the adequacy of the energy scales in describing the actual physical interactions at play. This question requires some prior knowledge of the expected mathematical relationship between interaction parameters and onset temperature.

(4) To provide a more comprehensive context for the present study, it is useful to compare the microphase separation seen in the authors' simulation with the micelle-like structures observed in recent simulated condensed/aggregated states of hydrophobic-polar (HP) model sequences in Statt et al., J Chem Phys 152, 075101 (2020) [see esp. Fig.6] and Wessén et al., J Phys Chem B 126, 9222-9245 (2022) [see, e.g., Fig.10].

(5) "Gyroid-like morphology" is mentioned several times in the manuscript (p.4, p.8, p.17, Fig.S3). This is apparently an interesting observation but a clear explanation is lacking. A more detailed and specific discussion, perhaps with additional graphical presentations, should be provided to demonstrate why the simulated condensed-phase ELP configurations are similar to the classical description of gyroid as in, e.g., Terrones & Mackay, Chem Phys Lett 207, 45-50 (1993) and Lambert et al., Phil Trans R Soc A 354, 2009-2023 (1996).

Comments on the revised manuscript:

The authors have adequately addressed my previous concerns.

Reviewer #1 (Public Review):

Most amino acids are stereoisomers in the L-enantiomer, but natural D-serine has also been detected in mammals and its levels shown to be connected to a number of different pathologies. Here, the authors convincingly show that D-serine is transported in the kidney by the neutral amino acid transporter ASCT2 and as a non-canonical substrate for the sodium-coupled monocarboxylate transporter SMCTs. Although both transport D-serine, this important study further shows in a mouse model for acute kidney injury that ASCT2 has the dominant role.

Strengths:

The paper combines proteomics, animal models, ex vivo transport analyses and in vitro transport assays using purified components. The exhaustive methods employed provide compelling evidence that both transporters can translocate D-serine in the kidney.

Weakness:

In the model for acute kidney injury the SMCTs proteins were not showing a significant change in expression levels and were rather analysed based on other, circumstantial evidence. Although its clear SMCTs can transport D-serine its physiological role is less obvious compared to ASCT2.

Reviewer #1 (Public Review):

Summary:

LRRK2 protein is familially linked to Parkinson's disease by the presence of several gene variants that all confer a gain-of-function effect on LRRK2 kinase activity.

The authors examine the effects of BDNF stimulation in immortalized neuron-like cells, cultured mouse primary neurons, hIPSC-derived neurons, and synaptosome preparations from the brain. They examine an LRRK2 regulatory phosphorylation residue, LRRK2 binding relationships, and measures of synaptic structure and function.

Strengths:

The study addresses an important research question: how does a PD-linked protein interact with other proteins, and contribute to responses to a well-characterized neuronal signalling pathway involved in the regulation of synaptic function and cell health?

They employ a range of good models and techniques to fairly convincingly demonstrate that BDNF stimulation alters LRRK2 phosphorylation and binding to many proteins. Some effects of BDNF stimulation appear impaired in (some of the) LRRK2 knock-out scenarios (but not all). A phosphoproteomic analysis of PD mutant Knock-in mouse brain synaptosomes is included.

Weaknesses:

The data sets are disjointed, conclusions are sweeping, and not always in line with what the data is showing. Validation of 'omics' data is very light. Some inconsistencies with the major conclusions are ignored. Several of the assays employed (western blotting especially) are likely underpowered, findings key to their interpretation are addressed in only one or other of the several models employed, and supporting observations are lacking.

As examples to aid reader interpretation:

(a) pS935 LRRK2 seems to go up at 5 minutes but goes down below pre-stimulation levels after (at times when BDNF-induced phosphorylation of other known targets remains very high). This is ignored in favour of discussion/investigation of initial increases, and the fact that BDNF does many things (which might indirectly contribute to initial but unsustained changes to pLRRK2) is not addressed.

(b) Drebrin coIP itself looks like a very strong result, as does the increase after BDNF, but this was only demonstrated with a GFP over-expression construct despite several mouse and neuron models being employed elsewhere and available for copIP of endogenous LRRK2. Also, the coIP is only demonstrated in one direction. Similarly, the decrease in drebrin levels in mice is not assessed in the other model systems, coIP wasn't done, and mRNA transcripts are not quantified (even though others were). Drebrin phosphorylation state is not examined.

(c) The large differences in the CRISPR KO cells in terms of BDNF responses are not seen in the primary neurons of KO mice, suggesting that other differences between the two might be responsible, rather than the lack of LRRK2 protein.

(d) No validation of hits in the G2019S mutant phosphoproteomics, and no other assays related to the rest of the paper/conclusions. Drebrin phosphorylation is different but unvalidated, or related to previous data sets beyond some discussion. The fact that LRRK2 binding occurs, and increases with BDNF stimulation, should be compared to its phosphorylation status and the effects of the G2019S mutation.

Reviewer #1 (Public Review):

Amason et al. investigated the formation of granulomas in response to Chromobacterium violaceum infection, aiming to uncover the cellular mechanisms governing the granuloma response. They identify spatiotemporal gene expression of chemokines and receptors associated with the formation and clearance of granulomas, with a specific focus on those involved in immune trafficking. By analyzing the presence or absence of chemokine/receptor RNA expression, they infer the importance of immune cells in resolving infection. Despite observing increased expression of neutrophil-recruiting chemokines, treatment with reparixin (an inhibitor of CXCR1 and CXCR2) did not inhibit neutrophil recruitment during infection. Focusing on monocyte trafficking, they found that CCR2 knockout mice infected with C. violaceum were unable to form granulomas, ultimately succumbing to infection.

The spatial transcriptomics data presented in the figures could be considered a valuable resource if shared, with the potential for improved and clarified analyses. The primary conclusion of the paper, that C. violaceum infection in the liver cannot be contained without macrophages, would benefit from clarification.

While the spatial transcriptomic data generated in the figures are interesting and valuable, they could benefit from additional information. The manual selection of regions of granulomas for analysis could use additional context - was the rest of the liver not sequenced, or excluded for other reasons? Including a healthy liver in the analysis could serve as a control for any lasting effects at the final time point of 21 days. Providing more context for the scalebars throughout the spatial analyses, such as whether the data are raw counts or normalized based on the number of reads per spatial spot, would be helpful for interpretation, as changes in expression could signal changes in the numbers of cells or changes in the gene expression of cells.

In Figure 4, qualitative measurements are valuable, but having an idea of the raw data for a few of the pursued chemokines/receptors would aid interpretation. It would also be beneficial to clarify whether the reported values are across all clusters and consider focusing on clusters with the greatest change in expression. Figures 5E and F would benefit from clarification regarding the x-axis units and whether the expression levels are summed across all clusters for each time point. Additionally, information on the sequencing depth of the samples would be helpful, particularly as shallow sequencing of RNA can result in poor capture of low-expression transcripts.

Regarding the conclusion of the essentiality of macrophages in granuloma formation, it may be prudent to further investigate the role of macrophages versus CCR2. Analyzing total cell counts in the liver after infection could provide insight into whether the decrease in the fraction of macrophages is due to decreased numbers or infiltration of other cell types. Consideration of experiments deleting macrophages directly, instead of CCR2, could provide more definitive evidence of the necessity of macrophage migration in containing infections.

Reviewer #1 (Public Review):

Pineda et al investigate the association of the hypothesis that Dux4, an embryonic transcription factor, expression in tumor cells is associated with immune evasion and resistance to immunotherapy. They analyze existing cohorts of bulk RNAseq sequenced tumors across cancer types to identify Dux4 expression and association with survival. They find that Dux4 expression is detected in a higher proportion of metastatic tumors compared to primary tumors, is associated with decreased immune infiltrate and a variety of immune metrics and previously nominated immune signatures, and do an in depth evaluation of a cohort of metastatic urothelial cell carcinoma, finding that Dux4 expression is associated with a more immunodeficient tumor microenvironment (desert or excluded microenvironment) and worse survival in this aPDL1 treated cohort. They then find that Dux4 expression is a major independent predictor of survival in this cohort using different types of survival analyses (KM, Cox PH, and random survival forests). With prior existing biological data supporting the hypothesis (in prior work, the senior author has demonstrated Dux4 expression causally suppresses MHC-I expression in interferon-gamma treated cell lines), the current work links Dux4 expression with less immune activity in clinical tumor samples and with survival in ICI treated urothelial carcinomas, and demonstrates that Dux4 expression provides independent information towards survival including other molecular and clinical characteristics (TMB, ECOG PS as the other strongest markers), and provides interesting resolution on landmark analyses with TMB and Dux4 expression providing greater informativeness at later survival landmarks (e.g. 1 year and later), while ECOG PS has strong informativeness already at earlier time points. This work provides impetus towards more mechanistic and functional dissection of the mechanism of Dux4-associated changes with the tumor microenvironment (e.g. in vivo mouse studies) as well as potential interventional studies (e.g. Dux4 as a target in combination therapies). What the work does not provide is additional resolution on the mechanism of how Dux4 may be associated with a more immunodeficient microenvironment.

The conclusions are generally well supported, but there are issues that would benefit from clarification and extension:

- The finding that Dux4 expression is detected in a higher proportion of metastatic tumors and at higher levels compared to TCGA samples (Fig 1BC) is striking. However, at least for one tumor type (melanoma), the TCGA cohort is comprised of mostly locoregional metastatic (n=81 primary and 367 metastatic tumors in the PanCan Atlas). Since there are annotations for primary and (locoregional) metastatic samples in TCGA, an analysis of the primary vs. locoregional metastasis vs distant metastatic samples seems reasonable and likely informative. The analysis of tumors with matched FFPE and flash frozen samples with hybrid probe capture and polyA sequencing, respectively is a nice validation to show that the difference in Dux4 expression is not due to differences in preservation of starting material/sequencing in the metastatic samples vs TCGA samples (S1BC).<br /> - The findings that Dux4 expression in the metastatic urothelial carcinoma setting is associated with a more immunodeficient microenvironment (Figure 2) is clear and unambiguous using multiple lines of data and analyses (bulk RNAseq, DUX4-positive vs DUX4-negative tumors, different immune cell and cytokine signatures; IHC showing an association with immune deserts and immune excluded phenotypes). However, this is an association and does not demonstrate causality.<br /> - The survival analyses (Fig 3,4,5) show fairly convincingly that Dux4 provide independent predictive information beyond clinical variables and TMB towards survival in the aPDL1 treated metastatic urothelial carcinoma cohort. However, the choice to split the cohort into Dux4 negative (defined as < 0.25 TPM) and Dux4 positive (> 1 TPM) while excluding a large number of patients (n=126 pts) that fall in between has significant impact on the rigor of conclusions. This would benefit from showing all the data (e.g. including the 3rd group of in-betweens in the survival analyses as a separate group).<br /> - The authors demonstrate that adding Dux4 to clinical markers and TMB results in an improved predictive model for survival, but there are a few questions regarding this model as a clinical biomarker<br /> o Is Dux4 expression better than other correlated immune signatures/markers (e.g. interferon gamma, T effector signature, overall immune infiltrate) in providing additional information?<br /> - The use of random survival forests to quantify the (predictive) marginal effect of Dux4+ vs Dux4- expression on survival in a non-parametric model as well as shed light on association with survival at different landmark times using Shapley values is quite interesting and well conducted.

Reviewer #1 (Public Review):

The authors have made a novel and important effort to distinguish and include different sources of active deformations for fitting C elegans embryo development: cyclic muscle contractions and actomyosion circumferential stresses. The combination and synchronisation of both contributions are, according to the model, responsible for different elongation rates, and can induce bending and torsion deformations, which are a priori not expected from purely contractile forces. The model can be applied to other growth processes in initially cylindrical shapes.

The tilt of the fibers is an important assumption of the model. However, fiber direction in Figure 3B is not fully clear for explaining the tilting. The fiber in 3B has not very much in common with the fibers in the color part of the figure. Also, is vector m supposed to be tangent to the fiber? In the figure does not seem to be so. It should be expected that alpha is a consequence of the deformation, not as an input parameter, as it seems in the tests of Figure 6A. How is the value of alpha chosen? According to Figure 6, torsion is expected for alpha>0, but for beta=pi/2 and alpha>0 no torsion may be obtained. In fact, it seems that torsion should appear when cos(beta)*sin(alpha)>0. As a consequence, value of beta should be given in Figure 6. Can the amount of torsion be tested as a function of alpha and beta?

The transfer of energy and deformation is a very interesting aspect of the paper, and also crucial for the model and predicting elongation. However, the modelling of this transfer remains very obscure and only explained in the Appendix. Some more details on how the transfer is selected should be given in the main text. Can the transfer of energy interpreted as a change of the relaxed reference configuration? Once a ratio of the energy transferred is fixed, the assumption on elongation distribution should be stated. (Uniformly? ) The authors should also define in the main text the factor g_a1, and explain how this value is computed from condition W_c=W_r .

Given the convoluted shape of the embryo in the egg, contact may be a crucial mechanism for determining growth and torsion. The model does not include this contact, and this limitation should be reflected in the article.

Minor comment:<br /> -Line 300: "we determine the optimal values for the activation parameters". the optimal with respect to which objective? Norm of difference between experimental and computational displacements? How this is quantified needs to be specified.

Reviewer #1 (Public Review):

Summary:

This study presents careful biochemical experiments to understand the relationship between LRRK2 GTP hydrolysis parameters and LRRK2 kinase activity. The authors report that incubation of LRRK2 with ATP increases the KM for GTP and decreases the kcat. From this they suppose an autophosphorylation process is responsible for enzyme inhibition. LRRK2 T1343A showed no change, consistent with it needing to be phosphorylated to explain the changes in G-domain properties. The authors propose that phosphorylation of T1343 inhibits kinase activity and influences monomer-dimer transitions.

Strengths:

Strengths of the work are the very careful biochemical analyses and interesting result for wild type LRRK2.

Weaknesses:

The conclusions related to involvement of a monomer-dimer transition are to this reviewer, premature and an independent method needs to be utilized to bolster this aspect of the story.

Reviewer #1 (Public Review):

The author studies a family of models for heritable epigenetic information, with a focus on enumerating and classifying different possible architectures. The key aspects of the paper are:

- Enumerate all 'heritable' architectures for up-to 4 constituents.<br /> - A study of whether permanent ("genetic") or transient ("epigenetic") perturbations lead to heritable changes<br /> - Enumerated the connectivity of the "sequence space" formed by these heritable architectures<br /> - Incorporating stochasticity, the authors explore stability to noise (transient perturbations)<br /> - A connection is made with experimental results on C elegans.

The study is timely, as there is a renewed interest in the last decade in non-genetic, heritable heterogeneity (e.g., from single-cell transcriptomics). Consequently, there is a need for a theoretical understanding of the constraints on such systems. There are some excellent aspects of this study: for instance, the attention paid to how one architecture "mutates" into another. Unfortunately, the manuscript as a whole does not succeed in formalising nor addressing any particular open questions in the field. Aside from issues in presentation and modelling choices (detailed below), it would benefit greatly from a more systematic approach rather than the vignettes presented.

## Terminology

The author introduces a terminology for networks of interacting species in terms of "entities" and "sensors" -- the former being nodes of a graph, and the latter being those nodes that receive inputs from other nodes. In the language of directed graphs, "entities" would seem to correspond to vertices, and "sensors" those vertices with positive indegree and outdegree. Unfortunately, the added benefit of redefining accepted terminology from the study of graphs and networks is not clear.

## Model

The model seems to suddenly change from Figure 4 onwards. While the results presented here have at least some attempt at classification or statistical rigour (i.e. Fig 4 D), there are suddenly three values associated with each entity ("property step, active fraction, and number"). Furthermore, the system suddenly appears to be stochastic. The reader is left unsure what has happened, especially after having made the effort to deduce the model as it was in Figs 1 through 3. No respite is to be found in the SI, either, where this new stochastic model should have been described in sufficient detail to allow one to reproduce the simulation.

## Perturbations

Inspired especially by experimental manipulations such as RNAi or mutagenesis, the author studies whether such perturbations can lead to a heritable change in network output. While this is naturally the case for permanent changes (such as mutagenesis), the author gives convincing examples of cases in which transient perturbations lead to heritable changes. Presumably, this is due the the underlying multistability of many networks, in which a perturbation can pop the system from one attractor to another.

Unfortunately, there appears to be no attempt at a systematic study of outcomes, nor a classification of when a particular behaviour is to be expected. Instead, there is a long and difficult-to-read description of numerical results that appear to have been sampled at random (in terms of both the architecture and parameter regime chosen). The main result here appears to be that "genetic" (permanent) and "epigenetic" (transient) perturbations can differ from each other -- and that architectures that share a response to genetic perturbation need not behave the same under an epigenetic one. This is neither surprising (in which case even illustrative evidence would have sufficed) nor is it explored with statistical or combinatorial rigour (e.g. how easy is it to mistake one architecture for another? What fraction share a response to a particular perturbation?)

As an additional comment, many of the results here are presented as depending on the topology of the network. However, each network is specified by many kinetic constants, and there is no attempt to consider the robustness of results to changes in parameters.

## DNA analogy

At two points, the author makes a comparison between genetic information (i.e. DNA) and epigenetic information as determined by these heritable regulatory architectures. The two claims the author makes are that (i) heritable architectures are capable of transmitting "more heritable information" than genetic sequences, and (ii) that, unlike DNA, the connectivity (in the sense of mutations) between heritable architectures is sparse and uneven (i.e. some architectures are better connected than others).

In both cases, the claim is somewhat tenuous -- in essence, it seems an unfair comparison to consider the basic epigenetic unit to be an "entity" (e.g., an entire transcription factor gene product, or an organelle), while the basic genetic unit is taken to be a single base-pair. The situation is somewhat different if the relevant comparison was the typical size of a gene (e.g., 1 kb).

Reviewer #1 (Public Review):

Summary:

This describes the molecular identity of the intermediate status of cranial neural crest cells (NCCs) during the initial delamination process. Taking advantage of single-cell RNA seq, the authors identify new populations of cells during EMT characterized by a specific set of gene expressions, including Dlc1. Promigratory cranial NCCs differentiate through different trajectories depending on their cell cycle phases but converge into a common progenitor, then differentiate into mesenchymal cells expressing region-specific genes.

Strengths:

Single-cell RNA seq data convincingly support what the authors claim. This is the first time to identify intermediate states between premigratory and migratory cranial NCCs. Silencing one of the marker genes, Dlc1, reduces the migratory activity of cranial NCCs. These findings deepen our understanding of the mechanism of EMT in general.

Comments on revised version:

Weaknesses:

None after substantial revision.

Reviewer #1 (Public Review):

In this manuscript, the authors report a molecular mechanism for recruiting syntaxin 17 (Syn17) to the closed autophagosomes through the charge interaction between enriched PI4P and the C-terminal region of Syn17. How to precisely control the location and conformation of proteins is critical for maintaining autophagic flux. Particularly, the recruitment of Syn17 to autophagosomes remains unclear. In this paper, the author describes a simple lipid-protein interaction model beyond previous studies focusing on protein-protein interactions. This represents conceptual advances.

Reviewer #1 (Public Review):

Li et al report that upon traumatic brain injury (TBI), Pvr signalling in astrocytes activates the JNK pathway and up-regulates the expression of the well-known JNK target MMP1. The FACS sort astrocytes, and carry out RNAseq analysis, which identifies pvr as well as genes of the JNK pathway as particularly up-regulated after TBI. They use conventional genetics loss of function, gain of function and epistasis analysis with and without TBI to verify the involvement of the JNK-MMP1 signalling pathway downstream of PVR. They also show that blocking endocytosis prolongs the involvement of this pathway in the TBI response.

The strengths are that multiple experiments are used to demonstrate that TBI in their hands damaged the BBB, induced apoptosis and increased MMP1 levels. The RNAseq analysis on FACS sorted astrocytes is nice and will be valuable to scientists beyond the confines of this paper. The functional genetic analysis is conventional, yet sound, and supports claims of JNK and MMP1 functioning downstream of Pvr in the TBI context.

For this revised version the authors have removed all the unsupported claims. This renders their remaining claims more solid. However, it has resulted in the loss of important cellular aspects of the response to TBI, limiting the scope and value of the work.

The main weakness is that novelty and insight are both rather limited. Others had previously published that both JNK signalling and MMP1 were activated upon injury, in multiple contexts (as well as the articles cited by the authors, they should also see Losada-Perez et al 2021). That Pvr can regulate JNK signalling was also known (Ishimaru et al 2004). The authors claim that the novelty was investigating injury responses in astrocytes in Drosophila. However, others had investigated injury responses by astrocytes in Drosophila before. It had been previously shown that astrocytes - defined as the Prospero+ neuropile glia, and also sharing evolutionary features with mammalian NG2 glia - respond to injury both in larval ventral nerve cords and in adult brains, where they proliferate regenerating glia and induce a neurogenic response (Kato et al 2011; Losada-Perez et al 2016; Harrison et al 2021; Simoes et al 2022). The authors argue that the novelty of the work is the investigation of the response of astrocytes to TBI. However, this is of somewhat limited scope. The authors mention that MMP1 regulates tissue remodelling, the inflammatory process and cancer. Exploring these functions further would have been an interesting addition, but the authors did not investigate what consequences the up-regulation of MMP1 after injury has in repair or regeneration processes.

The statistical analysis is incorrect in places, and this could affect the validity of some claims.

Altogether, this is an interesting and valuable addition to the repertoire of articles investigating neuron-glia communication and glial responses to injury in the Drosophila central nervous system (CNS). It is good and important to see this research area in Drosophila grow. This community together is building a compelling case for using Drosophila and its unparalleled powerful genetics to investigate nervous system injury, regeneration and repair, with important implications. Thus, this paper will be of interest to scientists investigating injury responses in the CNS using Drosophila, other model organisms (eg mice, fish) and humans.

Reviewer #1 (Public Review):

Dasguta et al. have dissected the role of Sema7a in fine tuning of a sensory microcircuit in the posterior lateral line organ of zebrafish. They attempt to also outline the different roles of a secreted verses membrane-bound form of Sema7a in this process. Using genetic perturbations and axonal network analysis, the authors show that loss of both Sema7a isoforms causes abnormal axon terminal structure with more bare terminals and fewer loops in contact with presynaptic sensory hair cells. Further, they show that loss of Sema7a causes decreased number and size of both the pre- and post-synapse. Finally, they show that overexpression of the secreted form of Sema7a specifically can elicit axon terminal outgrowth to an ectopic Sema7a expressing cell. Together, the analysis of Sema7a loss of function and overexpression on axon arbor structure is fairly thorough and revealed a novel role for Sema7a in axon terminal structure. However, the connection between different isoforms of Sema7a and the axon arborization needs to be substantiated. Furthermore, the effect of loss of Sema7a on the presynaptic cell is not ruled out as a contributing factor to the synaptic and axon structure phenotypes. These issues weaken the claims made by the authors including the statement that they have identified dual roles for the GPI-anchored verses secreted forms of Sema7a on synapse formation and as a chemoattractant for axon arborization respectively.

Reviewer #1 (Public Review):

Summary:

Through an unbiased genomewide KO screen, the authors identified loss of DBT to suppress MG132-mediated death of cultured RPE cells. Further analyses suggested that DBT reduces ubiquitinated proteins by promoting autophagy. Mechanistic studies indicated that DBT loss promotes autophagy via AMPK and its downstream ULK and mTOR signaling. Furthermore, loss of DBT suppresses polyglutamine- or TDP-43-mediated cytotoxicity and/or neurodegeneration in fly models. Finally, the authors showed that DBT proteins are increased in ALS patient tissues, compared to non-neurological controls.

Strengths:

The idea is novel, the evidence is convincing, and the data are clean. The findings have implications for human diseases.

Weaknesses:

None.

Reviewer #1 (Public Review):

Summary:

In this study, the authors distinguished afferent inputs to different cell populations in the VTA using dimensionality reduction approaches and found significantly distinct patterns between normal and drug treatment conditions. They also demonstrated negative correlations of the inputs induced by drugs with gene expression of ion channels or proteins involved in synaptic transmission and demonstrated the knockdown of one of the voltage-gated calcium ion channels caused decreased inputs.

Weaknesses:

(1) For quantifications of brain regions in this study, boundaries were based on the Franklin-Paxinos (FP) atlas according to previous studies (Beier KT et al 2015, Beier KT et al 2019). It has been reported significant discrepancies exist between the anatomical labels on the FP atlas and the Allen Brain Atlas (ref: Chon U et al., Nat Commun 2019). Although a summary of conversion is provided as a sheet, the authors need to describe how consistent or different the brain boundaries they defined in the manuscript with Allen Brain Atlas by adding histology images. Also, I wonder how reliable the annotations were for over a hundred of animals with manual quantification. The authors should briefly explain it rather than citing previous studies in the Material and Methods Section.

(2) Regarding the ellipsoids in the PC, although it's written in the manuscript that "Ellipsoids were centered at the average coordinate of a condition and stretched one standard deviation along the primary and secondary axes", it's intuitively hard to understand in some figures such as Figure 2O, P and Figure S1. The authors need to make their data analysis methods more accessible by providing source code to the public.

(3) In histology images (Figure 1B and 3K), the authors need to add dashed lines or arrows to guide the reader's attention.

(4) In Figure 2A and G, apparently there are significant differences in other brain regions such as NAcMed or PBN. If they are also statistically significant, the authors should note them as well and draw asterisks(*).

(5) In Figure 2N about the spatial distribution of starter cells, the authors need to add histology images for each experimental condition (i.e. saline, fluoxetine, cocaine, methamphetamine, amphetamine, nicotine, and morphine) as supplement figures.

(6) In the manuscript, it is necessary to explain why Cacna1e was selected among other calcium ion channels.

Reviewer #2 (Public Review):

The authors analysed functional MRI recordings of brain activity at rest, using state-of-the-art methods that reveal the diverse ways in which the information can be integrated in the brain. In this way, they found brain areas that act as (synergistic) gateways for the 'global workspace', where conscious access to information or cognition would occur, and brain areas that serve as (redundant) broadcasters from the global workspace to the rest of the brain. The results are compelling and consisting with the already assumed role of several networks and areas within the Global Neuronal Workspace framework. Thus, in a way, this work comes to stress the role of synergy and redundancy as complementary information processing modes, which fulfill different roles in the big context of information integration.<br /> In addition, to prove that the identified high-order interactions are relevant to the phenomenon of consciousness, the same analysis was performed in subjects under anesthesia or with disorders of consciousness (DOC), showing that indeed the loss of consciousness is associated with a deficient integration of information within the gateway regions.

However, there is still a standing issue that could be the basis for an improved analysis: the concepts of gateways and broadcasters allude to a directionality in the information flow. In fact, Figure 1 depicts Stage (i) and Stage (iii) as one-way processes. However, the identification of gateway and broadcaster regions relies on matrices that are symmetrical, i.e. they are not directed. Would it be possible to assess the gateway or broadcaster nature of a region taking into account the directionality of the information flow? In other words, if region X is a gateway, I would expect a synergistic relationship between the past of X,Y and present of Y (Y not being a gateway) towards the present of X; but not necessarily the other way around (i.e. the present of Y being less dependent on the past/present of X). A similar reasoning can be made for broadcasters.

Although regional differences in haemodynamics complicate attempts to map directed information flow from fMRI recordings, perhaps the IID framework could be leveraged to extract directed data (i.e., there are many atoms that are explicitly directed). As an avenue for future research, it would be interesting to discuss the feasibility or limitations of such analysis.

Also, there is something confusing in Figure 4B-C and its description. Awake should be similar to recovery (they are both awake, aren't they? Not much info is given, anyway); thus it seems counterintuitive that anesthesia minus awake looks so different than anesthesia minus recovery. The first is mostly blue-ish and the second is mostly red. Is it possible that Figure 4C is actually recovery minus anesthesia? That would make much more sense, also for Figure 4D. Please correct me if I am wrong.

Reviewer #1 (Public Review):

Qin et al. set out to investigate the role of mechanosensory feedback during swallowing and identify neural circuits that generate ingestion rhythms. They use Drosophila melanogaster swallowing as a model system, focusing their study on the neural mechanisms that control cibarium filling and emptying in vivo. They find that pump frequency is decreased in mutants of three mechanotransduction genes (nompC, piezo, and Tmc), and conclude that mechanosensation mainly contributes to the emptying phase of swallowing. Furthermore, they find that double mutants of nompC and Tmc have more pronounced cibarium pumping defects than either single mutants or Tmc/piezo double mutants. They discovered that the expression patterns of nompC and Tmc overlap in two classes of neurons, md-C and md-L neurons. The dendrites of md-C neurons warp the cibarium and project their axons to the subesophageal zone of the brain. Silencing neurons that express both nompC and Tmc leads to severe ingestion defects, with decreased cibarium emptying. Optogenetic activation of the same population of neurons inhibited filling of the cibarium and accelerated cibarium emptying. In the brain, the axons of nompC∩Tmc cell types respond during ingestion of sugar but do not respond when the entire fly head is passively exposed to sucrose. Finally, the authors show that nompC∩Tmc cell types arborize close to the dendrites of motor neurons that are required for swallowing and that swallowing motor neurons respond to the activation of the entire Tmc-GAL4 pattern.

Strengths:<br /> -The authors rigorously quantify ingestion behavior to convincingly demonstrate the importance of mechanosensory genes in the control of swallowing rhythms and cibarium filling and emptying<br /> -The authors demonstrate that a small population of neurons that express both nompC and Tmc oppositely regulate cibarium emptying and filling when inhibited or activated, respectively<br /> -They provide evidence that the action of multiple mechanotransduction genes may converge in common cell types

Weaknesses:<br /> -A major weakness of the paper is that the authors use reagents that are expressed in both md-C and md-L but describe the results as though only md-C is manipulated<br /> -Evidence that the defects they see in pumping can be specifically attributed to md-C is based on severing the labellum and allowing md-L neurons to degrade.<br /> -GRASP is known to be non-specific and prone to false positives when neurons are in close proximity but not synaptically connected. A positive GRASP signal supports but does not confirm direct synaptic connectivity between md-C/md-L axons and MN11/MN12.<br /> -MN11/MN12 LexA lines are not included in the manuscript and their expression patterns (shared with the reviewers in the author response) do not appear to contain any motor neurons. Double labeling with previously described MN11 and MN12 motor neuron Gal4 lines is needed to support the claim that these LexA lines in fact label MN11 and MN12.<br /> -As seen in Figure Supplement 2, the expression pattern of Tmc-GAL4 is broader than md-C alone. Therefore, the functional connectivity the authors observe between Tmc expressing neurons and MN11 and 12 cannot be traced to md-C alone<br /> -Example traces of md-C calcium imaging during ingestion in vivo are not included, and evidence that md-C neurons respond to mechanical force is lacking<br /> -A positive control (perhaps demonstrating that sugar sensory neurons respond to sucrose in this preparation) is needed to assess whether the lack of response to sucrose ex vivo in Figure 4K is informative<br /> -Proximity between md-C neurons and muscles is not evidence that they sense stretch<br /> -Reporting of posthoc tests needs to be improved throughout the manuscript, as it is not clear which comparisons are noted with asterisks in the figures.

Overall, this work convincingly shows that swallowing and swallowing rhythms are dependent on several mechanosensory genes. Qin et al. also characterize a candidate neuron, md-C, that is likely to provide mechanosensory feedback to pumping motor neurons, but the results they present here are not sufficient to assign this function to md-C alone. This work will have a positive impact on the field by demonstrating the importance of mechanosensory feedback to swallowing rhythms and providing a potential entry point for future investigation of the identity and mechanisms of swallowing central pattern generators.

Reviewer #1 (Public Review):

Original Review:

Bischoff et al present a carefully prepared study on a very interesting and relevant topic: the role of ion channels (here a Ca2+-activated K+ channel BK) in regulating mitochondrial metabolism in breast cancer cells. The potential impact of these and similar observations made in other tumor entities has only begun to be appreciated. That being said, the authors pursue in my view an innovative approach to understanding breast cancer cell metabolism.

Considering the following points would further strengthen the manuscript:

Methods:

(1) The authors use an extracellular Ca2+ concentration (2 mM) in their Ringer's solutions that is almost twice as high as the physiologically free Ca2+ concentration (ln 473). Moreover, the free Ca2+ concentration of their pipette solution is not indicated (ln 487).

(2) Ca2+I measurements: The authors use ATP to elicit intracellular Ca2+ signals. Is this then physiological stimulus for Ca2+ signaling in breast cancer? What is the rationale for using ATP? Moreover, it would be nice to see calibrated baseline values of Ca2+i

(3) Membrane potential measurements: It would be nice to see a calibration of the potential measurements; this would allow to correlate IV relationship with membrane potential. Without calibration it is hard to compare unless the identical uptake of the dye is shown.<br /> Do paxilline or IbTx also induce a depolarization?

(4) mito-potential measurements: Why did the authors use such a long time course and preincubated cells mit channel blockers overnight? Why did they not perform paired experiments and record the immediate effect of the BK channel blockers in the mito potential?

(5) MTT assay are also based on mitochondrial function - since modulation of mito function is at the core of this manuscript, an alternative method should be used.

Results:

(1) Fig. 5G: The number of BK "positive" mitoplasts is surprisingly low - how does this affect the interpretation? Did the authors attempt to record mitoBK current in the "whole-mitoplast" mode? How does the mitoBK current density compare with that of the plasma membrane? Is it possible to theoretically predict the number of mitoBK channels per mitochondrium to elicit the observed effects? Can these results be correlated with immuno-localization of mitoBK channels?

(2) There are also reports about other mitoK channels (e.g. Kv1.3, KCa3.1, KATP) playing an important role in mitochondrial function. Did the authors observe them, too? Can the authors speculate on the relative importance of the different channels? Is it known whether they are expressed organ-/tumor-specifically?

Comments on revised version:

The authors responded to all of my comments - except for one - in a satisfactory way so that I have no further concerns. The authors have prepared a very interesting piece of work that advances the field.

However, I disagree with respect to their interpretation of statistics. Individually analyzed cells are not the best biological replicate per se. In my view a true replicate requires the use of an independent batch of cells derived from a new passage. The statistical analysis can only based on the total number of n cells, if each replicate contributes the same number of cells. If this is not the case, the authors will have to calculate the average of each replicate first so that they are equally weighted.

Joint Public Review:

Neuropeptide signaling is an important component of nervous systems, where neuropeptides typically act via G-protein coupled receptors (GPCRs) to regulate many physiological and behavioral processes. Neuropeptides and their cognate GPCRs have been extensively characterized in bilaterian animals, revealing that a core set of neuropeptide signaling systems originated in common ancestors of extant Bilateria. Neuropeptides have also been identified in cnidarians, which are a sister group to the Bilateria. However, the GPCRs that mediate the effects of neuropeptides in cnidarians have not been identified.

In this paper the authors perform a phylogenetic analysis of GPCRs in metazoans and report that the orthologs of bilaterian neuropeptide receptors are not found in cnidarians. This indicates that neuropeptide signaling systems have largely evolved independently in cnidarians and bilaterians. To accomplish this, they generated a library of putative and known neuropeptides computationally identified in the genome of the cnidarian sea anemone Nematostella vectensis. These peptides were systematically screened for their ability to activate any of the 161 putative Nematostella GPCRs.

This work identified 31 validated GPCRs. These, together with GPCRs that cluster with them, were then used to demonstrate the independent expansion of GPCRs in cnidarian and bilaterian lineages. The authors then mapped validated receptors and ligands to the Nematostella single cell data to provide an overview of the cell types expressing these signaling genes. In addition, the authors have begun to analyze neuropeptide signaling networks in N. vectensis by showing potential signaling connections between cell types expressing neuropeptides and cell types expressing cognate receptors.

This work is the most extensive pharmacological characterization of neuropeptide GPCRs in a cnidarian to date and thus represents an important accomplishment, and is one that will improve our understanding of how peptidergic signaling evolved in animals and its impact on evolution of nervous systems. In addition, this impressive work transforms our knowledge of neuropeptide signaling systems in cnidarians and provides the foundations for extensive functional characterization neuropeptide systems in the context of nervous systems that exhibit radial symmetry, contrasting with the bilaterally symmetrical architecture of the majority of bilaterian nervous systems.

The reviewers did not detect any weaknesses in the work but asked that the authors comment on the following points, which they have done in the revised version.

(1) Clearly, other neuropeptide signaling systems in cnidarians remain to be discovered but this paper represents a huge step forward.

(2) There are limitations in what can be interpreted from single cell transcriptomic data but the data nevertheless provide the foundations for future studies involving i). detailed anatomical analysis of neuropeptide and neuropeptide receptor expression in N. vectensis using mRNA in situ hybridization and/or immunohistochemical methods and ii). functional analysis of the physiological/behavioral roles of neuropeptide signaling systems in N. vectensis.

Reviewer #1 (Public Review):

Summary:

In the resubmitted manuscript by Chen et al. entitled, "Retinal metabolism: Evidence for uncoupling of glycolysis and oxidative phosphorylation via Cori-, Cahill-, and mini-Krebs-cycle", the authors look to provide insight on retinal metabolism and substrate utilization but using a murine explant model with various pharmacological treatments in conjunction with metabolomics. The authors conclude that photoreceptors, a specific cell within the explant, which also includes retinal pigment epithelium (RPE) and many other types of cells, are able to uncouple glycolytic and Krebs-cycle metabolism via three different pathways: 1) the mini-Krebs-cycle, fueled by glutamine and branched-chain amino acids; 2) the alanine-generating Cahill-cycle; and 3) the lactate-releasing Cori-cycle. While the authors have toned down some of their bold conclusions made in the original manuscript, they did very little in the way of providing additional well-controlled experiments, including cell-specific treatments, genetic knockouts, or stable isotope tracing to support their conclusions. Rather, the authors proceed to speculate more without additional data. The major issues raised by this reviewer were not adequately addressed. As such, the conclusions continue to be highly speculative and not well supported with evidence.

Strengths of resubmission:

The resubmission toned down some of its bold statements.

Weaknesses of resubmission:

Major weaknesses of this study persist including lack of in vivo supporting data. Also, retinal explant culture metabolomics are done in neuroretina with RPE attached, which are metabolically active and can be altered by the treatments investigated herein, further confounding the claims made regarding the neuroretina. While including the RPE in the explant model is commended, it needs to be separated from the retina prior to metabolomics to get a better sense of each tissues' metabolism. Also, melanin within RPE will hinder immunofluorescence signal, so one cannot state that RPE do not express certain enzymes based solely on immunofluorescence. Pharmacologic treatments are not cell-specific as the enzymes are expressed in numerous cells within the retina and RPE, and/or the treatments have significant off-target effects (such as shikonin). So, it is difficult to ascertain that the metabolic changes are secondary to the effects on photoreceptors alone, which the authors claim. Additionally, the explants are taken at a very early age when photoreceptors are known to still be maturing. No mention or data is presented on how these metabolic changes are altered in retinal explants after photoreceptors have fully matured. Likewise, significant assumptions are made based on a single metabolomics experiment with no stable isotope tracing to support the pathways suggested. In vivo, stable-isotope retinal metabolomics are being done and have been done, so stating this technology is beyond our field is false. Therefore, the conclusions reached in this manuscript are still not supported.

Joint Public Review:

Bolivar et al. set out to explore whether four distinct neuronal subtypes within the peripheral nervous system exhibit varying potentials for axon regeneration following nerve injury. To investigate this question, they harnessed the power of four distinct reporter mouse models featuring fluorescent labeling of these neuronal subtypes. Their findings reveal that axons of nociceptor neurons exhibit faster regeneration than those of motor neurons, with mechanoreceptors, and proprioceptors displaying the slowest regeneration rate.

To delve into the molecular mechanisms underlying this divergence in regeneration potential, the authors employed the Ribotag technique in mice. This approach enabled them to dissect the differential translatomes of these four neuronal populations after nerve injury, comparing them to uninjured neurons. Their comprehensive expression profiling data uncovers a remarkably heterogeneous response among these neuron subtypes to axon injury.

To focus on one identified target with a mechanistic experiment as a proof of concept, their analysis highlights a striking upregulation of MED12 in proprioceptors, leading to the hypothesis that this molecule may play an inhibitory role, contributing to the comparatively slower regeneration of proprioceptor axons when compared to other neuronal subtypes. This hypothesis gains support from their in vitro model, where siRNA-mediated downregulation of MED12 results in a significant increase in neurite outgrowth in proprioceptive neurons after plating in cell culture dishes.

Overall, this is an interesting study, and in conjunction with similar work from others will be highly valuable for neurobiologists studying how to modulate the regeneration of axons from distinct neuronal subtypes. The quality of data presentation appears to be very good in general, and the manuscript is appropriately written.

Comments on revised version:

Because there are multiple explanations for the differential regeneration responses, the authors have provided further discussion about how regeneration may be regulated in vitro and in vivo. The detection of a gene, Med12, which is unregulated in proprioceptive neurons, but not nociceptive and mechanoceptors, gives support to the existence of specific programs of responses in the peripheral nervous system after injury. Further investigation is needed to define this responsiveness in detail.

Another response is the role of neurotrophins and their receptors. The authors have considered outcomes as a result of different Trk receptor signaling and also the effect of TGFbeta and IL6 as cytokine modulators. Add to this list is the possibility that axon guidance molecules and downstream substates may also play a role.

The original title was considered to be too broad and did not explain all the mechanistic aspects of this study. Therefore a revised title "Neuron-specific RNA-sequencing reveals different response in peripheral neurons after nerve injury" was used. It is appropriately suitable for the results reported in this manuscript.

Reviewer #1 (Public Review):

Summary:

The authors are interested in the developmental origin of the neurons of the cerebellar nuclei. They identify a population of neurons with a specific complement of markers that originate in a distinct location from where cerebellar nuclear precursor cells have been thought to originate that show distinct developmental properties. The cerebellar nuclei have been well studied in recent years both to understand their development and through an evolutionary lens, which supports the importance of this study. The discovery of a new germinal zone giving rise to a new population of CN neurons is an exciting finding, and it enriches our understanding of cerebellar development, which has previously been quite straightforward, where cerebellar inhibitory cells arise from the ventricular zone and the excitatory cells arise from the rhombic lip.

Strengths:

One of the strengths of the manuscript is that the authors use a wide range of technical approaches, including transgenic mice that allow them to disentangle the influence of distinct developmental organizers such as ATOH.<br /> Their finding of a novel germinal zone and a novel population of CN neurons is important for developmental neuroscientists, and cerebellar neuroscientists.

Weaknesses:

One important question raised by this work is what these newly identified cells eventually become in the adult cerebellum. Are they excitatory or inhibitory? Do they correspond to a novel cell type or perhaps one of the cell classes that have been recently identified in the cerebellum (e.g. Fujita et al., eLife, 2020)? Understanding this would significantly bolster the impact of this manuscript.

The major weakness of the manuscript is that it is written for a very specialized reader who has a strong background in cerebellar development, making it hard to read for a general audience. It's challenging to follow the logic of some of the experiments as well as to contextualize these findings in the field of cerebellar development.

Reviewer #1 (Public Review):

Summary:

This interesting study explores the mechanism behind an increased susceptibility of daf-18/PTEN mutant nematodes to paralyzing drugs that exacerbate cholinergic transmission. The authors use state-of-the-art genetics and neurogenetics coupled with locomotor behavior monitoring and neuroanatomical observations using gene expression reporters to show that the susceptibility occurs due to low levels of DAF-18/PTEN in developing inhibitory GABAergic neurons early during larval development (specifically, during the larval L1 stage). DAF-18/PTEN is convincingly shown to act cell-autonomously in these cells upstream of the PI3K-PDK-1-AKT-DAF-16/FOXO pathway, consistent with its well-known role as an antagonist of this conserved signaling pathway. The authors exclude a role for the TOR pathway in this process and present evidence implicating selectivity towards developing GABAergic neurons. Finally, the authors show that a diet supplemented with a ketogenic body, β-hydroxybutyrate, which also counteracts the PI3K-PDK-1-AKT pathway, promoting DAF-16/FOXO activity, partially rescues the proper development (morphology and function) of GABAergic neurons in daf-18/PTEN mutants, but only if the diet is provided early during larval development. This strongly suggests that the critical function of DAF-18/PTEN in developing inhibitory GABAergic neurons is to prevent excessive PI3K-PDK-1-AKT activity during this critical and particularly sensitive period of their development in juvenile L1 stage worms. Whether or not the sensitivity of GABAergic neurons to DAF-18/PTEN function is a defining and widespread characteristic of this class of neurons in C. elegans and other animals, or rather a particularity of the unique early-stage GABAergic neurons investigated remains to be determined.

Strengths:

The study reports interesting and important findings, advancing the knowledge of how daf-18/PTEN and the PI3K-PDK-1-AKT pathway can influence neurodevelopment, and providing a valuable paradigm to study the selectivity of gene activities towards certain neurons. It also defines a solid paradigm to study the potential of dietary interventions (such as ketogenic diets) or other drug treatments to counteract (prevent or revert?) neurodevelopment defects and stimulate DAF-16/FOXO activity.

Weaknesses:

(1 )Insufficiently detailed methods and some inconsistencies between Figure 4 and the text undermine the full understanding of the work and its implications.

The incomplete methods presented, the imprecise display of Figure 4, and the inconsistency between this figure and the text, make it presently unclear what are the precise timings of observations and treatments around the L1 stage. What exactly do E-L1 and L1-L2 mean in the figure? The timing information is critical for the understanding of the implications of the findings because important changes take place with the whole inhibitory GABAergic neuronal system during the L1 stage into the L2 stage. The precise timing of the events such as neuronal births and remodelling events are well-described (e.g., Figure 2 in Hallam and Jin, Nature 1998; Fig 7 in Mulcahy et al., Curr Biol, 2022). Likewise, for proper interpretation of the implication of the findings, it is important to describe the nature of the defects observed in L1 larvae reported in Figure 1E - at present, a representative figure is shown of a branched commissure. What other types of defects, if any, are observed in early L1 larvae? The nature of the defects will be informative. Are they similar or not to the defects observed in older larvae?

(2) The claim of proof of concept for a reversal of neurodevelopment defects is not fully substantiated by data.

The authors state that the work "constitutes a proof of concept of the ability to revert a neurodevelopmental defect with a dietary intervention" (Abstract, Line 56), however, the authors do not present sufficient evidence to distinguish between a "reversal" or prevention of the neurodevelopment defect by the dietary intervention. This clarification is critical for therapeutic purposes and claims of proof-of-concept. From the best of my understanding, reversal formally means the defect was present at the time of therapy, which is then reverted to a "normal" state with the therapy. On the other hand, prevention would imply an intervention that does not allow the defect to develop to begin with, i.e., the altered or defective state never arises. In the context of this study, the authors do not convincingly show reversal. This would require showing "embryonic" GABAergic neuron defects or showing convincing data in newly hatched L1 (0-1h), which is unclear if they do so or not, as I have failed to find this information in the manuscript. Again, the method description needs to be improved and the implications can be very different if the data presented in Figure 2D-E regard newly born L1 animals (0-1h) or L1 animals at say 5-7h after hatching. This is critical because the development of the embryonically-born GABAergic DD neurons, for instance, is not finalized embryonically. Their neurites still undergo outgrowth (albeit limited) upon L1 birth (see DataS2 in Mulcahy et al., Curr Biol 2022), hence they are susceptible to both committing developmental errors and to responding to nutritional interventions to prevent them. In contrast to embryonic GABAergic neurons, embryonic cholinergic neurons (DA/DB) do not undergo neurite outgrowth post-embryonically (Mulcahy et al., Curr Biol 2022), a fact which could provide some mechanistic insight considering the data presented. However, neurites from other post-embryonically-born neurons also undergo outgrowth post-embryonically, but mostly during the second half of the L1 stage following their birth up to mid-L2, with significant growth occurring during the L1-L2 transition. These are the cholinergic (VA/VB and AS neurons) and GABAergic (VD) neurons. The fact that AS neurons undergo a similar amount of outgrowth as VD neurons is informative if VD neurons are or are not susceptible to daf-18/PTEN activity. Independently, DD neurons are still quite unique on other aspects (see below), which could also bring insight into their selective response.

Finally, even adjusting the claim to "constitutes a proof-of-concept of the ability of preventing a neurodevelpmental defect with a dietary intervention" would not be completely precise, because it is unclear how much this work "constitutes a proof of concept". This is because, unless I misunderstood something, dietary interventions are already applied to prevent neurodevelopment defects, such as when folic acid supplementation is recommended to pregnant women to prevent neural tube defects in newborns.

(3) The data presented do not warrant the dismissal of DD remodeling as a contributing factor to the daf-18/PTEN defects.

Inhibitory GABAergic DD neurons are quite unique cells. They are well-known for their very particular property of remodeling their synaptic polarity (DD neurons switch the nature of their pre- and post-synaptic targets without changing their wiring). This process is called DD remodeling. It starts in the second half of the L1 stage and finishes during the L2 stage. Unfortunately, the fact that the authors find a specific defect in early GABAergic neurons (which are very likely these unique DD neurons) is not explored in sufficient detail and depth. The facts that these neurons are not fully developed at L1, that they still undergo limited neurite growth, and that they are poised for striking synaptic plasticity in a few hours set them apart from the other explored neurons, such as early cholinergic neurons, which show a more stable dynamics and connectivity at L1 (see Mulcahy et al., Curr Biol 2022).

The authors use their observation that daf-18/PTEN mutants present morphological defects in GABAergic neurons prior to DD remodeling to dismiss the possibility that the DAF-18/PTEN-dependent effects are "not a consequence of deficient rearrangement during the early larval stages". However, DD remodeling is just another cell-fate-determined process and as such, its timing, for instance, can be affected by mutations in genes that affect cell fates and developmental decisions, such as daf-18 and daf-16, which affect developmental fates such as those related with the dauer fate. Specifically, the authors do not exclude the possibility that the defects observed in the absence of either gene could be explained by precocious DD remodeling. Precocious DD remodeling can occur when certain pathways, such as the lin-14 heterochronic pathway, are affected. Interestingly, lin-14 has been linked with daf-16/FOXO in at least two ways: during lifespan determination (Boehm and Slack, Science 2005) and in the L1/L2 stages via the direct negative regulation of an insulin-like peptide gene ins-33 (Hristova et al., Mol Cell Bio 2005). It is likely that the prevention of DD dysfunction requires keeping insulin signaling in check (downregulated) in DD neurons in early larval stages, which seems to coincide with the critical timing and function of daf-18/PTEN. Hence, it will be interesting to test the involvement of these genes in the daf-18/daf-16 effects observed by the authors.

Discussion on the impact of the work on the field and beyond:

The authors significantly advance the field by bringing insight into how DAF-18/PTEN affects neurodevelopment, but fall short of understanding the mechanism of selectivity towards GABAergic neurons, and most importantly, of properly contextualizing their findings within the state-of-the-art C. elegans biology.

For instance, the authors do not pinpoint which type of GABAergic neuron is affected, despite the fact that there are two very well-described populations of ventral nerve cord inhibitory GABAergic neurons with clear temporal and cell fate differences: the embryonically-born DD neurons and the post-embryonically-born VD neurons. The time point of the critical period apparently defined by the authors (pending clarifications of methods, presentation of all data, and confirmation of inconsistencies between the text and figures in the submitted manuscript) could suggest that DAF-18/PTEN is required in either or both populations, which would have important and different implications. An effect on DD neurons seems more likely because an image is presented (Figure 2D) of a defect in an L1 daf-18/PTEN mutant larva with 6 neurons (which means the larva was processed at a time when VD neurons were not yet born or expressing pUnc-47, so supposedly it is an image of a larva in the first half of the L1 stage (0-~7h?)). DD neurons are also likely the critical cells here because the neurodevelopment errors are partially suppressed when the ketogenic diet is provided at an "early" L1 stage, but not later (e.g., from L2-L3, according to the text, L2-L4 according to the figure? ).

This study brings important contributions to the understanding of GABAergic neuron development in C. elegans, but unfortunately, it is justified and contextualized mostly in distantly-related fields - where the study has a dubious impact at this stage rather than in the central field of the work (post-embryonic development of C. elegans inhibitory circuits) where the study has stronger impact. This study is fundamentally about a cell fate determination event that occurs in a nutritionally-sensitive developmental stage (post-embryonic L1 larval stage) yet the introduction and discussion are focused on more distantly related problems such as excitatory/inhibitory (E/I) balance, pathophysiology of human diseases, and treatments for them. Whereas speculation is warranted in the discussion, the reduced in-depth consideration of the known biology of these neurons and organisms weakens the impact of the study as redacted. For instance, the critical role of DAF-18/PTEN seems to occur at the early L1 larval stage, a stage that is particularly sensitive to nutritional conditions. The developmental progression of L1 larvae is well-known to be sensitive to nutrition - eg, L1 larvae arrest development in the absence of food, something that is explored in nematode labs to synchronize animals at the L1 stage by allowing embryos to hatch into starvation conditions (water). Development resumes when they are exposed to food. Hence, the extensive postembryonic developmental trajectory that GABAergic neurons need to complete is expected to be highly susceptible to nutrition. Is it? The sensitivity towards the ketogenic diet intervention seems to favor this. In this sense, the attribution of the findings to issues with the nutrition-sensitive insulin-like signaling pathway seems quite plausible, yet this possibility seems insufficiently considered and discussed.

Finally, the fact that imbalances in excitatory/inhibitory (E/I) inputs are linked to Autism Spectrum Disorders (ASD) is used to justify the relevance of the study and its findings. Maybe at this stage, the speculation would be more appropriate if restricted to the discussion. In order to be relevant to ASD, for instance, the selectivity of PTEN towards inhibitory neurons should occur in humans too. However, at present, the E/I balance alteration caused by the absence of daf-18/PTEN in C. elegans could simply be a coincidence due to the uniqueness of the post-embryonic developmental program of GABAergic neurons in C. elegans. To be relevant, human GABAergic neurons should also pass through a unique developmental stage that is critically susceptible to the PI3K-PDK1-AKT pathway in order for DAF-18/PTEN to have any role in determining their function. Is this the case? Hence, even in the discussion, where the authors state that "this study provides universally relevant information on.... the mechanisms underlying the positive effects of ketogenic diets on neuronal disorders characterized by GABA dysfunction and altered E/I ratios", this claim seems unsubstantiated as written particularly without acknowledging/mentioning the criteria that would have to be fulfilled and demonstrated for this claim to be true.

Reviewer #1 (Public Review):

This study presents valuable observations of white matter organisation from diffusion MRI and two types of synchrotron imaging in both monkeys and mice. Cross-modality comparisons are interesting as the different methods are able to probe anatomical structures at different length scales, from single axons in high-resolution synchrotron (ESRF) imaging, to clusters of axons in lower-resolution synchrotron (DEXY) data, to axon populations at the mm-scale in diffusion MRI. By acquiring all modalities in monkey and mouse ex vivo samples, the authors can observe principles of fibre organisation, and characterise how fibre characteristics, such as tortuosity and micro-dispersion, vary across select brain regions and in healthy tissue versus a demyelination model. The results are solid, though some statements (in the abstract/discussion) do not appear to be fully supported, and statistical tests would help confirm whether tissue characteristics are similar/different between different conditions.

One very interesting result is the observation of apparent laminar organisation of fibres in ex vivo monkey white matter samples. DESY data from the corpus callosum shows fibres with two dominant orientations (one L-R, one slightly inclined), clustered in laminar structures within this major fibre bundle. Thanks to the authors providing open data, I was able to look through the raw DESY volume and observe regions with different "textures" (different orientations) in the described laminar arrangement. That this organisation can be observed by eye, as well as by structure tensor, is fairly convincing. As not all readers will download the data themselves, the manuscript could benefit from additional figures/videos to demonstrate (1) the quality of the DESY data and (2) a more 3D visualisation of the laminar structures (where the coronal plane shows convincing columnar structure or stripes). Similarly in Figure 5A, though this nicely depicts two populations with different orientations, it is somewhat difficult to see the laminar structure in the current image.

ESRF data of the centrum semiovale (CS) contributes evidence for similar laminar structures in a crossing fibre region, where primarily AP fibres are shown to cluster in 3 laminar structures. As above, further visualisations of the ESRF volume in the CS (as shown in Figure 4E) would be of value (e.g. showing consistency across the 4 volumes, 2D images showing stripey/columnar patterns along different axes, etc).

A key limitation of this result is that, though the DESY data from the CC seems convincing, the same structures were not observed in high-resolution synchrotron (ESRF) data of the same tissue sample in the corpus callosum. This seems surprising and the manuscript does not provide a convincing explanation for this inconsistency. The authors argue that this is due to the limited FOV of the ESRF data (~200x200x800 microns). However, the observed laminar structures in DESY are ~40 microns thick, and ERSF data from the CST suggests laminar thicknesses in the range of 5-40 microns with a similar FOV. This suggests the ERSF FOV would be sufficient to capture at least a partial description of the laminar organisation. Further, the DESY data from the CC shows columnar variations along the LR axis, which we might expect to be observed along the long axis of the ESFR volume of the same sample. Additional analyses or explanations to reconcile these apparently conflicting observations would be of value. For example, the authors could consider down-sampling the ESRF data in an appropriate manner to make it more similar to the DESY data, and running the same analysis, to see if the observed differences are related to resolution (i.e. the thinner laminar structures cluster in ways that they look like a thicker laminar structure at lower resolution), or crop the DESY data to the size of the ESRF volume, to test whether the observed differences can be explained by differences in FOV.

Laminar structures were not observed in mouse data, though it is unclear if this is due to anatomical differences or somewhat related to differences in data quality across species.

The authors further quantify various other characteristics of the white matter, such as micro-dispersion, tortuosity, and maximum displacement. Notably, the microscopic FA calculated via structure tensor is fairly consistent across regions, though not modalities. When fibre orientations are combined across the sample, they are shown to produce similar FODs to dMRI acquired in the same tissue, which is reassuring. As noted in the text, the estimates of tortuosity and max displacement are dependent on the FOV over which they are calculated. Calculating these metrics over the same FOV, or making them otherwise invariant to FOV, could facilitate more meaningful comparisons across samples and/or modalities.

Though the results seem solid, some statements, particularly in the abstract and discussion, do not seem to be fully supported by the data. For example, the abstract states "Our findings revealed common principles of fibre organisation in the two species; small axonal fasciculi and major bundles formed laminar structures with varying angles, according to the characteristics of major pathways.", though the results show "no strong indication within the mouse CC of the axonal laminar organisation observed in the monkey". Similarly, the introduction states: "By these means, we demonstrated a new organisational principle of white matter that persists across anatomical length scales and species, which governs the arrangement of axons and axonal fasciculi into sheet-like laminar structures." Further comments on the text are provided below.

One observation not notably discussed in the paper is that the spherical histograms of Figure 3E/H appear to have an anisotropic spread of the white points about 0,0. It would be interesting if the authors could comment on whether this could be interpreted as the FOD having asymmetric dispersion and if so, whether the axis of dispersion relates to the fibre orientations of the laminar structures.

A limitation of the study is that it considers only small ex vivo tissue samples from two locations in a single postmortem monkey brain and slightly larger regions of mouse brain tissue. Consequently, further evidence from additional brain regions and subjects would be required to support more generalised statements about white matter organisation across the brain.

Given the monkey results, the mouse study (section 2.5 onwards) lacks some motivation. In particular, it is unclear why a demyelination model was studied and if/how this would link to the laminar structure observed in the monkey data. Further, it is unclear how comparable tortuosity/max deviation values are across species, considering the differences in data quality and relative resolution, given that the presented results show these values are very modality-dependent.

The paper introduces a new method of "scale-space" parameters for structure tensors. Since, to my understanding, this is the first description of the method, some simple validation of the method would be welcomed. Further, the same scale parameters are not used across monkeys and mice, with a larger kernel used in mice (Table 2) which is surprising given their smaller brain size. Some explanation would be helpful.

Reviewer #1 (Public Review):

Summary:

The work in the manuscript titled " Altered firing output of VIP interneurons and early dysfunctions in CA1 hippocampal circuits in the 3xTg mouse model of Alzheimer's disease" utilized patch-clamp techniques to explore the electrophysiological characteristics of VIP interneurons in the early stages of AD using the 3xTg mouse model. The study revealed that VIP interneurons exhibited prolonged action potentials and reduced firing rates. These changes could not be attributed to modifications in input signals or morphological transformations. The authors attributed aberrant VIP activity to the accumulation of beta-amyloid in those interneurons.

The decreased frequency of VIP inhibitory events was associated with no observed changes in excitatory drive to these interneurons. Consequently, heightened activity in the general population of CA1 interneurons was observed during a decision-making task and an object recognition test. In light of these findings, the authors concluded that the altered firing patterns of VIP interneurons may initiate early-stage dysfunction in hippocampal CA1 circuits, potentially influencing the progression of AD pathology.

Strengths:

Overall the work is novel and moves the field of Alzheimer's disease forward in a significant way. The manuscript reports a novel concept of aberrant activity in VIP interneurons during the early stages of AD thus contributing to dysfunctions of the CA1 microcircuit. This results in the enhancement of the inhibitory tone on the primary cells of CA1. Thus, the disinhibition by VIP interneurons of Principal Cells is dampened. The manuscript was skillfully composed, and the study was of strong scientific rigor featuring well-designed experiments. Necessary controls were present. Both sexes were included.

Limitations:

(1) The authors attributed aberrant circuit activity to the accumulation of "Abeta intracellularly" inside IS-3 cells. That is problematic. 6E10 antibody recognizes amyloid plaques in addition to Amyloid Precursor Protein (APP) as well as the C99 fragment. There are no plaques at the ages 3xTg mice were examined. Thus, the staining shown in Figure 1a is of APP/C99 inside neurons, not abeta accumulations in neurons. At the ages of 3-6 months, 3xTg starts producing abeta oligomers and potentially tau oligomers as well (Takeda et al., 2013 PMID: 23640054; Takeda et al., 2015 PMID: 26458742 and others). Emerging literature suggests that abeta and tau oligomers disrupt circuit function. Thus, a more likely explanation of abeta and tau oligomers disrupting the activity of VIP neurons is plausible.

(2) Authors suggest that their animals do not exhibit loss of synaptic connections and show Figure 3d in support of that suggestion. However, imaging with confocal microscopy of 70-micron thick sections would not allow the resolution of pre- and post-synaptic terminals. More sensitive measures such as electron microscopy or array tomography are the appropriate techniques to pursue. It is important for the authors to either remove that data from the manuscript or address the limitations of their technique in the discussion section. There is a possibility of loss of synaptic connections in their mouse model at the ages examined.

Reviewer #1 (Public Review):

Summary:

The paper of Mao et al. expands the genetic toolset that was previously developed by the Rao lab (Denfg et al 2019) to introduce the conditional KO or downregulation of neurotransmission components in Drosophila. The authors then use these tools to investigate neurotransmission in the the clock neurons of the Drosophila brain. They first test some known components and then analyze the contribution of the CNMa neuropeptide and its receptor to the circadian behavior. The results indicate that CNMA acts from a subset of DN1ps (dorsal clock neurons) to set the phase of the morning peak of locomotor activity in light:dark cycles, with an advanced morning activity in the absence of the neuropeptide. Interestingly, the receptor for the PDF neuropeptide appears to be acting in some of the CNMa neurons to control morning activity.

Strengths/weaknesses:

This is clearly a very useful new set of tools to restrict the manipulation of these components to specific neuronal populations, and overall (see specific points below), the paper is convincing to show that the tools indeed allow to efficiently and specifically eliminate neuropeptides/receptors from subsets of neurons. The analysis of the CNMa function in the clock network reveals a new and interesting function for CNMa in the control of morning anticipation in LD conditions. This function appears to depend on CNMA_expressing DN1ps.

Comment on revised version:

I believe that the authors properly addressed the main points that were raised in my comment on version 1.

Reviewer #1 (Public Review):

Summary:

In this paper, the authors present new tools to collect and process information from the biomedical literature that could be typically used in a meta-analytic framework. The tools have been specifically developed for the neuroimaging literature. However, many of their functions could be used in other fields. The tools mainly enable to downloading of batches of paper from the literature, extracting relevant information along with meta-data, and annotating the data. The tools are implemented in an open ecosystem that can be used from the command line or Python.

Strengths:

The tools developed here are really valuable for the future of large-scale analyses of the biomedical literature. This is a very well-written paper. The presentation of the use of the tools through several examples corresponding to different scientific questions really helps the readers to foresee the potential application of these tools.

Weaknesses:

The tools are command-based and store outcomes locally. So users who prefer to work only with GUI and web-based apps may have some difficulties. Furthermore, the outcomes of the tools are constrained by inherent limitations in the scientific literature, in particular, here the fact that only a small portion of the publications have full text openly available.

Reviewer #1 (Public Review):

Summary:

In this work, Xie, Prescott and colleagues have reevaluated the role of Nav1.7 in nociceptive sensory neurons excitability. They find that nociceptors can make use of different sodium channel subtypes to reach equivalent excitability. The existence of this degeneracy is critical to understanding the neuronal physiology under normal and pathological conditions and could explain why Nav subtype-selective drugs have failed in clinical trials. More concretely, nociceptor repetitive spiking relies on Nav1.8 at DIV0 (and probably under normal conditions in vivo), but on Nav1.7 and Nav1.3 at DIV4-7 (and after inflammation in vivo).

The conclusions of this paper are mostly well supported by data, and these findings should be of broad interest to scientists working on pain, drug development, neuronal excitability and ion channels.

Strengths:

The authors have employed elegant electrophysiology experiments (including specific pharmacology and dynamic clamp) and computational simulations to study the excitability of a subpopulation of DRGs that would very likely match with nociceptors (they take advantage of using transgenic mice to detect Nav1.8-expressing neurons). They make a strong point showing the degeneracy that occurs at the ion channel expression level in nociceptors, adding this new data to previous observations in other neuronal types. They also demonstrate that the different Nav subtypes functionally overlap and are able to interchange their "typical" roles in action potential generation. As Xie, Prescott and colleagues argue, the functional implications of the degenerate character of nociceptive sensory neurons excitability need to be seriously taken into account regarding drug development and clinical trials with Nav subtype-selective inhibitors.

In this revised version, the quality of the manuscript has been visibly improved. In my opinion, the questions and concerns raised by reviewers have been addressed clearly. After a detailed reading of this version and the comments to the reviewers, I have no additional comments or criticisms.

Reviewer #1 (Public Review):

Summary:

In Ryu et al., the authors use a cortical mouse astrocyte culture system to address the functional contribution of astrocytes to circadian rhythms in the brain. The authors' starting point is transcriptional output from serum-shocked culture, comparative informatics with existing tools and existing datasets. After fairly routine pathway analyses, they focus on the calcium homeostasis machinery and one gene, Herp, in particular. They argue that Herp is rhythmic at both mRNA and protein levels in astrocytes. They then use a calcium reporter targeted to the ER, mitochondria, or cytosol and show that Herp modulates calcium signaling as a function of circadian time. They argue that this occurs through the regulation of inositol receptors. They claim that the signaling pathway is clock-controlled by a limited examination of Bmal1 knockout astrocytes. Finally, they switch to calcium-mediated phosphorylation of the gap junction protein Connexin 43 but do not directly connect HERP-mediated circadian signaling to these observations. While these experiments address very important questions related to the critical role of astrocytes in regulating circadian signaling, the mechanistic arguments for HERP function, its role in circadian signaling through inositol receptors, the connection to gap junctions, and ultimately, the functional relevance of these findings is only partially substantiated by experimental evidence.

Strengths:

- The paper provides useful datasets of astrocyte gene expression in circadian time.

- Identifies HERP as a rhythmic output of the circadian clock.

- Demonstrates the circadian-specific sensitivity of ATP -> calcium signaling.

- Identifies possible rhythms in both Connexin 43 phosphorylation and rhythmic movement of calcium between cells.

Weaknesses:

- It is not immediately clear why the authors chose to focus on Ca2+ homeostasis or Herp from their initial screens as neither were the "most rhythmic" pathways in their primary analyses.

- It would have been interesting (and potentially important) to know whether various methods of cellular synchronization would also render HERP rhythmic (e.g., temperature, forskolin, etc). If Herp is indeed relatively astrocyte-specific and rhythmic, it should be easy to assess its rhythmicity in vivo.

- The authors show that Herp suppression reduces ATP-mediated suppression of calcium whereas it initially increases Ca2+ in the cytosol and mitochondria and then suppresses it. The dynamics of the mitochondrial and cytosolic responses are not discussed in any detail and it is unclear what their direct relationship is to Herp-mediated ER signaling. What is the explanation for Herp (which is thought to be ER-specific) to calcium signaling in other organelles?

- What is the functional significance of promoting ATP-mediated suppression of calcium in ER?

- The authors then nicely show that the effect of ATP is dependent on intrinsic circadian timing but do not explain why these effects are antiphase in cytosol or mitochondria. Moreover, the ∆F/F for calcium in mitochondria and cytosol both rise, cross the abscissa, and then diminish - strongly suggesting a biphasic signaling event. Therefore, one wonders whether measuring the area under the curve is the most functionally relevant measurement of the change.

- Why are mitochondrial and cytosolic calcium not also demonstrated for Bmal1 KO astrocytes?

- The authors claim that Herp acts by regulating the degradation of ITPRs but this hypothesis - rather central to the mechanisms proposed in this study - is not experimentally substantiated.

- There is no clear demonstration of the functional relevance of the circadian rhythms of ATP-mediated calcium signaling.

Reviewer #1 (Public Review):

This is an interesting and well-written paper reporting on a novel approach to studying cerebellar function based on the idea of selective recruitment using fMRI. The study is well-designed and executed. Analyses are sound and results are properly discussed. The paper makes a significant contribution to broadening our understanding of the role of the cerebellum in human behavior.

- While the authors provide a compelling case for the link between BOLD and the cerebellar cortical input layer, there remains considerable unexplained variance. Perhaps the authors could elaborate a bit more on the assumption that BOLD signals mainly reflect the input side of the cerebellum (see for example King et al., elife. 2023 Apr 21;12:e81511).

- The current approach does not appear to take the non-linear relationships between BOLD and neural activity into account.

- The authors may want to address a bit more the issue of closed loops as well as the underlying neuroanatomy including the deep cerebellar nuclei and pontine nuclei in the context of their current cerebello-cortical correlational approach. But also the contribution of other brain areas such as the basal ganglia and hippocampus.

- What about the direct projections of mossy fibers to the DCN that actually bypasses the cerebellar cortex?

Reviewer #1 (Public Review):

In this work, the authors set out to ask whether the MYRF family of transcription factors, represented by myrf-1 and myrf-2 in C. elegans, have a role in the temporally controlled expression of the miRNA lin-4. The precisely timed onset of lin-4 expression in the late L1 stage is known to be a critical step in the developmental timing ("heterochronic") pathway, allowing worms to move from the L1 to the L2 stage of development. Despite the importance of this step of the pathway, the mechanisms that control the onset of lin-4 expression are not well understood.

Overall, the paper provides convincing evidence that MYRF factors have a key role in promoting lin-4 expression in young larvae. Using state-of-the-art techniques (knock-in reporters and conditional alleles), the authors show that MYRF factors are essential for lin-4 activation and act cell-autonomously. Results using some unusual gain-of-function alleles are supported by consistent results using other approaches. The authors also provide evidence supporting the idea that MYRF factors activate lin-4 by directly activating its promoter. Because these results are indirect test of this, further experiments will be necessary to conclusively determine whether lin-4 is indeed a direct target of MYRF factors. myrf-1 and myrf-2 likely function redundantly to activate lin-4; potential complex interactions between these two genes will be an interesting area for future work.

Overall, the paper's results are convincing. The important findings on miRNA regulation and the control of developmental timing will make this work of interest to a broad range of developmental biologists.

Reviewer #2 (Public Review):

Summary:

Shotgun data have been analysed to obtain fungal and bacterial organisms abundance. Through their metabolic functions and through co-occurrence networks, a functional relationship between the two types of organisms can be inferred. By means of metabolomics, function-related metabolites are studied in order to deepen the fungus-bacteria synergy.

Strengths:

Data obtained in bacteria correlate with data from other authors.<br /> The study of metabolic "interactions" between fungi and bacteria is quite new.<br /> The inclusion of metabolomics data to support the results is a great contribution.

Weaknesses:

All my concerns have been clarified

Reviewer #1 (Public Review):

The manuscript by Mullen et al. investigated the gene expression changes in cancer cells treated with the DHODH inhibitor brequinar (BQ), to explore the therapeutic vulnerabilities induced by DHODH inhibition. The study found that BQ treatment causes upregulation of antigen presentation pathway (APP) genes and cell surface MHC class I expression, mechanistically which is mediated by the CDK9/PTEFb pathway triggered by pyrimidine nucleotide depletion. The combination of BQ and immune checkpoint therapy demonstrated a synergistic (or additive) anti-cancer effect against xenografted melanoma, suggesting the potential use of BQ and immune checkpoint blockade as a combination therapy in clinical therapeutics.

The interesting findings in the present study include demonstrating a novel cellular response in cancer cells induced by DHODH inhibition. However, whether the increased antigen presentation by DHODH inhibition actually contributed to the potentiation of the efficacy of immune-check blockade (ICB) is not directly examined is the limitation of the study. Moreover, the mechanism of the increased antigen presentation pathway by pyrimidine depletion mediated by CDK9/PTEFb was not validated by genetic KD or KO targeting by CDK9/PTEFb pathways. Finally, high concentrations of BQ have been reported to show off-target effects, sensitizing cancer cells to ferroptosis, and the authors should discuss whether the dose used in the in vivo study reached the ferroptotic sensitizing dose or not.

Comment on the revised version:

In their response letter, the authors appropriately addressed the reviewer's comments.

However, it is unfortunate that these comments are not reflected in the main text. Consequently, readers may encounter the same questions. Therefore, the reviewer recommends mentioning them in the discussion or limitations of the study, even if briefly, to address readers' concerns. Especially, addressing the comments such as the dosage of BQ being lower than the reported pro-ferroptotic dose (PMID 37407687), and the lack of examining potential impact of immune cell depletion on the efficacy of BQ treatment would be necessary for considering the proposed mechanism. The latter limitation is also raised by the other reviewer.

Reviewer #1 (Public Review):

Summary:

This manuscript by Warfvinge et al. reports the results of CITE-seq to generate single-cell multi-omics maps from BM CD34+ and CD34+CD38- cells from nine CML patients at diagnosis. Patients were retrospectively stratified by molecular response after 12 months of TKI therapy using European Leukemia Net (ELN) recommendations. They demonstrate heterogeneity of stem and progenitor cell composition at diagnosis, and show that compared to optimal responders, patients with treatment failure after 12 months of therapy demonstrate increased frequency of molecularly defined primitive cells at diagnosis. These results were validated by deconvolution of an independent previously published dataset of bulk transcriptomes from 59 CML patients. They further applied a BCR-ABL-associated gene signature to classify primitive Lin-CD34+CD38- stem cells as BCR:ABL+ and BCR:ABL-. They identified variability in the ratio of leukemic to non-leukemic primitive cells between patients, showed differences in expression of cell surface markers and determined that a combination of CD26 and CD35 cell surface markers could be used to prospectively isolate the two populations. The relative proportion of CD26-CD35+ (BCR:ABL-) primitive stem cells was higher in optimal responders compared to treatment failures, both at diagnosis and following 3 months of TKI therapy.

Strengths:

The studies are carefully conducted and the results are very clearly presented. The data generated will be a valuable resource for further studies. The strengths of this study are the application of single-cell multi-omics using CITE-Seq to study individual variations in stem and progenitor clusters at diagnosis that are associated with good versus poor outcomes in response to TKI treatment. These results were confirmed by deconvolution of a historical bulk RNAseq data set. Moreover, they are also consistent with a recent report from Krishnan et al. and are a useful confirmation of those results. The major new contribution of this study is the use of gene expression profiles to distinguish BCR-ABL+ and BCR-ABL- populations within CML primitive stem cell clusters and then applying antibody-derived tag (ADT) data to define molecularly identified BCR:ABL+ and BCR-ABL- primitive cells by expression of surface markers. This approach allowed them to show an association between the ratio of BCR-ABL+ vs BCR-ABL- primitive cells and TKI response and study dynamic changes in these populations following short-term TKI treatment.

Weaknesses:

The number of samples studied by CITE-Seq is limited. However, the authors have confirmed their key observations in additional samples. The BCR-ABL+ versus BCR-ABL- status of cells was not confirmed by direct sequencing for BCR-ABL. However, we recognize that the methodologies to perform these analyses on single cells is still evolving and the authors have shown that CD26 and CD35 expression can consistently identify BCR-ABL+ versus BCR-ABL- cells. It will be of interest to learn whether the GEP and surface markers identified here can distinguish BCR-ABL+ primitive stem cells later in the course of TKI treatment.

Reviewer #1 (Public Review):

This is an important manuscript that links gene expression to genetic variants and regions of open chromatin. The mechanisms of genetic gene regulation are essential to understanding how standing genetic variation translates to function and phenotype. This data set has the ability to add substantial insight into the field. In particular, the authors show how the relationships between variants, chromatin, and genes are spatially constrained by topologically associated domains.

Reviewer #1 (Public Review):

Cell type deconvolution is one of the early and critical steps in the analysis and integration of spatial omic and single cell gene expression datasets, and there are already many approaches proposed for the analysis. Sang-aram et al. provide an up-to-date benchmark of computational methods for cell type deconvolution.

In doing so, they provide some (perhaps subtle) additional elements that I would say are above the average for a benchmarking study: i) a full Nextflow pipeline to reproduce their analyses; ii) methods implemented in Docker containers (which can be used by others to run their datasets); iii) a fairly decent assessment of their simulator compared to other spatial omics simulators. A key aspect of their results is that they are generally very concordant between real and synthetic datasets. And, it is important that they authors include an appropriate "simpler" baseline method to compare against and surprisingly, several methods performed below this baseline. Overall, this study also has the potential to also set the standard of benchmarks higher, because of these mentioned elements.

The only weakness of this study that I can readily see is that this is a very active area of research and we may see other types of data start to dominate (CosMx, Xenium) and new computational approaches will surely arrive. The Nextflow pipeline will make the the prospect of including new reference datasets and new computational methods easier.

Reviewer #1 (Public Review):

The authors primary objective in this study was to identify differences between patients with preeclampsia and normal patients with respect to the placental syncytiotrophoblast extracellular vesicle proteome.

A strength of this study is that the authors identified novel STB-EV protein markers that are more abundant in the placenta of patients with preeclampsia compared with normal controls. This contributes a little more to what is already known about STB-EV markers and preeclampsia. If these markers can be shown to be more abundant in maternal plasma of preeclampsia patients, it would be very useful for identifying patients who are at high risk for developing early-onset preeclampsia.

Weaknesses include:<br /> (1) The small sample size. There were only 6 patients in the study group and 6 normal controls. However, this can be considered as a pilot study.<br /> (2) The normal controls were not matched with the study patients and the authors did not state how the controls were selected.<br /> (3) The authors state that the placenta samples were obtained at the time of elective cesarean section. However, it is likely that all the preeclampsia patients were delivered for clinical indications rather than electively. This should be clarified.

Reviewer #1 (Public Review):

The authors set out to use structural biology (cryo-em), SPR and complement convertase assays to understand the mechanism(s) by which ISG65 dampens the cytotoxicity/cellular clearance to/of trypanosmes opsonised with C3b by the innate immune system.

The cryo-EM structure adds significantly the the author's previous crystallographic data because the latter was limited to the C3d sub-domain of C3b. Further, the in vitro convertase assay adds an additional functional dimension to this study.

The authors have achieved their aims and the results support their conclusions.

The role of complement in immunity to T. brucei (or lack thereof) has been a significant question in molecular parasitology for over 30 years. The identification of ISG65 as the C3 receptor and now this study providing mechanistic insights represents a major advance in the field.

The authors have appropriately put their results into perspective with other recent reports on the role of ISG65.

Reviewer #1 (Public Review):

The goal of this study is to understand the allosteric mechanism of overall activity regulation in an anaerobic ribonucleotide reductase (RNR) that contains an ATP-cone domain. Through cryo-EM structural analysis of various nucleotide-bound states of the RNR, the mechanism of dATP inhibition is found to involve order-disorder transitions in the active site. These effects appear to prevent binding of substrate and a radical transfer needed to initiate the reaction.

Strengths of the manuscript include the comprehensive nature of the work - including both numerous structures of different forms of the RNR and detailed characterization of enzyme activity to establish the parameters of dATP inhibition. The manuscript has been improved in a revision by performing additional experiments to help corroborate certain aspects of the study. But these new experiments do not address all of the open questions about the structural basis for mechanism. Additionally, some questions about the strength of biochemical data and fit of binding or kinetic curves to data that were raised by other referees still remain. Some experimental observations are not consistent with the proposed model. For example, why does dATP enhance Gly radical formation when the proposed mechanism of dATP inhibition involves disorder in the Gly radical domain?

The work is impactful because it reports initial observations about a potentially new mode of allosteric inhibition in this enzyme class. It also sets the stage for future work to understand the molecular basis for this phenomenon in more detail.

Reviewer #3 (Public Review):

In the revised manuscript by Maio et al, the authors examined the bioenergetic mechanisms involved in the delayed migration of DC's during Mtb infection. The authors performed a series of in vitro infection experiments including bioenergetic experiments using the Agilent Seahorse XF, and glucose uptake and lactate production experiments. Also, data from SCENITH is included in the revised manuscript as well as some clinical data. This is a well written manuscript and addresses an important question in the TB field. A remaining weakness is the use of dead (irradiated) Mtb in several of the new experiments and claims where iMtb data were used to support live Mtb data. Another notable weakness lies in the author's insistence on asserting that lactate is the ultimate product of glycolysis, rather than acknowledging a large body of historical data in support of pyruvate's role in the process. This raises a perplexing issue highlighted by the authors: if Mtb indeed upregulates glycolysis, one would expect that inhibiting glycolysis would effectively control TB. However, the reality contradicts this expectation. Lastly, the examination of the bioenergetics of cells isolated from TB patients undergoing drug therapy, rather than studying them at their baseline state is a weakness.

Reviewer #1 (Public Review):

Summary:

Transposable Elements (TEs) are exogenously acquired DNA regions that have played important roles in the evolutional acquisition of various biological functions. TEs may have been important in the evolution of the immune system, but their role in thymocytes has not been fully clarified.

Using the human thymus scRNA dataset, the authors suggest the existence of cell type-specific TE functions in the thymus. In particular, it is interesting to show that there is a unique pattern in the type and expression level of TEs in thymic antigen-presenting cells, such as mTECs and pDCs, and that they are associated with transcription factor activities. Furthermore, the authors suggested that TEs may be non-redundantly regulated in expression by Aire, Fezf2, and Chd4, and that some TE-derived products are translated and present as proteins in thymic antigen-presenting cells. These findings provide important insights into the evolution of the acquired immune system and the process by which the thymus acquires its function as a primary lymphoid tissue.

Strengths:

(1) By performing single-cell level analysis using scRNA-seq datasets, the authors extracted essential information on heterogeneity within the cell population. It is noteworthy that this revealed the diversity of expression not only of known autoantigens but also of TEs in thymic antigen-presenting cells.

(2) The attempt to use mass spectrometry to confirm the existence of TE-derived peptides is worthwhile, even if the authors did not obtain data on as many transcripts as expected.

(3) The use of public data sets and the clearly stated methods of analysis improved the transparency of the results.

Weaknesses:

(1) The authors sometimes made overstatements largely due to the lack or shortage of experimental evidence.

For example in Figure 4, the authors concluded that thymic pDCs produced higher copies of TE-derived RNAs to support the constitutive expression of type-I interferons in thymic pDCs, unlike peripheral pDCs. However, the data was showing only the correlation between the distinct TE expression pattern in pDCs and the abundance of dsRNAs. We are compelled to say that the evidence is totally too weak to mention the function of TEs in the production of interferon. Even if pDCs express a distinct type and amount of TE-derived transcripts, it may be a negligible amount compared to the total cellular RNAs. How many TE-derived RNAs potentially form the dsRNAs? Are they over-expressed in pDCs?<br /> The data interpretation requires more caution to connect the distinct results of transcriptome data to the biological significance.

We contend that our manuscript combines the attributes of a research article (novel concepts) and a resource article (datasets of TEs implicated in various aspects of thymus function). The critical strength of our work is that it opens entirely novel research perspectives. We are unaware of previous studies on the role of TEs in the human thymus. The drawback is that, as with all novel multi-omic systems biology studies, our work provides a roadmap for a multitude of future mechanistic studies that could not be realized at this stage. Indeed, we performed wet lab experiments to validate some but not all conclusions: i) presentation of TE-derived MAPs by TECs and ii) formation of dsRNAs in thymic pDCs. In response to Reviewer #1, we performed supplementary analyses to increase the robustness of our conclusions. Also, we indicated when conclusions relied strictly on correlative evidence and clarified the hypotheses drawn from our observations. Regarding the Reviewer's questions about TE-derived dsRNAs, LINE, LTR, and SINE elements all have the potential to generate dsRNAs, given their highly repetitive nature and bi-directional transcription (1). As ~32% of TE subfamilies are overexpressed in pDCs, we hypothesized that these TE sequences might form dsRNA structures in these cells. To address the Reviewer's concerns regarding the amount of TE-derived RNAs among total cellular RNAs, we also computed the percentage of reads assigned to TEs in the different subsets of thymic APCs (see Reviewer 1 comment #4).<br /> ------

I appreciate the authors' efforts to improve the quality of this valuable paper. The additional data proposed by the authors enhanced the possibility that the non-negligible amount of RNAs in pDCs is derived from TE elements. Their biological roles and significance will be demonstrated in future research.

(2) Lack of generality of specific examples. This manuscript discusses the whole genomic picture of TE expression. In addition, one good way is to focus on the specific example to clearly discuss the biological significance of the acquisition of TEs for the thymic APC functions and the thymic selection.

In Figure 2, the authors focused on ETS-1 and its potential target genes ZNF26 and MTMR3, however, the significance of these genes in NK cell function or development is unclear. The authors should examine and discuss whether the distinct features of TEs can be found among the genomic loci that link to the fundamental function of the thymus, e.g., antigen processing/presentation.

We thank the Reviewer for this highly relevant comment. We investigated the genomic loci associated with NK cell biology to determine if ETS1 peaks would overlap with TE sequences in protein-coding genes' promoter region. Figure 2h illustrates two examples of ETS1 significant peaks overlapping TE sequences upstream of PRF1 and KLRD1. PRF1 is a protein implicated in NK cell cytotoxicity, whereas KLRD1 (CD94) dimerizes with NKG2 and regulates NK cell activation via interaction with the nonclassical MHC-I molecule HLA-E (2, 3). Thus, we modified the section of the manuscript addressing these results to include these new analyses: "Finally, we analyzed publicly available ChIP-seq data of ETS1, an important TF for NK cell development (4), to confirm its ability to bind TE sequences. Indeed, 19% of ETS1 peaks overlap with TE sequences (Figure 2g). Notably, ETS1 peaks overlapped with TE sequences (Figure 2h, in red) in the promoter regions of PRF1 and KLRD1, two genes important for NK cells' effector functions (2, 3)."<br /> ------

I am convinced by the authors' explanation that TE elements may contribute to the functions of NK cells.<br /> However, since I have understood that the main topic of this paper is about the thymus and thymic antigen-presenting cells, the mention of NK cells seems abrupt and unconnected to me. NK cells are a type of innate lymphocyte that arise in the bone marrow, and thymus is dispensable for their development and function. The readers might expect to find something more fundamental regarding the function of the thymus and immunological tolerance.

(3) Since the deep analysis of the dataset yielded many intriguing suggestions, why not add a discussion of the biological reasons and significance? For example, in Figure 1, why is TE expression negatively correlated with proliferation? cTEC-TE is mostly postnatal, while mTEC-TE is more embryonic. What does this mean?

We thank the Reviewer for this comment. To our knowledge, the relationship between cell division and transcriptional activity of TEs has not been extensively studied in the literature. However, a recent study has shown that L1 expression is induced in senescent cells. We therefore added the following sentences to our Discussion: "The negative correlation between TE expression and cell cycle scores in the thymus is coherent with recent data showing that transcriptional activity of L1s is increased in senescent cells (5). A potential rationale for this could be to prevent deleterious transposition events during DNA replication and cell division." We also added several discussion points regarding the regulation of TEs by KZFPs to answer concerns raised by Reviewer 2 (see Reviewer 2 comment #1).<br /> ------

I agree on the possibility suggested by the authors.

(4) To consolidate the experimental evidence about pDCs and TE-derived dsRNAs, one option is to show the amount of TE-derived RNA copies among total RNAs. The immunohistochemistry analysis in Figure 4 requires additional data to demonstrate that overlapped staining was not caused by technical biases (e.g. uneven fixation may cause the non-specifically stained regions/cells). To show this, authors should have confirmed not only the positive stainings but also the negative staining (e.g. CD3, etc.). Another possible staining control was showing that non-pDC (CD303- cell fractions in this case) cells were less stained by the ds-RNA probe.

We thank the Reviewer for this suggestion. We computed the proportion of reads in each cell assigned to two groups of sequences known to generate dsRNAs: TEs and mitochondrial genes (1). These analyses showed that the proportion of reads assigned to TEs is higher in pDCs than other thymic APCs by several orders of magnitude (~20% of all reads). In contrast, reads derived from mitochondrial genes had a lower abundance in pDCs. We included these results in Figure 4 - figure supplement 2 and included the following text in the Results section "To evaluate if these dsRNAs arise from TE sequences, we analyzed in thymic APC subsets the proportion of the transcriptome assigned to two groups of genomic sequences known as important sources of dsRNAs, TEs and mitochondrial genes (1). Strikingly, whereas the percentage of reads from mitochondrial genes was typically lower in pDCs than in other thymic APCs, the proportion of the transcriptome originating from TEs was higher in pDCs (~22%) by several orders of magnitude (Figure 4 - figure supplement 2)." As a negative control for the immunofluorescence experiments, we used CD123- cells. Indeed, flow cytometry analysis of the magnetically enriched CD303+ fraction was around 90% pure, as revealed by double staining with CD123 and CD304 (two additional markers of pDCs): CD123- cells were also CD304-/lo, showing that these cells are non- pDCs. Thus, we decided to compare the dsRNA signal between CD123+ cells (pDCs) and CD123- cells (non-pDCs). The difference between CD123+ and CD123- cells was striking (Figure 4d).<br /> ------

Although the technical concerns about immunostaining were not resolved, it is understandable that it would be difficult to rerun the experiment since the authors used the precious human thymi as the experimental material. Immunostaining co-staining requires careful interpretation so that careful experimental setup is needed.

Reviewer #1 (Public Review):

Summary:

The authors use insights into the dynamics of the PKA kinase domain, obtained by NMR experiments, to inform MD simulations that generate an energy landscape of PKA kinase domain conformational dynamics.

Strengths:

The authors integrate strong experimental data through the use of state-of-the-art MD studies and derive detailed insights into allosteric communication in PKA kinase. Comparison of wt kinase with a mutant (F100A) shows clear differences in the allosteric regulation of the two proteins. These differences can be rationalized by NMR and MD results. During the revision process, the authors have addressed the reviewers' comments adequately and have improved the accessibility of the manuscript to a wider audience.

Reviewer #1 (Public Review):

Summary:

The authors have implemented Optimal Transport algorithm in GromovMatcher for comparing LC/MS features from different datasets. This paper gains significance in the proteomics field for performing meta-analysis of LC/MS data.

Strengths:

The main strength is that GromovMatcher acheives significant performance metrics compared to other existing methods. The authors have done extensive comparisons to claim that GromovMatcher performs well.

Weaknesses:

The authors might need to add the limitation of datasets and thus have tested/validated their tool using simulated data in the abstract as well.

Reviewer #1 (Public Review):

Theoretical principles of viscous fluid mechanics are used here to assess likely mechanisms of transport in the ER. A set of candidate mechanisms is evaluated, making good use of imaging to represent ER network geometries. Evidence is provided that the contraction of peripheral sheets provides a much more credible mechanism than the contraction of individual tubules, junctions, or perinuclear sheets.

The work has been conducted carefully and comprehensively, making good use of underlying physical principles. There is a good discussion of the role of slip; sensible approximations (low volume fraction, small particle size, slender geometries, pragmatic treatment of boundary conditions) allow tractable and transparent calculations; clear physical arguments provide useful bounds; stochastic and deterministic features of the problem are well integrated.

There are just a couple of areas where more discussion might be warranted, in my view.

(1) The energetic cost of tubule contraction is estimated, but I did not see an equivalent estimate for the contraction of peripheral sheets. It might be helpful to estimate the energetic cost of viscous dissipation in generated flows at higher frequencies. The mechanism of peripheral sheet contraction is unclear: do ATP-driven mechanisms somehow interact with thermal fluctuations of membranes?

(2) Mutations are mentioned in the abstract but not (as far as I could see) later in the manuscript. It would be helpful if any consequences for pathologies could be developed in the text.

Reviewer #1 (Public Review):

Summary:

In this paper, proteomics analysis of the plasma of human subjects that underwent an exercise training regime consisting of a combination of endurance and resistance exercise led to the identification of several proteins that were responsive to exercise training. Confirming previous studies, many exercise-responsive secreted proteins were found to be involved in the extra-cellular matrix. The protein CD300LG was singled out as a potential novel exercise biomarker and the subject of numerous follow-up analyses. The levels of CD300LG were correlated with insulin sensitivity. The analysis of various open-source datasets led to the tentative suggestion that CD300LG might be connected with angiogenesis, liver fat, and insulin sensitivity. CD300LG was found to be most highly expressed in subcutaneous adipose tissue and specifically in venular endothelial cells. In a subset of subjects from the UK Biobank, serum CD300LG levels were positively associated with several measures of physical activity - particularly vigorous activity. In addition, serum CD300LG levels were negatively associated with glucose levels and type 2 diabetes. Genetic studies hinted at these associations possibly being causal. Mice carrying alterations in the CD300LG gene displayed impaired glucose tolerance, but no change in fasting glucose and insulin. Whether the production of CD300LG is changed in the mutant mice is unclear.

Strengths:

The specific proteomics approach conducted to identify novel proteins impacted by exercise training is new. The authors are resourceful in the exploitation of existing datasets to gain additional information on CD300LG.

Weaknesses:

While the analyses of multiple open-source datasets are necessary and useful, they lead to relatively unspecific correlative data that collectively insufficiently advance our knowledge of CD300LG and merely represent the starting point for more detailed investigations. Additional more targeted experiments of CD300LG are necessary to gain a better understanding of the role of CD300LG and the mechanism by which exercise training may influence CD300LG levels. One should also be careful to rely on external data for such delicate experiments as mouse phenotyping. Can the authors vouch for the quality of the data collected?

Reviewer #1 (Public Review):

Summary:

This manuscript presented a useful toolkit designed for CyTOF data analysis, which integrates 5 key steps as an analytical framework. A semi-supervised clustering tool was developed, and its performance was tested in multiple independent datasets. The tool was compared to human experts as well as supervised and unsupervised methods.

Strengths:

The study employed multiple independent datasets to test the pipeline. A new semi-supervised clustering method was developed.

Weaknesses:

The examination of the whole pipeline is incomplete. Lack of descriptions or justifications for some analyses.

Reviewer #1 (Public Review):

Summary:

This study identifies new types of interactions between Drosophila gustatory receptor neurons (GRNs) and shows that these interactions influence sensory responses and behavior. The authors find that HCN, a hyperpolarization-activated cation channel, suppresses the activity of GRNs in which it is expressed, preventing those GRNs from depleting the sensillum potential, and thereby promoting the activity of neighboring GRNs in the same sensilla. HCN is expressed in sugar GRNs, so HCN dampens the excitation of sugar GRNs and promotes the excitation of bitter GRNs. Impairing HCN expression in sugar GRNs depletes the sensillum potential and decreases bitter responses, especially when flies are fed on a sugar-rich diet, and this leads to decreased bitter aversion in a feeding assay. The authors' conclusions are supported by genetic manipulations, electrophysiological recordings, and behavioral assays.

Strengths:

(1) Non-synaptic interactions between neurons that share an extracellular environment (sometimes called "ephaptic" interactions) have not been well-studied, and certainly not in the insect taste system. A major strength of this study is the new insight it provides into how these interactions can impact sensory coding and behavior.

(2) The authors use many different types of genetic manipulations to dissect the role of HCN in GRN function, including mutants, RNAi, overexpression, ectopic expression, and neuronal silencing. Their results convincingly show that HCN impacts the sensillum potential and has both cell-autonomous and nonautonomous effects that go in opposite directions. There are a couple of conflicting or counterintuitive results, but the authors discuss potential explanations.

(3) Experiments comparing flies raised on different food sources suggest an explanation for why the system may have evolved the way that it did: when flies live in a sugar-rich environment, their bitter sensitivity decreases, and HCN expression in sugar GRNs helps to counteract this decrease.

Weaknesses/Limitations:

(1) The genetic manipulations were constitutive (e.g. Ih mutations, RNAi, or misexpression), and depleting Ih from birth could lead to compensatory effects that change the function of the neurons or sensillum. Using tools to temporally control Ih expression could help to confirm the results of this study.

(2) The behavioral experiment shows a striking loss of bitter sensitivity, but it was only conducted for one bitter compound at one concentration. It is not clear how general this effect is. The same is true for some of the bitter GRN electrophysiological experiments that only tested one compound and concentration.

(3) Several experiments using the Gal4/UAS system only show the Gal4/+ control and not the UAS/+ control (or occasionally neither control). Since some of the measurements in control flies seem to vary (e.g., spiking rate), it is important to compare the experimental flies to both controls to ensure that any observed effects are in fact due to the transgene expression.

(4) I was surprised that manipulations of sugar GRNs (e.g. Ih knockdown, Gr64a-f deletion, or Kir silencing) can impact the sensillum potential and bitter GRN responses even in experiments where no sugar was presented. I believe the authors are suggesting that the effects of sugar GRN activity (e.g., from consuming sugar in the fly food prior to the experiment) can have long-lasting effects, but it wasn't entirely clear if this is their primary explanation or on what timescale those long-lasting effects would occur. How much / how long of a sugar exposure do the flies need for these effects to be triggered, and how long do those effects last once sugar is removed?

(5) The authors mention that HCN may impact the resting potential in addition to changing the excitability of the cell through various mechanisms. It would be informative to record the resting potential and other neuronal properties, but this is very difficult for GRNs, so the current study is not able to determine exactly how HCN affects GRN activity.

Reviewer #1 (Public Review):

Summary:

The authors demonstrate that it is possible to carry out eQTL experiments for the model eukaryote S. cerevisiae, in "one pot" preparations, by using single-cell sequencing technologies to simultaneously genotype and measure expression. This is a very appealing approach for investigators studying genetic variation in single-celled and other microbial systems, and will likely inspire similar approaches in non-microbial systems where comparable cell mixtures of genetically heterogeneous individuals could be achieved.

Strengths:

While eQTL experiments have been done for nearly two decades (the corresponding author's lab are pioneers in this field), this single-cell approach creates the possibility for new insights about cell biology that would be extremely challenging to infer using bulk sequencing approaches. The major motivating application shown here is to discover cell occupancy QTL, i.e. loci where genetic variation contributes to differences in the relative occupancy of different cell cycle stages. The authors dissect and validate one such cell cycle occupancy QTL, involving the gene GPA1, a G-protein subunit that plays a role in regulating the mating response MAPK pathway. They show that variation at GPA1 is associated with proportional differences in the fraction of cells in the G1 stage of the cell cycle. Furthermore, they show that this bias is associated with differences in mating efficiency.

Weaknesses:

While the experimental validation of the role of GPA1 variation is well done, the novel cell cycle occupancy QTL aspect of the study is somewhat underexploited. The cell occupancy QTLs that are mentioned all involve loci that the authors have identified in prior studies that involved the same yeast crosses used here. It would be interesting to know what new insights, besides the "usual suspects", the analysis reveals. For example, in Cross B there is another large effect cell occupancy QTL on Chr XI that affects the G1/S stage. What candidate genes and alleles are at this locus? And since cell cycle stages are not biologically independent (a delay in G1, could have a knock-on effect on the frequency of cells with that genotype in G1/S), it would seem important to consider the set of QTLs in concert.

Reviewer #1 (Public Review):

The manuscript investigates the role of the membrane-deforming cytoskeletal regulator protein Abba in cortical development and its potential implications for microcephaly. It is a valuable contribution to the understanding of Abba's role in cortical development. The strengths and weaknesses identified in the manuscript are outlined below:

Clinical Relevance:

The authors identified a patient with microcephaly and a patient with an intellectual disability harboring a mutation in the Abba variant (R671W) adding a clinically relevant dimension to the study.

Mechanistic Insights:

The study offers valuable mechanistic insights into the development of microcephaly by elucidating the role of Abba in radial glial cell proliferation, radial fiber organization, and the migration of neuronal progenitors. The identification of Abba's involvement in the cleavage furrow during cell division, along with its interaction with Nedd9 and positive influence on RhoA activity, adds depth to our understanding of the molecular processes governing cortical development. Though the reported results establish the novel interaction between Abba and Nedd9, the authors have not addressed whether the mutant protein loses this interaction and whether that results in the observed effects.

In Vivo Validation:

The overexpression of mutant Abba protein (R671W) resulting in phenotypic similarities to Abba knockdown effects supports the significance of Abba in cortical development.

Reviewer #1 (Public Review):

Summary:

This work proposes a new method, DyNetCP, for inferring dynamic functional connectivity between neurons from spike data. DyNetCP is based on a neural network model with a two-stage model architecture of static and dynamic functional connectivity.

This work evaluates the accuracy of the synaptic connectivity inference and shows that DyNetCP can infer the excitatory synaptic connectivity more accurately than a state-of-the-art model (GLMCC) by analyzing the simulated spike trains. Furthermore, it is shown that the inference results obtained by DyNetCP from large-scale in-vivo recordings are similar to the results obtained by the existing methods (jitter-corrected CCG and JPSTH). Finally, this work investigates the dynamic connectivity in the primary visual area VISp and in the visual areas using DyNetCP.

Strengths:

The strength of the paper is that it proposes a method to extract the dynamics of functional connectivity from spike trains of multiple neurons. The method is potentially useful for analyzing parallel spike trains in general, as there are only a few methods (e.g. Aertsen et al., J. Neurophysiol., 1989, Shimazaki et al., PLoS Comput Biol 2012) that infer the dynamic connectivity from spikes. Furthermore, the approach of DyNetCP is different from the existing methods: while the proposed method is based on the neural network, the previous methods are based on either the descriptive statistics (JSPH) or the Ising model.

Weaknesses:

Although the paper proposes a new method, DyNetCP, for inferring the dynamic functional connectivity, its strengths are neither clear nor directly demonstrated in this paper. That is, insufficient analyses are performed to support the usefulness of DyNetCP.

First, this paper attempts to show the superiority of DyNetCP by comparing the performance of synaptic connectivity inference with GLMCC (Figure 2). However, the improvement in the synaptic connectivity inference does not seem to be convincing. While this paper compares the performance of DyNetCP with a state-of-the-art method (GLMCC), there are several problems with the comparison. For example:

(1) This paper focused only on excitatory connections (i.e., ignoring inhibitory neurons).

(2) This paper does not compare with existing neural network-based methods (e.g., CoNNECT: Endo et al. Sci. Rep. 2021; Deep learning: Donner et al. bioRxiv, 2024).

(3) Only a population of neurons generated from the Hodgkin-Huxley model was evaluated.

Thus, the results in this paper are not sufficient to conclude the superiority of DyNetCP in the estimation of synaptic connections. In addition, this paper compares the proposed method with the standard statistical methods Jitter-corrected CCG (Figure 3) and JPSTH (Figure 4). Unfortunately, these results do not show the superiority of the proposed method. It only shows that the results obtained by the proposed method are consistent with those obtained by the existing methods (CCG or JPSTH). This paper also compares the proposed method with standard statistical methods, such as jitter-corrected CCG (Figure 3) and JPSTH (Figure 4). It only shows that the results obtained by the proposed method are consistent with those obtained by the existing methods (CCG or JPSTH), which does not show the superiority of the proposed method.

In summary, although DyNetCP has the potential to infer synaptic connections more accurately than existing methods, the paper does not provide sufficient analysis to make this claim. It is also unclear whether the proposed method is superior to the existing methods for estimating functional connectivity, such as jitter-corrected CCG and JPSTH. Thus, the strength of DyNetCP is unclear.

Reviewer #1 (Public Review):

A subclass of inhibitory heterotrimeric guanine nucleotide-binding protein subunits, GNAI, has been implicated in sensory hair cell formation, namely the establishment of hair bundle (stereocilia) orientation and staircase formation. However, the former role of hair bundle orientation has only been demonstrated in mutants expressing pertussis toxin, which blocks all GNAI subunits, but not in mutants with a single knockout of any of the Gnai genes, suggesting that there is a redundancy among various GNAI proteins in this role. Using various conditional mutants, the authors concluded that GNAI3 is the primary GNAI proteins required for hair bundle morphogenesis, whereas hair bundle orientation requires both GNAI2 and GNAI3.

Strength

Various compound mutants were generated to decipher the contribution of individual GNAI1, GNAI2, GNAI3 and GNAIO in the establishment of hair bundle orientation and morphogenesis. The study is thorough with detailed quantification of hair bundle orientation and morphogenesis, as well as auditory functions.

The revised manuscript has clarified the phenotypic differences raised between the Gnai2/3 double mutants and Ptx mutant phenotypes and resolved the weakness pointed out in the previous submission. These results further illustrate the dynamic requirement of Gnai/O in hair bundle establishment and is an important contribution to the field.

Reviewer #2 (Public Review):

In this manuscript, the authors analyze the shapes of cerebral cortices from several primate species, including subgroups of young and old humans, to characterize commonalities in patterns of gyrification, cortical thickness, and cortical surface area. The authors state that the observed scaling law shares properties with fractals, where shape properties are similar across several spatial scales. One way the authors assess this is to perform a "cortical melting" operation that they have devised on surface models obtained from several primate species. The authors also explore differences in shape properties between brains of young (~20 year old) and old (~80) humans. A challenge the authors acknowledge struggling with in reviewing the manuscript is merging "complex mathematical concepts and a perplexing biological phenomenon." This reviewer remains a bit skeptical about whether the complexity of the mathematical concepts being drawn from are justified by the advances made in our ability to infer new things about the shape of the cerebral cortex.

(1) The series of operations to coarse-grain the cortex illustrated in Figure 1 produces image segmentations that do not resemble real brains. The process to assign voxels in downsampled images to cortex and white matter is biased towards the former, as only 4 corners of a given voxel are needed to intersect the original pial surface, but all 8 corners are needed to be assigned a white matter voxel. The reason for introducing this bias (and to the extent that it is present in the authors' implementation) is not provided. The authors provide an intuitive explanation of why thickness relates to folding characteristics, but ultimately an issue for this reviewer is, e.g., for the right-most panel in Figure 2b, the cortex consists of several 4.9-sided voxels and thus a >2 cm thick cortex. A structure with these morphological properties is not consistent with the anatomical organization of typical mammalian neocortex.

(2) For the comparison between 20-year-old and 80-year-old brains, a well-documented difference is that the older age group possesses more cerebral spinal fluid due to tissue atrophy, and the distances between the walls of gyri becomes greater. This difference is born out in the left column of Figure 4b. It seems this additional spacing between gyri in 80 year olds requires more extensive down-sampling (larger scale values in Figure 4a) to achieve a similar shape parameter K as for the 20 year olds. The authors assert that K provides a more sensitive measure (associated with a large effect size) than currently used ones for distinguishing brains of young vs. old people. A more explicit, or elaborate, interpretation of the numbers produced in this manuscript, in terms of brain shape, might make this analysis more appealing to researchers in the aging field.

(3) In the Discussion, it is stated that self-similarity, operating on all length scales, should be used as a test for existing and future models of gyrification mechanisms. Given the lack of association between the abstract mathematical parameters described in this study and explicit properties of brain tissue and its constituents, it is difficult to envision how the coarse-graining operation can be used to guide development of "models of cortical gyrification."

(4) There are several who advocate for analyzing cortical mid-thickness surfaces, as the pial surface over-represents gyral tips compared to the bottoms of sulci in the surface area. The authors indicate that analyses of mid-thickness representations will be taken on in future work, but this seems to be a relevant control for accepting the conclusions of this manuscript.

Reviewer #1 (Public Review):

In this study, Lin et al developed a protocol termed HIF-Clear, to perform tissue clearing and labelling on large-scale FFPE mouse brain specimens. They have optimized protocols for dewaxing and adequate delipidation of FFPE tissues to enable deep immunolabelling, even for whole mouse brains. This was useful for the study of disease models such as in an astrocytoma model to evaluate spatial architecture of the tumour and its surrounding microenvironment. It was also used in a traumatic brain injury model to quantify changes in vasculature density and differences in monoaminergic innervation. They have also demonstrated the potential of multi-round immunolabelling using photobleaching, as well as expansion microscopy with FFPE samples using Hif Clear.

Comments on revised version:

The revised manuscript by Lin et al is much improved with a more detailed methods description. There are only a few minor comments for the authors that are still valid:

- Some procedures, including the basic HIF-Clear protocol, seem to produce marked tissue expansion that is not mentioned in the manuscript. Users should take this fact into consideration when making measurements.<br /> - The authors have provided a comparison between mouse and human brain samples in Figure S12. However, it is misleading to mention that the "fluorescent signals are comparable at varying depth" as the figure clearly showed a lack of continuous staining especially for SMI312 at 900um depth, and human brain tissue showed considerably increased background signal (likely due to endogenous lipofuscin which has autofluorescent properties). Also, This is difficult to assess in the present design of the experiment because, at different depths, the tissue and the antigen may change themselves... making it difficult to make a direct staining comparison with other depths.

Reviewer #1 (Public Review):

Summary:

Working memory is imperfect - memories accrue errors over time and are biased towards certain identities. For example, previous work has shown memory for orientation is more accurate near the cardinal directions (i.e., variance in responses is smaller for horizontal and vertical stimuli) while being biased towards diagonal orientations (i.e., there is a repulsive bias away from horizontal and vertical stimuli). The magnitude of errors and biases increase the longer an item is held in working memory and when more items are held in working memory (i.e., working memory load is higher). Previous work has argued that biases and errors could be explained by increased perceptual acuity at cardinal directions. However, these models are constrained to sensory perception and do not explain how biases and errors increase over time in memory. The current manuscript builds on this work to show how a two-layer neural network could integrate errors and biases over a memory delay. In brief, the model includes a 'sensory' layer with heterogenous connections that lead to the repulsive bias and decreased error in the cardinal directions. This layer is then reciprocally connected with a classic ring attractor layer. Through their reciprocal interactions, the biases in the sensory layer are constantly integrated into the representation in memory. In this way, the model captures the distribution of biases and errors for different orientations that have been seen in behavior and their increasing magnitude with time. The authors compare the two-layer network to a simpler one-network model, showing that the one-model network is harder to tune and shows an attractive bias for memories that have lower error (which is incompatible with empirical results).

Strengths:

The manuscript provides a nice review of the dynamics of items in working memory, showing how errors and biases differ across stimulus space. The two-layer neural network model is able to capture the behavioral effects as well as relate to neurophysiological observations that memory representations are distributed across the sensory cortex and prefrontal cortex.

The authors use multiple approaches to understand how the network produces the observed results. For example, analyzing the dynamics of memories in the low-dimensional representational space of the networks provides the reader with an intuition for the observed effects.

As a point of comparison with the two-layer network, the authors construct a heterogenous one-layer network (analogous to a single memory network with embedded biases). They argue that such a network is incapable of capturing the observed behavioral effects but could potentially explain biases and noise levels in other sensory domains where attractive biases have lower errors (e.g., color).

The authors show how changes in the strength of Hebbian learning of excitatory and inhibitory synapses can change network behavior. This argues for relatively stronger learning in inhibitory synapses, an interesting prediction.

The manuscript is well-written. In particular, the figures are well done and nicely schematize the model and the results.

Weaknesses:

Despite its strengths, the manuscript does have some weaknesses.

First, as far as we can tell, behavioral data is only presented in schematic form. This means some of the nuances of the effects are lost. It also means that the model is not directly capturing behavioral effects. Therefore, while providing insight into the general phenomenon, the current manuscript may be missing some important aspects of the data.

Relatedly, the models are not directly fit to behavioral data. This makes it hard for the authors to exclude the possibility that there is a single network model that could capture the behavioral effects. In other words, it is hard to support the authors' conclusion that "....these evolving errors...require network interaction between two distinct modules." (from the abstract, but similar comments are made throughout the manuscript). Such a strong claim needs stronger evidence than what is presented. Fitting to behavioral data could allow the authors to explore the full parameter space for both the one-layer and two-layer network architectures.

In addition, directly comparing the ability of different model architectures to fit behavioral data would allow for quantitative comparison between models. Such quantitative comparisons are currently missing from the manuscript.

To help broaden the impact of the paper, it would be helpful if the authors provided insight into how the observed behavioral biases and/or network structures influence cognition. For example, previous work has argued that biases may counteract noise, leading to decreased variance at certain locations. Is there a similar normative explanation for why the brain would have repulsive biases away from commonly occurring stimuli? Are they simply a consequence of improved memory accuracy? Why isn't this seen for all stimulus domains?

Previous work has found both diffusive noise and biases increase with the number of items in working memory. It isn't clear how the current model would capture these effects. The authors do note this limitation in the Discussion, but it remains unclear how the current model can be generalized to a multi-item case.

The role of the ring attractor memory network isn't completely clear. There is noise added in this stage, but how is this different from the noise added at the sensory stage? Shouldn't these be additive? Is the noise necessary? Similarly, it isn't clear whether the memory network is necessary - can it be replaced by autapses (self-connections) in the sensory network to stabilize its representation? In short, it would be helpful for the authors to provide an intuition for why the addition of the memory network facilitates the repulsive bias.

Overall:

Overall, the manuscript was successful in building a model that captured the biases and noise observed in working memory. This work complements previous studies that have viewed these effects through the lens of optimal coding, extending these models to explain the effects of time in memory. In addition, the two-layer network architecture extends previous work with similar architectures, adding further support to the distributed nature of working memory representations.

Reviewer #1 (Public Review):

Summary:

A cortico-centric view is dominant in the study of the neural mechanisms of consciousness. This investigation represents the growing interest in understanding how subcortical regions are involved in conscious perception. To achieve this, the authors engaged in an ambitious and rare procedure in humans of directly recording from neurons in the subthalamic nucleus and thalamus. While participants were in surgery for the placement of deep brain stimulation devices for the treatment of essential tremor and Parkinson's disease, they were awakened and completed a perceptual-threshold tactile detection task. The authors identified individual neurons and analyzed single-unit activity corresponding with the task phases and tactile detection/perception. Among the neurons that were perception-responsive, the authors report changes in firing rate beginning ~150 milliseconds from the onset of the tactile stimulation. Curiously, the majority of the perception-responsive neurons had a higher firing rate for missed/not perceived trials. In summary, this investigation is a valuable addition to the growing literature on the role of subcortical regions in conscious perception.

Strengths:

The authors achieved the challenging task of recording human single-unit activity while participants performed a tactile perception task. The methods and statistics are clearly explained and rigorous, particularly for managing false positives and non-normal distributions. The results offer new detail at the level of individual neurons in the emerging recognition of the role of subcortical regions in conscious perception.

Weaknesses:

"Nonetheless, it remains unknown how the firing rate of subcortical neurons changes when a stimulus is consciously perceived." (lines 76-77) The authors could be more specific about what exactly single-unit recordings offer for interrogating the role of subcortical regions in conscious perception that is unique from alternative neural activity recordings (e.g., local field potential) or recordings that are used as proxies of neural activity (e.g., fMRI).

Related comment for the following excerpts:

"After a random delay ranging from 0.5 to 1 s, a "respond" cue was played, prompting participants to verbally report whether they felt a vibration or not. Therefore, none of the reported analyses are confounded by motor responses." (lines 97-99).

"These results show that subthalamic and thalamic neurons are modulated by stimulus onset, irrespective of whether it was reported or not, even though no immediate motor response was required." (lines 188-190).

"By imposing a delay between the end of the tactile stimulation window and the subjective report, we ensured that neuronal responses reflected stimulus detection and not mere motor responses." (lines 245-247).

It is a valuable feature of the paradigm that the reporting period was initiated hundreds of milliseconds after the stimulus presentation so that the neural responses should not represent "mere motor responses". However, verbal report of having perceived or not perceived a stimulus is a motor response and because the participants anticipate having to make these reports before the onset of the response period, there may be motor preparatory activity from the time of the perceived stimulus that is absent for the not perceived stimulus. The authors show sensitivity to this issue by identifying task-selective neurons and their discussion of the results that refer to the confound of post-perceptual processing. Still, direct treatment of this possible confound would help the rigor of the interpretation of the results.

"When analyzing tactile perception, we ensured that our results were not contaminated with spurious behavior (e.g. fluctuation of attention and arousal due to the surgical procedure)." (lines 118-117).

Confidence in the results would be improved if the authors clarified exactly what behaviors were considered as contaminating the results (e.g., eye closure, saccades, and bodily movements) and how they were determined.

The authors' discussion of the thalamic neurons could be more precise. The authors show that only certain areas of the thalamus were recorded (in or near the ventral lateral nucleus, according to Figure S3C). The ventral lateral nucleus has a unique relationship to tactile and motor systems, so do the authors hypothesize these same perception-selective neurons would be active in the same way for visual, auditory, olfactory, and taste perception? Moreover, the authors minimally interpret the location of the task, sensory, and perception-responsive neurons. Figure S3 suggests these neurons are overlapping. Did the authors expect this overlap and what does it mean for the functional organization of the ventral lateral nucleus and subthalamic nucleus in conscious perception?

"We note that, 6 out of 8 neurons had higher firing rates for missed trials than hit trials, although this proportion was not significant (binomial test: p = 0.145)." (lines 215-216).

It appears that in the three example neurons shown in Figure 4, 2 out of 3 (#001 and #068) show a change in firing rate predominantly for the missed stimulations. Meanwhile, #034 shows a clear hit response (although there is an early missed response - decreased firing rate - around 150 ms that is not statistically significant). This is a counterintuitive finding when compared to previous results from the thalamus (e.g., local field potentials and fMRI) that show the opposite response profile (i.e., missed/not perceived trials display no change or reduced response relative to hit/perceived trials). The discussion of the results should address this, including if these seemingly competing findings can be rectified.

The authors report 8 perception-responsive neurons, but there are only 5 recording sites highlighted (i.e., filled-in squares and circles) in Figures S3C and 4D. Was this an omission or were three neurons removed from the perception-responsive analysis?

Could the authors speak to the timing of the responses reported in Figure 4? The statistically significant intervals suggested both early (~160-200ms) to late responses (~300ms). Some have hypothesized that subcortical regions are early - ahead of cortical activation that may be linked with conscious perception. Do these results say anything about this temporal model for when subcortical regions are active in conscious perception?

Reviewer #1 (Public Review):

Summary:

People with Parkinson's disease often experience a variety of nonmotor symptoms, the biological bases of which remain poorly understood. Johansson et al began to study potential roles of the dorsal raphe nucleus (DRN) degeneration in the pathophysiology of neuropsychiatric symptoms in PD.

Strengths:

Boi et al validated a transgenic reporter mouse line that can reliably label dopaminergic neurons in the DRN. This brain region shows severe neurodegeneration and has been proposed to contribute to the manifestation of neuropsychiatric symptoms in PD. Using this mouse line (and others), Boi and colleagues characterized electrophysiological and morphological phenotypes of dopaminergic and serotoninergic neurons in the raphe nucleus. This study involved very careful topographical registration of recorded neurons to brain slices for post hoc immunohistochemical validation of cell identity, making it an elegant and thorough piece of work.

In relevance to PD pathophysiology, the authors evaluated the physiological and morphological changes of DRN serotoninergic and dopaminergic neurons after a partial loss of nigrostriatal dopamine neurons, which serves as a mouse model of early parkinsonian pathology. Moreover, the authors identified a series of physiological and morphological changes of subtypes of DRN neurons that depend on nigral dopaminergic neurodegeneration, LC noradrenergic neurodegeneration, or both. Indeed this works highlights the importance of LC noradrenergic degeneration in PD pathophysiology.

Overall, this is a well-designed study with high significance to the Parkinson's research field.

Reviewer #1 (Public Review):

Summary:

This is a study on the role of the retrosplenial cortex (RSC) and the hippocampus in working memory. Working memory is a critical cognitive function that allows temporary retention of information for task execution. The RSC, which is functionally and anatomically connected to both primary sensory (especially visual) and higher cognitive areas, plays a key role in integrating spatial-temporal context and in goal-directed behaviors. However, the specific contributions of the RSC and the hippocampus in working memory-guided behaviors are not fully understood due to a lack of studies that experimentally disrupt the connection between these two regions during such behaviors.

In this study, researchers employed eArch3.0 to silence hippocampal axon terminals in the RSC, aiming to explore the roles of these brain regions in working memory. Experiments were conducted where animals with silenced hippocampal axon terminals in the RSC performed a delayed non-match to place (DNMP) task. The results indicated that this manipulation impaired memory retrieval, leading to decreased performance and quicker decision-making in the animals. Notably, the authors observed that the effects of this impairment persisted beyond the light-activation period of the opsin, affecting up to three subsequent trials. They suggest that disrupting the hippocampal-RSC connection has a significant and lasting impact on working memory performance.

Strengths:

They conducted a study exploring the impact of direct hippocampal inputs into the RSC, a region involved in encoding spatial-temporal context and transferring contextual information, on spatial working memory tasks. Utilizing eArch3.0 expressed in hippocampal neurons via the viral vector AAV5-hSyn1-eArch3.0, they aimed to bilaterally silence hippocampal terminals located at the RSC in rats pre-trained in a DNMP task. They discovered that silencing hippocampal terminals in the RSC significantly decreased working memory performance in eArch+ animals, especially during task interleaving sessions (TI) that alternated between trials with and without light delivery. This effect persisted even in non-illuminated trials, indicating a lasting impact beyond the periods of direct manipulation. Additionally, they observed a decreased likelihood of correct responses following TI trials and an increased error rate in eArch+ animals, even after incorrect responses, suggesting an impairment in error-corrective behavior. This contrasted with baseline sessions where no light was delivered, and both eArch+ and control animals showed low error rates.

Weaknesses:

While I agree with the authors that the role of hippocampal inputs to the RSC in spatial working memory is understudied and merits further investigation, I find that the optogenetic experiment, a core part of this manuscript that includes viral injections, could be improved. The effects were rather subtle, rendering some of the results barely significant and possibly too weak to support major conclusions. Additionally, no mechanistic investigation was conducted beyond referencing previous reports to interpret the core behavioral phenotypes.

Reviewer #1 (Public Review):

Summary:

The manuscript entitled "Staphylococcus aureus counters organic acid anion-mediated inhibition of peptidoglycan cross-linking through robust alanine racemase activity" by Panda, S et al. reports an extensive biochemical analysis of the result from a Tn screen that identified alr1 as being required for acetic acid tolerance. In the end, they demonstrate that reduced D-Ala pools in the ∆alr1 mutant lead to a drastic reduction in D-Ala-D-Ala dipeptide. They show that this is due to the ability of organic acid anions to limit the D-Ala-D-Ala ligase enzyme Ddl. They demonstrate that:

(1) Acetate exposure in the ∆alr1 results in reduced D-Ala-D-Ala dipeptide, but not the monomers.

(2) Acetate can bind to purified Ddl in vitro.

(3) This binding results in reduced enzyme activity.

(4) Other organic acid anions such as lactate, proprionate, and itaconitate can also inhibit Ddl.

The experiments are clearly described and logically laid out. I have only a few minor comments to add.

Strengths:

The most significant strength is the exceptional experimental data that supports the authors' hypotheses.

Weaknesses:

Only minor weaknesses were identified by this reviewer.

(1) Which allele is alr1, the one upstream of MazEF or the one in the Lysine biosynthetic operon?

(2) Figure 3B. Where does the C3N2 species come from in the WT and why is it absent in the mutants? It is about 25% of the total dipeptide pool.

(3) Figure 3D could perhaps be omitted. I understand that the authors attained statistical significance in the fitness defect, but biologically this difference is very minor. One would have to look at the isotopomer distribution in the Dat overexpressing strain to make sure that increased flux actually occurred since there are other means of affecting activity (e.g. allosteric modulators).

(4) In Figure 4A, why is the complete subunit UDP-NAM-AEKAA increasing in each strain upon acetate challenge if there was such a stark reduction in D-Ala-D-Ala, particularly in the ∆alr1 mutant? For that matter, why are the levels of UDP-NAM-AEKAA in the ∆alr1 mutant identical to that of WT with/out acetate?

(5) Figure 4B. Is there no significant difference between ddl and murF transcripts between WT and ∆alr1 under acetate stress? This comparison was not labeled if the tests were done.

(6) Although tricky, it is possible to measure intracellular acetate. It might be of interest to know where in the Ddl inhibition curve the cells actually are.

Reviewer #2 (Public Review):

The authors aimed at elucidating the development of high altitude polycythemia which affects mice and men staying in a hypoxic atmosphere at high altitude (hypobaric hypoxia; HH). HH causes increased erythropoietin production which stimulates the production of red blood cells. The authors hypothesize that increased production is only partially responsible for exaggerated red blood cell production, i.e. polycythemia, but that decreased erythrophagocytosis in the spleen contributes to high red blood cells counts.

The main strength of the study is the use of a mouse model exposed to HH in a hypobaric chamber. However, not all of the reported results are convincing due to some smaller effects which one may doubt to result in the overall increase in red blood cells as claimed by the authors. Moreover, direct proof for reduced erythrophagocytosis is compromised due to a strong spontaneous loss of labelled red blood cells, although effects of labelled E. coli phagocytosis are shown.

Comments on latest version:

The authors have partly addressed my comments.

(1) The response to my question regarding unchanged MCH is a kind of "hand waiving" - maybe it would require substantially more extensive work to clarify this issue

(2) The moderate if not marginal difference in normal vs splenectomy argues against a significant role of the spleen - even if the difference was slightly larger in HH

(3) There is still overinterpretation of data. My Q was: Is the reduced phagocytic capacity in Fig 4B significant? Response: "This is indicative of a diminished phagocytic capacity, particularly when contrasted<br /> with NN conditions." I guess that is a "no"

(4) I assume my question with respect to bi- or trivalent iron chelators was misunderstood.

In general, as indicated above, it is an interesting hypothesis which is corroborated by data in several instances. Maybe the scientific community should decide whether it is all in all conclusive.

Reviewer #1 (Public Review):

A descriptive manuscript investigating the ability of a peptide, implicated in development, to induce signalling responses indicative of immunity. The work clearly documents the ability of the synthetic peptide to induce these responses, and open future work to link this back to physiology.

Comments on revised version:

Congratulations to the authors for the improvements to the manuscript.

I still have reservations, as raised by other reviewers, about whether the outputs observed can definitively be classified as immune/defence outputs without assaying an impact upon microbial growth. Indeed, this is challenging to address as many of the outputs are shared by multiple pathways. This is especially the case here as the peptide could have different effects in different tissues or cells with different expression levels of the receptors (e.g. hypothetically - no expression = no effect; weak expression - cell wall loosening and susceptibility; high expression - strong response and 'defence' response). I do however appreciate that the authors have toned down some of the conclusions regarding the defence response and also they included further reference to outputs also being from developmental pathways.

Reviewer #1 (Public Review):

This study presents valuable data on effector proteins (=virulence factors) used by the bacterial pathogen Legionella pneumophila that target host vesicle trafficking GTPases during infection. The evidence supporting the claims of the authors is robust, and the data suggest a sophisticated interplay between multiple effectors with the goal of temporarily exploiting host cell Rab10 during infection.

The authors have done a nice job addressing my earlier concerns. I have no further criticism about the revised paper.

Reviewer #1 (Public Review):

Summary:

This study brings new information about the function of serotonin-gated ion channels 5-HT3AR, by describing the conformational changes undergoing during ligands binding. These results can be potentially extrapolated to other members of the Cys-loop ligand-gated ion channels. By combining fluorescence microscopy with electrophysiological recordings, the authors investigate structural changes inside and outside the orthosteric site elicited by agonists, partial agonists, and antagonists. The results are convincing and correlate well with the observations from cryo-EM structures. The work will be of important significance and broad interest to scientists working on channel biophysics but also drug development targeting ligand-gated ion channels.

Strengths:

The authors present an elegant and well-designed study to investigate the conformational changes on 5-HT3AR where they combine electrophysiological and fluorometry recordings. They determined four positions suitable to act as sensors for the conformational changes of the receptor: two inside and two outside the agonist binding site. They make a strong point showing how antagonists produce conformational changes inside the orthosteric site similarly as agonists do but they failed to spread to the lower part of the ECD, in agreement with previous studies and Cryo-EM structures. They also show how some loss-of-function mutant receptors elicit conformational changes (changes in fluorescence) after partial agonist binding but failed to produce measurable ionic currents, pointing to intermediate states that are stabilized in these conditions. The four fluorescence sensors developed in this study may be good tools for further studies on characterizing drugs targeting the 5-HT3R. The major conclusions of the manuscript seem well justified.

Weaknesses:

Weaknesses have been very well addressed during the review process.

Joint Public Review:

Summary:

This important manuscript investigates a subpopulation of glutamatergic neurons in the suprammamillary nucleus that projects to the pre-optic hypothalamus area (SuM-VGLUT2+::POA). First, they define the neural circuitry of these neurons, which contact many stress/threat-associated brain regions. Then they employ fibre photometry to measure the activity of these neurons during various threatening tasks and find the responses correlate well with threat stimuli. Finally, they stimulate these neurons and find multiple lines of evidence that mice find this aversive and will act to avoid receiving this stimulation. In sum, they provide solid evidence that this neuronal population represents a new node in stress response circuitry that allows the animal to produce flexible behaviours in response to stress, which will be of interest to neuroscientists across several sub-fields.

Strengths:

Overall this is a solid manuscript tackling an important question. Coping with stress by an animal in danger is essential for survival. This manuscript identifies a novel population of neurons in the murine supramamillary nucleus (SuM) projecting to the pre-optic hypothalamus area among other regions that is involved in this important process. The evidence to support the conclusions is solid.

Specific strengths:

• The topic is novel.

• The manuscript follows a logical structure and neatly moves through the central story. Several potential alternate interpretations are well-controlled for.

• The manuscript employs an array of different tasks to provide converging evidence for their conclusions.

• The authors provide excellent evidence of the specificity of the function of this neuronal population, both from anatomical studies and from behavioural studies (e.g. demonstrating that activity of gabaergic neurons in the same region does not correlate with behaviours in the same way).

• The study is well-powered (sample sizes are good) and the effects are convincing.

Weaknesses:

* Not all of the reviewer comments were addressed in the manuscript itself, although this was acknowledged in the author's responses to reviewers. One key example is as follows:

* The authors did not entirely address comments related to rigor but they at least acknowledged it. For example, in multiple places they argue that WT, purchased mice are probably not different in baseline behavior compared to Vgltu2-IRES-Cre because it is unlikely that adding the IRES-Cre will change behavior. However, they do not acknowledge that transgenic lines are not from the exact same genetic background and generation number, and there is ample evidence in the literature that transgenic mice on a B6J background can differ in basal phenotypes from one another and B6J. In one place they show some basal behavior, at least in heat map form though not quantified. Had the authors decided to apply this more pervasively, it would have made the story even more compelling in terms of a stress/threat-induced phenotype.

Comments on revised version from the Reviewing Editor:

The authors have done a thorough job of answering the reviewer queries, and a good job of explaining why they have not answered a particular point. Indeed, there is so much additional information in response to the reviewers that I hope readers of the manuscript will read the reviews and responses as well! I think they add a lot.

Reviewer #1 (Public Review):

Summary:

The authors describe that the endocytic pathway is crucial for ColI fibrillogenesis. ColI is endocytosed by fibroblasts, prior to exocytosis and formation of fibrils, which can include a mixture of endogenous/nascent ColI chains and exogenous ColI. ColI uptake and fibrillogenesis are regulated by circadian rhythm as described by the authors in 2020, thanks to the dependence of this pathway on circadian-clock-regulated protein VPS33B. Cells are capable of forming fibrils with recently endocytosed ColI when nascent chains are not available. Previously identified VPS33B is demonstrated not to have a role in endocytosis of ColI, but to play a role in fibril formation, which the authors demonstrate by showing the loss of fibril formation in VPS33B KO, and an excess of insoluble fibrils - along-side a decrease in soluble ColI secretion - in VPS33B overexpression conditions. A VPS33B binding protein VIPAS39 is also shown to be required for fibrillogenesis and to colocalise with ColI. The authors thus conclude that ColI is internalised into endosomal structures within the cell, and that ColI, VPS33B, and VIPA39 are co-trafficked to the site of fibrillogenesis, where along with ITGA11, which by mass spectrometric analysis is shown to be regulated by VPS33B levels, ColI fibrils are formed. Interestingly, in involved human skin sections from idiopathic pulmonary fibrosis (IPF) patients, ITGA11 and VPS33B expression is increased compared to healthy tissue, while in patient-derived fibroblasts, uptake of fluorescently-labelled ColI is also increased. This suggests that there may be a significant contribution of endocytosis-dependent fibrillogenesis in the formation of fibrotic and chronic wound-healing diseases in humans.

Strengths:

This is an interesting paper that contributes an exciting novel understanding of the formation of fibrotic disease, which despite its high occurrence, still has no robust therapeutic options. The precise mechanisms of fibrillogenesis are also not well understood, so a study devoted to this complex and key mechanism is well appreciated. The dependence of fibrillogenesis on VPS33B and VIPA39 is convincing and robust, while the distinction between soluble ColI secretion and insoluble fibrillar ColI is interesting and informative.

Weaknesses:

There are a number of limitations to this study in its current state. Inhibition of ColI uptake is performed using Dyngo4a, which although proposed as an inhibitor of Clathrin-dependent endocytosis is known to be quite un-specific. This may not be a problem however, as the endocytic mechanism for ColI also does not seem to be well defined in the literature, in fact, the principle mechanism described in the papers referred to by the authors is that of phagocytosis. It would be interesting to explore this important part of the mechanism further, especially in relation to the intracellular destination of ColI. The circadian regulation does not appear as robust as the authors' last paper, however, there could be a larger lag between endocytosis of ColI and realisation of fibrils. The authors state that the endocytic pathway is the mechanism of trafficking and that they show ColI, VPS33B, and VIPA39 are co-trafficked. However, the only link that is put forward to the endosomes is rather tenuously through VPS33B/VIPA39. There is no direct demonstration of ColI localisation to endosomes (ie. immunofluorescence), and this is overstated throughout the text. Demonstrating the intracellular trafficking and localisation of ColI, and its actual relationship to VPS33B and VIPA39, followed by ITGA11, would broaden the relevance of this paper significantly to incorporate the field of protein trafficking. Finally, the "self-formation" of ColI fibrils is discussed in relation to the literature and the concentration of fluorescently-tagged ColI, however as the key message of the paper is the fibrillogenesis from exocytosed colI, I do not feel like it is demonstrated to leave no doubt. Specific inhibition of intracellular trafficking steps, or following the progressive formation of ColI fibrils over time by immunofluorescence would demonstrate without any further doubt that ColI must be endocytosed first, to form fibrils as a secondary step, rather than externally-added ColI being incorporated directly to fibrils, independent of cellular uptake.

Reviewer #1 (Public Review):

Summary:

The authors quantitatively describe the complex binding equilibria of BRAF and its inhibitors resulting in some cases in the paradoxical activation of BRAF dimer when bound to ATP competitive inhibitors. The authors use a biophysical tour de force involving FRET binding assays, NMR, kinase activity assays, and DEER spectroscopy.

Strengths:

The strengths of the study are the beautifully conducted assays that allow for a thorough characterization of the allostery in this complex system. Additionally, the use of F-NMR and DEER spectroscopy provides important insights into the details of the process.

The resulting model for binding of inhibitors and dimerization (Figure 4) is very helpful.

Weaknesses:

This is a complex system and its communication is inherently challenging. It might be of interest to the broader readership to understand the implications of the model for drug development and therapy.

Reviewer #1 (Public Review):

Summary:

Szathmary and colleagues explore the parabolic growth regime of replicator evolution. Parabolic growth occurs when nucleic acid strain separation is the rate limiting step of the replication process which would have been the case for non-enzymatic replication of short oligonucleotide that could precede the emergence of ribozyme polymerases and helicases. The key result is that parabolic replication is conducive to the maintenance of genetic diversity, that is, coexistence of numerous master sequences (the Gause principle does not apply). Another important finding is that there is no error threshold for parabolic replication except for the extreme case of zero fidelity.

Strengths:

I find both the analytic and the numerical results to be quite convincing and well described. The results of this work are potentially important because they reveal aspects of a realistic evolutionary scenario for the origin of replicators.

Weaknesses:

There are no obvious technical weaknesses. It can be argued that the results represent an incremental advance because many aspects of parabolic replication have been explored previously (the relevant publications are properly cited). Obviously, the work is purely theoretical, experimental study of parabolic replication is due. In the opinion of this reviewer, though, these are understandable limitations that do not actually detract from the value of this work.

Reviewer #1 (Public Review):

This paper identifies GABA cells in the preoptic hypothalamus and others in the posterior hypothalamus which are involved in REM sleep rebound (the increase in REM sleep) after selective REM sleep deprivation. By calcium photometry, these preoptic cells are most active during REM, and show more calcium signals during REM deprivation, suggesting they respond to "REM pressure". Inhibiting these cells ontogenetically diminishes REM sleep. The optogenetic and photometry work is carried out to a high standard, the paper is well written, and the findings are interesting and enhance our understanding of REM sleep regulation. The new findings make it clear that as for the circuitry that regulates NREM sleep, REM sleep circuitry is also quite distributed in the brain. It is unclear if there is a true "REM center". The study of mechanisms of catching up on lost sleep (sleep homeostasis), has previously focused on NREM sleep, where various circuits have been identified. That there is a special mechanism that also tracks time awake and compensates with REM sleep is intriguing.

In a broader context, the existence of REM rebound suggests that REM sleep must have a function, otherwise why catch up on it. There is a lot of literature that suggests REM contributes to emotional processing, for example. The new findings deepen our appreciation of REM regulation. As REM sleep is often disturbed in stress (e.g. post-traumatic stress disorder) and in depression, understanding more about REM regulation could ultimately aid treatments for people living with these conditions.

Reviewer #2 (Public Review):

Summary:

The authors describe and quantify a phenomenon in the CA1 and CA3 of the hippocampus that they call aberrant Ca2+ micro-waves. Micro-waves are sometimes seen during 2-photon calcium imaging of populations of neurons under certain conditions. They are spatially confined slow calcium events that start in a few cells and slowly spread to neighboring groups of cells. This phenomenon has been uttered between researchers in the field at conferences, but no one has taken the time to carefully capture and quantify micro-waves and pin down the causes. The authors show that micro-waves are dependent on the viral titre of the genetically encoded calcium indicators (GECIs), the genetic promoter (synapsin), the neuronal subtype (granule cells in the dentate gyrus do not produce micro-waves and they are not seen in the neocortex), and the density of GECI expression. The authors should be commended for their work and for raising awareness to all labs doing any form of calcium imaging in populations of neurons. The authors also come up with alternative approaches to avoid artifactual micro-waves such as reducing the transduction titre (1:2 dilution of virus) and a transduction method employing sparser and cre-dependent GECI expression in principal cells using a CaMKII promoter.

Strengths:

The micro-waves reported in the paper were robustly observed across 4 laboratories and 3 different countries with various experimenters and calcium imaging set-ups. This adds significant strength to the work.

The age of mice used covered a broad range (from 6 to 43 weeks). This is a strength because it covers most ages that are used in labs that regularly do calcium imaging.

Another strength is they used different GCaMP variants (GCaMP6m, GCaMP6s, GCaMP7f), as well as a red indicator: RCaMP. This shows the micro-waves are not an issue with any particular GECI, as the authors suggest.

The authors include many movies of micro-waves. This is extremely useful for researchers in the field to view them in real-time so they can identify them in their own data.

They provide a useful table with specific details of the virus injected, titre, dilution, and other information along with the incidence of micro-waves. A nice look-up table for researchers to see if their viral strategy is associated with a high or low incidence of micro-waves.

Weaknesses:

The effect of mico-waves on single cell function was not analyzed. It would be useful, for example, if we knew the influence of micro-waves on place fields. Can a place cell still express a place field in a hippocampus that produces micro-waves? What effect might a microwave passing over a cell have on its place field? Mice were not trained in these experiments, so the authors do not have the data. However, they do briefly discuss these ideas.

Reviewer #1 (Public Review):

The manuscript by Oleh et al. uses in vitro electrophysiology and compartmental modeling (via NEURON) to investigate the expression and function of HCN channels in mouse L2/3 pyramidal neurons. The authors conclude that L2/3 neurons have developmentally regulated HCN channels, the activation of which can be observed when subjected to large hyperpolarizations. They further conclude via blockade experiments that HCN channels in L2/3 neurons influence cellular excitability and pathway-specific EPSP kinetics, which can be neuromodulated. While the authors perform a wide range of slice physiology experiments, concrete evidence that L2/3 cells express functionally relevant HCN channels is limited. There are serious experimental design caveats and confounds that make drawing strong conclusions from the data difficult. Furthermore, the significance of the findings is generally unclear, given modest effect sizes and a lack of any functional relevance, either directly via in vivo experiments or indirectly via strong HCN-mediated changes in known operations/computations/functions of L2/3 neurons.

Specific points:

(1) The interpretability and impact of this manuscript are limited due to numerous methodological issues in experimental design, data collection, and analysis. The authors have not followed best practices in the field, and as such, much of the data is ambiguous and/or weak and does not support their interpretations (detailed below). Additionally, the authors fail to appropriately explain their rationale for many of their choices, making it difficult to understand why they did what they did. Furthermore, many important references appear to be missing, both in terms of contextualizing the work and in terms of approach/method. For example, the authors do not cite Kalmbach et al 2018, which performed a directly comparable set of experiments on HCN channels in L2/3 neurons of both humans and mice. This is an unacceptable omission. Additionally, the authors fail to cite prior literature regarding the specificity or lack thereof of Cs+ in blocking HCN. In describing a result, the authors state "In line with previous reports, we found that L2/3 PCs exhibited an unremarkable amount of sag at 'typical' current commands" but they then fail to cite the previous reports.

(2) A critical experimental concern in the manuscript is the reliance on cesium, a nonspecific blocker, to evaluate HCN channel function. Cesium blocks HCN channels but also acts at potassium channels (and possibly other channels as well). The authors do not acknowledge this or attempt to justify their use of Cs+ and do not cite prior work on this subject. They do not show control experiments demonstrating that the application of Cs+ in their preparation only affects Ih. Additionally, the authors write 1 mM cesium in the text but appear to use 2 mM in the figures. In later experiments, the authors switch to ZD7288, a more commonly used and generally accepted more specific blocker of HCN channels. However, they use a very high concentration, which is also known to produce off-target effects (see Chevaleyre and Castillo, 2002). To make robust conclusions, the authors should have used both blockers (at accepted/conservative concentrations) for all (or at least most) experiments. Using one blocker for some experiments and then another for different experiments is fraught with potential confounds.

(3) A stronger case could be made that HCN is expressed in the somatic compartment of L2/3 cells if the authors had directly measured HCN-isolated currents with outside-out or nucleated patch recording (with appropriate leak subtraction and pharmacology). Whole-cell voltage-clamp in neurons with axons and/or dendrites does not work. It has been shown to produce erroneous results over and over again in the field due to well-known space clamp problems (see Rall, Spruston, Williams, etc.). The authors could have also included negative controls, such as recordings in neurons that do not express HCN or in HCN-knockout animals. Without these experiments, the authors draw a false equivalency between the effects of cesium and HCN channels, when the outcomes they describe could be driven simply by multiple other cesium-sensitive currents. Distortions are common in these preparations when attempting to study channels (see Williams and Womzy, J Neuro, 2011). In Fig 2h, cesium-sensitive currents look too large and fast to be from HCN currents alone given what the authors have shown in their earlier current clamp data. Furthermore, serious errors in leak subtraction appear to be visible in Supplementary Figure 1c. To claim that these conductances are solely from HCN may be misleading.

(4) The authors present current-clamp traces with some sag, a primary indicator of HCN conductance, in Figure 2. However, they do not show example traces with cesium or ZD7288 blockade. Additionally, the normalization of current injected by cellular capacitance and the lack of reporting of input resistance or estimated cellular size makes it difficult to determine how much current is actually needed to observe the sag, which is important for assessing the functional relevance of these channels. The sag ratio in controls also varies significantly without explanation (Figure 6 vs Figure 7). Could this variability be a result of genetically defined subgroups within L2/3? For example, in humans, HCN expression in L2/3 varies from superficial and deep neurons. The authors do not make an effort to investigate this. Regardless of inconsistencies in either current injection or cell type, the sag ratio appears to be rather modest and similar to what has already been reported previously in other papers.

(5) In the later experiments with ZD7288, the authors measured EPSP half-width at greater distances from the soma. However, they use minimal stimulation to evoke EPSPs at increasingly far distances from the soma. Without controlling for amplitude, the authors cannot easily distinguish between attenuation and spread from dendritic filtering and additional activation and spread from HCN blockade. At a minimum, the authors should share the variability of EPSP amplitude versus the change in EPSP half-width and/or stimulation amplitudes by distance. In general, this kind of experiment yields much clearer results if a more precise local activation of synapses is used, such as dendritic current injection, glutamate uncaging, sucrose puff, or glutamate iontophoresis. There are recording quality concerns here as well: the cell pictured in Figure 3a does not have visible dendritic spines, and a substantial amount of membrane is visible in the recording pipette. These concerns also apply to the similar developmental experiment in 6f-h, where EPSP amplitude is not controlled, and therefore, attenuation and spread by distance cannot be effectively measured. The outcome, that L2/3 cells have dendritic properties that violate cable theory, seems implausible and is more likely a result of variable amplitude by proximity.

(6) Minimal stimulation used for experiments in Figures 3d-i and Figures 4g-h does not resolve the half-width measurement's sensitivity to dendritic filtering, nor does cesium blockade preclude only HCN channel involvement. Example traces should be shown for all conditions in 3h; the example traces shown here do not appear to even be from the same cell. These experiments should be paired (with and without cesium/ZD). The same problem appears in Figure 4, where it is not clear that the authors performed controls and drug conditions on the same cells. 4g also lacks a scale bar, so readers cannot determine how much these measurements are affected by filtering and evoked amplitude variability. Finally, if we are to believe that minimal stimulation is used to evoke responses of single axons with 50% fail rates, NMDA receptor activation should be minimal to begin with. If the authors wish to make this claim, they need to do more precise activation of NMDA-mediated EPSPs and examine the effects of ZD7288 on these responses in the same cell. As the data is presented, it is not possible to draw the conclusion that HCN boosts NMDA-mediated responses in L2/3 neurons.

(7) The quality of recordings included in the dataset has concerning variability: for example, resting membrane potentials vary by >15-20 mV and the AP threshold varies by 20 mV in controls. This is indicative of either a very wide range of genetically distinct cell types that the authors are ignoring or the inclusion of cells that are either unhealthy or have bad seals.

(8) The authors make no mention of blocking GABAergic signaling, so it must be assumed that it is intact for all experiments. Electrical stimulation can therefore evoke a mixture of excitatory and inhibitory responses, which may well synapse at very different locations, adding to interpretability and variability concerns.

(9) The investigation of serotonergic interaction with HCN channels produces modest effect sizes and suffers the same problems as described above.

(10) The computational modeling is not well described and is not biologically plausible. Persistent and transient K channels are missing. Values for other parameters are not listed. The model does not seem to follow cable theory, which, as described above, is not only implausible but is also not supported by the experimental findings.

Taken together, there are serious methodological and analytical concerns that need to be addressed before the authors' claims can be supported. Combined with the small effect sizes and high data variability throughout the paper, this makes it hard to see how the manuscript could make a strong contribution to advancing our understanding of L2/3 cortical pyramidal neuron function.