Reviewer #2 (Public Review):

In this manuscript, Aydemir et al. utilized the large TEDDY study and examined the effect of previously identified tri-SNP in the HLA-DRA gene on the risk of type 1 diabetes (T1D) and celiac disease (CD). They confirmed the protective effect of the tri-SNP haplotype "101" on T1D development. Meanwhile, the same haplotype appeared to be positively associated with risk for CD and the development of CD autoimmunity. The authors further explored the molecular effect of different tri-SNP haplotypes. They proposed that C4A and C4B might be the downstream target.

Overall, the study is rigorously conducted with proper statistical methods applied. The tri-SNP could be used as an additional risk factor when estimating T1D and celiac disease susceptibility in genetic screening. However, how this locus be incorporated into the current scheme of genetic screening is not discussed and is unlikely to be straightforward.

Reviewer #2 (Public Review):

In this study, Aso and Rubin generated new split-GAL4 lines to label Drosophila mushroom body output neurons (MBONs) that previously lacked specific GAL4 drivers. The MBONs represent the output channels for the mushroom body (MB), a computational center in the fly brain. Prior research identified 21 types of typical MBONs whose dendrites exclusively innervate the MB and 14 types of atypical MBONs whose dendrites also innervate brain regions outside the MB. These MBONs transmit information from the MB to other brain areas and form recurrent connections to dopaminergic neurons whose axonal terminals innervate the MB. Investigating the functions of the MBONs is crucial to understanding how the MB processes information and regulates behavior. The authors previously established a collection of split-GAL4 lines for most of the typical MBONs and one atypical MBON. That split-GAL4 collection has been an invaluable tool for researchers studying the MB. This work extends their previous effort by generating additional driver lines labeling the MBON types not covered by the previous split-GAL4 collection. Using these new driver lines, the authors also activated the labeled MBONs using optogenetics and assessed their role in learning, locomotion, and valence coding. The expression patterns of the new split-GAL4 lines and the behavioral analysis presented in this manuscript are generally convincing. I believe that these new lines will be a valuable resource for the fly community.

Reviewer #2 (Public Review):

Summary:

This manuscript is part of the Wright lab's ongoing studies that investigate whether the bumblebee B. terrestris can detect the presence of pesticides when feeding. Previously, they showed that B. terrestris cannot detect neonicotinoids and would prefer food containing neonicotinoids (Kessler et al. 2015). However, in that paper, they showed that B. terrestris cannot taste neonicotinoids but did not provide evidence on why B. terrestris prefer food containing neonicotinoids. In the current paper, the authors continue to suggest that B. terrestris cannot taste neonicotinoids as well as another insecticide, sulfoxaflor, based on additional behavioral experiments and electrophysiological experiments focusing on specific GRNs. While the data from these experiments continue to suggest that B. terrestris cannot taste these insecticides using their mouthparts, whether B. terrestris can actually perceive these insecticides, and why this species prefers food containing these compounds is still unknown.

Strengths:

The authors provided additional evidence that B. terrestris cannot taste neonicotinoids with their mouthparts.

Weaknesses:

There are too many overgeneralizations in the manuscript and parts of it are written in a way that seems to sound combative towards studies from other groups that came to slightly different conclusions from their previous paper.

Reviewer #2 (Public Review):

Summary:<br /> This main purpose of this investigation was to 1) compare the effects of a single knockout (sKO) of Numb or a double knockout (dKO) of Numb and NumbL on ex-vivo physiological properties of the extensor digitorium longus (EDL) muscle in C57BL/6NCrl mice; and 2) analyze protein complexes isolated from C2C12 myotubes via immunoprecipitation and LC/MS/MS for potential Numb binding partners. The main findings are 1) the muscles from sKO and dKO were significantly weaker with little difference between the sKO and dKO lines, indicating the reduced force is mainly due to the inactivation of the Numb gene; and 2) there were 11 potential Numb binding proteins that were identified and cytoskeletal specific proteins including Septin 7.

Strengths:<br /> Strait-forward yet elegant design to help determine the important role the Numb has in skeletal muscle.

Weaknesses: There were a limited number of samples (3-6) that were used for the physiological experiments; however, there was a very large effect size in terms of differences in muscle tension development between the induced KO models and the controls.

Reviewer #2 (Public Review):

Summary:

This article takes an expansive look at the potential role of DUX4 in cancer treatment and prognosis, including its correlation with other key biomarkers, the potential for cancer to be resistant to treatment, and risk prediction.

Strengths:

The primary strength of this work is the breadth of the analyses. The authors have linked DUX4 to not just one but multiple points in the trajectory of cancer, which increases the face validity of their conclusion that DUX4 is meaningfully related to the course of a cancer as well as the prognosis for a patient.

Statistically, the authors have taken care to properly validate their findings using appropriate bootstrapping and testing strategies.

Weaknesses:

Several weaknesses are noted. First, there is little-to-no description of the underlying sample population. It is only stated that "several large cohorts of patients with different metastatic cancers" were analyzed, and that a cohort of patients with advanced urothelial cancer was used for estimating associations with clinical outcomes. Lacking is information on the sampling mechanism, inclusion/exclusion criteria, treatment modalities, the definition of 'time = 0', the number of events observed, or even the sample size. Knowledge about the underlying study design would help explain some counterintuitive results, e.g. that the hazard of death among patients with Stage IV cancer is half that of those with Stage I cancer (Table 1); presumably this is not because Stage IV is actually protective but rather an artifact of the sampling scheme for these data. Second, the definition of negative versus positive DUX4 expression varies throughout the paper. In Figure 2A and Figure 3A, it is defined as >1 TPM vs. <= 1 TPM; in Figure 3C, it is defined as >1 TPM vs. < 0.5 TPM; in Figure 4A and Figure 5A, it is defined as >1 TPM vs. < 0.25 TPM; in Figure S1C it is partitioned into four groups, with boundaries defined at 0.25 TPM, 1 TPM, and 5 TPM. If categorization is needed, a rationale should be provided (ideally prospectively and not based upon the observed data, so as to avoid the perception of forking paths analyses), and it should be consistently applied. Third and finally, data seem to be occasionally excluded without rationale. For example, as mentioned above, the Cox model presented in Figure 4A seems to exclude all patients with DUX4 TPM between 0.25 and 1. Figure 3C excludes patients with either DUX4 TPM between 0.5 and 1 and/or with TMB in the lowest quartile (although the latter decision was ostensibly to control for TMB confounding, there are more appropriate ways to do so that don't result in loss of data, e.g. a stratified KM plot). Excluding patients based upon a particular region of the covariate space makes interpreting the resulting model awkward.

Reviewer #2 (Public Review):

Summary: In this manuscript, the authors performed single cell RNA-sequencing of cells from the penises of healthy and diabetes mellitus model (STZ injection-based) mice, identified *Lbh* as a marker of penis pericytes, and report that penis-specific overexpression of *Lbh* is sufficient to rescue erectile function in diabetic animals. In public human single cell RNA-sea datasets, the authors report that *LBH* is similarly specific to pericytes and down regulated in diabetic patients. Additionally, the authors report discovery of CRYAB and VIM1 as protein interacting partners with LBH.

The authors contributions are of interest to the erectile dysfunction community and their *Lbh* overexpression experiments are especially interesting and well-conducted. However, claims in the manuscript regarding the specificity of *Lbh* as a pericyte marker, the mechanism by which *Lbh* overexpression rescues erectile function, cell-cell interactions impaired by diabetes, and protein-interaction partners require qualification or further evidence to justify.

Major claims and evidence:

1. Marker gene specificity and quantification: One of the authors' major contributions is the identification of *Lbh* as a marker of pericytes in their data. The authors present qualitative evidence for this marker gene relationship, but it is unclear from the data presented if *Lbh* is truly a specific marker gene for the pericyte lineage (either based on gene expression or IF presented in Fig. 2D, E). Prior results (see Tabula Muris Consortium, 2018) suggest that *Lbh* is widely expressed in non-pericyte cell types, so the claims presented in the manuscript may be overly broad. Even if *Lbh* is not a globally specific marker, the authors' subsequent intervention experiments argue that it is still an important gene worth studying.<br /> 2. Cell-cell communication and regulon activity changes in the diabetic penis: The authors present cell-cell communication analysis and TF regulon analysis in Fig 3 and report differential activities in healthy and DM mice. These results are certainly interesting, however, no statistical analyses are performed to justify claimed changes in the disease state and no validations are performed. It is therefore challenging to interpret these results, and the relevant claims do not seem well supported.<br /> 3. Rescue of ED by Lbh overexpression: This is a striking and very interesting result that warrants attention. By simple overexpression of the pericyte marker gene Lbh, the authors report rescue of erectile function in diabetic animals. While mechanistic details are lacking, the phenomenon appears to have a large effect size and the experiments appear sophisticated and well conducted. If anything, the authors appear to underplay the magnitude of this result.<br /> 4. Mechanistic claims for rescue of ED by Lbh overexpression: The authors claim that cell type-specific effects on MPCs are responsible for the rescue of erectile function induced by Lbh overexpression. This causal claim is unsupported by the data, which only show that Lbh overexpression influences MPC performance. In vivo, it's likely that Lbh is being over expressed by diverse cell types, any of which could be the causal driver of ED rescue. In fact, the authors report rescue of cell type abundance in endothelial cells and neuronal cells. Therefore, it cannot be concluded that MPC effects alone or in principal are responsible for ED rescue.<br /> 5. Protein interaction data: The authors claim that CRYAB and VIM1 are novel interacting partners of LBH. However, the evidence presented (2 blots in Fig. 6A,B) lack the relevant controls. It is possible that CRYAB and VIM1 are cross-reactive with the anti-LBH antibody or were not washed out completely. The abundance of bands on the Coomassie stain in Fig. 6A suggests that either event is plausible. Therefore, the evidence presented is insufficient to support the claim that CRYAB and VIM1 are protein interacting partners of LBH.

**Impact**: These data will trigger interest in Lbh as a target gene within the erectile dysfunction community.

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.

Their discussion addresses some of the unexpected results, such as the reduced expression of HO-1 under hypoxia but due to the above mentioned limitations much of the discussion remains hypothetical.

In response to the reviewers´comments the authors extensively tried to address the points that were raised. They provided additional data, removed figures from the initial manuscript and referred to ongoing or further work. Nevertheless, not all questions could be answered leaving some hand-waiving and hypothetical explanations for some unexpected results.

Reviewer #2 (Public Review):

The work presented here by Morgun et al is performed in the context of vaccine development, a field especially active in the context of tuberculosis (TB). The generation of a new vaccine either enhancing or replacing the 100-year-old BCG is urgently needed.

Most subunit vaccines integrate protein antigens formulated with adjuvants and there are few examples of the performance of subunit vaccines integrating lipid antigens. Considering the hydrophobic and lipid nature of the mycobacterial cell envelope studies, assessing the suitability of mycobacterial lipids in vaccine formulations may contribute to generate new vaccines to tackle the disease.

The mycobacterial lipid antigens under study are mycolic acids (MA), which are located at the cell wall covalently linked to arabinogalactan. These lipids carry extremely long chain fatty acids of up to 60-90 carbons.

The group has previously shown that formulating MA into micellar nanocarriers and vaccinating mice intranasally it could activate CD1-restricted T cells. However, this formulation did not allow for the incorporation of protein antigens.

This work is novel, and it brings new data of high relevance for the TB vaccine field pointing to alternative formulations and antigens and immune mechanisms.

Authors assay different routes of vaccination but the main results are obtained using non-conventional vaccination routes. Although, it maybe out of the scope of the paper, no protection studies are provided.

Reviewer #2 (Public Review):

Summary: This paper takes on the important topic of preprocessing of single cell/nuclei RNA-seq prior to testing for differential gene expression. However, the manuscript has a number of critical weaknesses.

Strengths: This is an important topic and a key dataset for illustration.

Weaknesses: A major contribution is the use of the authors' own inhouse pipeline for data preparation (scFLOW), but this software is unpublished since 2021 and consequently not yet refereed. It isn't reasonable to take this pipeline as being validated in the field.

The authors claim that Mathys' analysis didn't use batch correction prior to analysis and claim that such processing is routine in the field, but the only citation they give is to the above-mentioned scFLOW. Batch correction for DEG analysis isn't the field standard, for example, Bryois et al. (2022) PMID: 35915177 doesn't perform batch correction. Whether or not to do such preprocessing is certainly arguable, but the authors need to argue it, not presuppose it.

The authors spend considerable effort in discounting the pseudoreplication analysis of Mathys. It is well understood that this analysis yields a lot of false positives, but Mathys only used this approach for removing genes, not as a valid test in and of itself. They also worry that the significant findings in Mathys' paper are influenced by the number of cells of each type. I'm sure it is since power is a function of sample size, but is this a bad thing? It seems odd that their approach is not influenced by sample size.

Reviewer #2 (Public Review):

Summary:<br /> The authors use previously characterised FRET methods to measure distances between intracellular segments of ASIC and with the membrane. The distances are measured across different conditions and at multiple positions in a very complete study. The picture that emerges is that the N- and C-termini do not associate.

Strengths:<br /> Good controls, good range of measurements, advanced, well-chosen and carefully performed FRET measurements. The paper is a technical triumph. Particularly, given the weak fluorescence of ANAP, the extent of measurements and the combination with TETAC is noteworthy.

The distance measurements are largely coherent and favour the interpretation that the N and C terminus are not close together as previously claimed.

Weaknesses:<br /> One difficulty is that we do not have a positive control for what binding of something to either N- or C-terminus would look like (either in FRET or otherwise).

One limitation that is not mentioned is the unroofing. The concept of interaction with intracellular domains is being examined. But the authors use unroofing to measure the positions, fully disrupting the cytoplasm. Thus it is not excluded that the unroofing disrupts that interaction. This should be mentioned as a possible (if unlikely) limitation.

Reviewer #2 (Public Review):

This manuscript by Nagel et al provides a comprehensive examination of the chemical composition of mouse urine (an important source of semiochemicals) across strain and sex, and correlates these differences with functional responses of vomeronasal sensory neurons (an important sensory population for detecting chemical social cues). The strength of the work lies in the careful and comprehensive imaging and chemical analyses, the rigor of quantification of functional responses, and the insight into the relevance of olfactory work on lab-derived vs wild-derived mice.

With regards to the chemical analysis, the reader should keep in mind that a difference in the concentration of a chemical across strain or sex does not necessarily mean that that chemical is used for chemical communication. In the most extreme case, the animals may be completely insensitive to the chemical. Thus, the fact that the repertoire of proteins and volatiles could potentially allow sex and/or strain discrimination, it is unclear to what degree both are used in different situations.

Reviewer #2 (Public Review):

The Xerces Blue is an iconic species, now extinct, that is a symbol for invertebrate conservation. Using genomic sequencing of century-old specimens of the Xerces Blue and its closest living relatives, the authors hypothesize about possible genetic indicators of the species' demise. Although the limited range and habitat destruction are the most likely culprits, it is possible that some natural reasons have been brewing to bring this species closer to extinction.

The importance of this study is in its generality and applicability to any other invertebrate species. The authors find that low effective population size, high inbreeding (for tens of thousands of years), and higher fraction of deleterious alleles characterize the Xerces colonies prior to extinction. These signatures can be captured from comparative genomic analysis of any target species to evaluate its population health.

It should be noted that it remains unclear if these genomic signatures are indeed predictive of extinction, or populations can bounce back given certain conditions and increase their genetic diversity somehow.

Methods are detailed and explained well, and the study could be replicated. I think this is a solid piece of work. Interested researchers can apply these methods to their chosen species and eventually, we will assemble datasets to study extinction process in many species to learn some general rules.

Reviewer #2 (Public Review):

Summary:<br /> In this work, Yuasa et al. aimed to study the spatial resolution of modulations in alpha frequency oscillations (~10Hz) within the human occipital lobe. Specifically, the authors examined the receptive field (RF) tuning properties of alpha oscillations, using retinotopic mapping and invasive electroencephalogram (iEEG) recordings. The authors employ established approaches for population RF mapping, together with a careful approach to isolating and dissociating overlapping, but distinct, activities in the frequency domain. Whereby, the authors dissociate genuine changes in alpha oscillation amplitude from other superimposed changes occurring over a broadband range of the power spectrum. Together, the authors used this approach to test how spatially tuned estimated RFs were when based on alpha range activity, vs. broadband activities (focused on 70-180Hz). Consistent with a large body of work, the authors report clear evidence of spatially precise RFs based on changes in alpha range activity. However, the size of these RFs were far larger than those reliably estimated using broadband range activity at the same recording site. Overall, the work reflects a rigorous approach to a previously examined question, for which improved characterization leads to improved consistency in findings and some advance of prior work.

Strengths:<br /> Overall, the authors take a careful and well-motivated approach to data analyses. The authors successfully test a clear question with a rigorous approach and provide strong supportive findings. Firstly, well-established methods are used for modeling population RFs. Secondly, the authors employ contemporary methods for dissociating unique changes in alpha power from superimposed and concomitant broadband frequency range changes. This is an important confound in estimating changes in alpha power not employed in prior studies. The authors show this approach produces more consistent and robust findings than standard band-filtering approaches. As noted below, this approach may also account for more subtle differences when compared to prior work studying similar effects.

Weaknesses:<br /> -Theoretical framing: The authors frame their study as testing between two alternative views on the organization, and putative functions, of occipital alpha oscillations: i) alpha oscillation amplitude reflects broad shifts in arousal state, with large spatial coherence and uniformity across cortex; ii) alpha oscillation amplitude reflects more specific perceptual processes and can be modulated at local spatial scales. However, in the introduction this framing seems mostly focused on comparing some of the first observations of alpha with more contemporary observations. Therefore, I read their introduction to more reflect the progress in studying alpha oscillations from Berger's initial observations to the present. I am not aware of a modern alternative in the literature that posits alpha to lack spatially specific modulations. I also note this framing isn't particularly returned to in the discussion. A second important variable here is the spatial scale of measurement. It follows that EEG based studies will capture changes in alpha activity up to the limits of spatial resolution of the method (i.e. limited in ability to map RFs). This methodological distinction isn't as clearly mentioned in the introduction, but is part of the author's motivation. Finally, as noted below, there are several studies in the literature specifically addressing the authors question, but they are not discussed in the introduction.

-Prior studies: There are important findings in the literature preceding the author's work that are not sufficiently highlighted or cited. In general terms, the spatio-temporal properties of the EEG/iEEG spectrum are well known (i.e. that changes in high frequency activity are more focal than changes in lower frequencies). Therefore, the observations of spatially larger RFs for alpha activities is highly predicted. Specifically, prior work has examined the impact of using different frequency ranges to estimate RF properties, for example ECoG studies in the macaque by Takura et al. NeuroImage (2016) [PubMed: 26363347], as well as prior ECoG work by the author's team of collaborators (Harvey et al., NeuroImage (2013) [PubMed: 23085107]), as well as more recent findings from other groups (Luo et al., (2022) BioRxiv: https://doi.org/10.1101/2022.08.28.505627). Also, a related literature exists for invasively examining RF mapping in the time-voltage domain, which provides some insight into the author's findings (as this signal will be dominated by low-frequency effects). The authors should provide a more modern framing of our current understanding of the spatial organization of the EEG/iEEG spectrum, including prior studies examining these properties within the context of visual cortex and RF mapping. Finally, I do note that the author's approach to these questions do reflect an important test of prior findings, via an improved approach to RF characterization and iEEG frequency isolation, which suggests some important differences with prior work.

-Statistical testing: The authors employ many important controls in their processing of data. However, for many results there is only a qualitative description or summary metric. It appears very little statistical testing was performed to establish reported differences. Related to this point, the iEEG data is highly nested, with multiple electrodes (observations) coming from each subject, how was this nesting addressed to avoid bias?

Reviewer #2 (Public Review):

Summary:<br /> The authors proposed that grid cells may be aligned by simpler, 1D attractors, and they showed that the structure and the representational space of an attractor network can be two different topological objects.

Strengths:<br /> It is very interesting that the toroidal topology of the population activity (the representational space) and the structure of the attractor network do not necessarily to be the same. The authors carried out extensive computational modeling to support such evidence. The results presented by the authors in this study could have an impact in the grid cell field, which will motivate future experimental studies to examine the detailed structure of the grid cell population.

Weaknesses:<br /> The authors mentioned that "the recurrent collateral structure defines the geometry of the manifold..." and pointed out that this assumption is wrong. I am afraid this claim is too strong. The Gardner Torus paper showed evidence of the 2D CAN exists in the EC as a possible substrate of the grid pattern. Do the authors mean here that even the population activity in the grid cells show the torus structure, it does not necessarily mean that the grid cells form a 2D CAN? I understand that from the computational modeling view, it is doable to find counter-examples (like the 1D attractor network) in which the representational space is a torus but the structure is different. However, from the experimental view, do you expect that the grid cell network is a low-dimensional attractor network? To prove this, is there any evidence from the experimental data?

Reviewer #2 (Public Review):

Summary:<br /> Mitochondria in synapses are important to support functional needs, such as local protein translation and calcium buffering. Thus, they may be strategically localized to maximize functional efficiency. In this study, the authors examine whether a correlation exists between the positioning of mitochondria and the structure or function of dendritic spines in the visual cortex of a ferret. Unexpectedly, the authors found no correlation between structural measures of synaptic strength to mitochondria positioning, which may indicate that they are not localized only because of the local energy needs. Instead, the authors discover that mitochondria are positioned preferably in spines that display heterogeneous responses, showing that they are localized to support specific functional needs probably distinct from ATP output.

Strengths:<br /> The thorough analysis provides a yet unprecedented insight into the correlation between synaptic tuning and mitochondrial positioning in the visual cortex in vivo.

Weaknesses:<br /> The study defined 1 μm and 5 μm as short and extended ranges relative to the synapse and examined the correlation between mitochondria volume and multiple parameters within that defined range. Results showed that mitochondria display preferences towards spines that respond differently to visual stimuli or areas with low local calcium activity. However, it is not known whether this mitochondria preference is a cause or a result of spine heterogeneity. It will be interesting to see the correlation of spine volume relative to mitochondrial positioning in 1µm and 5µm ranges around mitochondria.

Analysis of this study suggested that mitochondrial volume does not correlate with the structural measure of synaptic strength (e.g. spine volume and post-synaptic density (PSD) area). However, the authors did not examine whether mitochondrial volume correlates to synaptic transmission frequency or plasticity. It may still be possible that mitochondria are localized in positions that exhibit a high frequency of transmission or a high degree of plasticity. Future studies will have to determine the underlying cause of mitochondria positioning preference.

Reviewer #2 (Public Review):

Hagihara et al. conducted a study investigating the correlation between decreased brain pH, increased brain lactate, and poor working memory. They found altered brain pH and lactate levels in animal models of neuropsychiatric and neurodegenerative disorders. Their study suggests that poor working memory performance may predict higher brain lactate levels.

However, the study has some significant limitations. One major concern is that the authors examined whole-brain pH and lactate levels, which might not fully represent the complexity of disease states. Different brain regions and cell types may have distinct protein and metabolite profiles, leading to diverse disease outcomes. For instance, certain brain regions like the hippocampus and nucleus accumbens exhibit opposite protein/signaling pathways in neuropsychiatric disease models.

Moreover, the memory tests used in the study are specific to certain brain regions, but the authors did not measure lactate levels in those regions. Without making lactate measurements in brain-regions and cell types involved in these diseases, any conclusions regarding the role of lactate in CNS diseases is premature.

Additionally, evidence suggests that exogenous treatment with lactate has positive effects, such as antidepressant effects in multiple disease models (Carrard et al., 2018, Carrard et al., 2021, Karnib et al., 2019, Shaif et al., 2018). It also promotes learning, memory formation, neurogenesis, and synaptic plasticity (Suzuki et al., 2011, Yang et al., 2014, Weitian et al., 2015, Dong et al., 2017, El Hayek et al. 2019, Wang et al., 2019, Lu et al., 2019, Lev-Vachnish et a.l, 2019, Descalzi G et al., 2019, Herrera-López et al., 2020, Ikeda et al., 2021, Zhou et al., 2021,Roumes et al., 2021, Frame et al., 2023, Akter et al., 2023).

In conclusion, the relevance of total brain pH and lactate levels as indicators of the observed correlations is controversial, and evidence points towards lactate having more positive rather than negative effects. It is important that the authors perform studies looking at brain-region-specific concentrations of lactate and that they modulate lactate levels (decrease) in animal models of disease to validate their conclusions. it is also important to consider the above-mentioned studies before concluding that "altered brain pH and lactate levels are rather involved in the underlying pathophysiology of some patients with neuropsychiatric disorders" and that "lactate can serve as a potential therapeutic target for neuropsychiatric disorders".

Reviewer #2 (Public Review):

The manuscript presents compelling evidence for the role of the zona incerta area of the brain in regulating movement and sensory stimuli in mice. The study uses an appropriate and validated methodology in line with the current state-of-the-art, including optogenetic manipulation and recording of single-unit activity. The authors' claims and conclusions are well-supported by their data, which includes a comprehensive review of previous research on the zona incerta. Overall, the manuscript provides solid evidence for the role of the zona incerta in regulating movement and sensory processing.

Major strengths and weaknesses of the methods and results.<br /> The zona incerta has many integrative functions that link sensory stimuli with motor responses to guide behavior.<br /> The study explored the activation of zona incerta GABAergic neurons during cued avoidance tasks and found that these neurons activate during goal-directed avoidance movement. Optogenetic manipulation of these neurons affected movement speed and performance during active avoidance tasks.<br /> The findings suggest that the zona incerta area of the brain plays a significant role in regulating movement and responding to salient auditory tones in association with movement in mice. The evidence presented is fundamental and provides a comprehensive review of previous research on the zona incerta and its involvement in various behaviors and sensory processing.

The article is very well written, with a correct hypothesis and a cutting-edge methodology to achieve the expected objectives. Moreover, they use statistical rigorous approaches in the analysis of the results. Also, analyzes are performed using scripts that automate all aspects of data analysis, ensuring their objectivity. The results are very novel, and provide solid evidence for the role of the zona incerta in regulating movement and sensory processing.

Reviewer #2 (Public Review):

Previous studies have extensively explored the rules by which patterned inputs from the two eyes are combined in visual cortex. Here the authors explore these rules for un-patterned inputs (luminance flicker) at both the level of cortex, using Steady-State Visual Evoked Potentials (SSVEPs) and at the sub-cortical level using pupillary responses. They find that the pattern of binocular combination differs between cortical and sub-cortical levels with the cortex showing less dichoptic masking and somewhat more binocular facilitation.

Importantly, the present results with flicker differ markedly from those with gratings (Hou et al., 2020, J Neurosci, Baker and Wade 2017 cerebral cortex, Norcia et al, 2000 Neuroreport, Brown et al., 1999, IOVS. When SSVEP responses are measured under dichoptic conditions where each eye is driven with a unique temporal frequency, in the case of grating stimuli, the magnitude of the response in the fixed contrast eye decreases as a function of contrast in the variable contrast eye. Here the response increases by varying (small) magnitudes. The authors favor a view that cortex and perception pool binocular flicker inputs approximately linearly using cells that are largely monocular. The lack of a decrease below the monocular level when modulation strength increase is taken to indicate that previously observed normalization mechanism in pattern vision does not play a substantial role in the processing of flicker. The authors present of computational model of binocular combination that captures features of the data when fit separately to each data set. Because the model has no frequency dependence and is based on scalar quantities, it cannot make joint predictions for the multiple experimental conditions which one of its limitations.

A strength of the current work is the use of frequency-tagging of both pupil and EEG responses to measure responses for flicker stimuli at two anatomical levels of processing. Flicker responses are interesting but have been relatively neglected. The tagging approach allows one to access responses driven by each eye, even when the other eye is stimulated which is a great strength. The tagging approach can be applied at both levels of processing at the same time when stimulus frequencies are low, which is an advantage as they can be directly compared. The authors demonstrate the versatility of frequency tagging in a novel experimental design which may inspire other uses, both within the present context and others. A disadvantage of the tagging approach for studying sub-cortical dynamics via pupil responses is that it is restricted to low temporal frequencies given the temporal bandwidth of the pupil. The inclusion of a behavioral measure and a model is also a strength, but there are some limitations in the modeling (see below).

The authors suggest in the discussion that luminance flicker may preferentially drive cortical mechanisms that are largely monocular and in the results that they are approximately linear in the dichoptic cross condition (no effect of the fixed contrast stimulus in the other eye). By contrast, prior research using dichoptic dual frequency flickering stimuli has found robust intermodulation (IM) components in the VEP response spectrum (Baitch and Levi, 1988, Vision Res; Stevens et al., 1994 J Ped Ophthal Strab; France and Ver Hoeve, 1994, J Ped Ophthal Strab; Suter et al., 1996 Vis Neurosci). The presence of IM is a direct signature of binocular interaction and suggests that at least under some measurement conditions, binocular luminance combination is "essentially" non-linear, where essential implies a point-like non-linearity such as squaring of excitatory inputs. The two views are in striking contrast.

In this revised manuscript, the addition of Figure 8, which shows more complete response spectra, partially addresses this issue. However, it also raises new questions. Critically, intermodulation (IM) has to be generated at or after a point of binocular combination, as it is a mixture of the two monocular frequencies and the monocular frequencies can only mix after a point of binocular combination.

In equations 1 and 2 and in the late summation and two-stage models of Meese et al (2006), there are divisive binocular cross-links prior to a summation block. This division is a form of binocular interaction. Do equations 1 and 2 generate IM on their own with parameters used for the overall modeling? Multiplication of two inputs clearly does, as the authors indicate in their toy model. If not, then a different binocular summation rule than the one expressed in equation 3 needs to be considered to produce IM.

The discussion considers flicker processing as manifest in the EEG to be largely monocular, given the relative lack of binocular facilitation and suppression effects. And yet there is robust IM. These are difficult to reconcile as it stands. The authors suggest that their generic modeling framework can predict IM, but can it predict IM with the parameters used to fit the data, e.g. with very low values of the weight of interocular suppression and no other binocular non-linearity?

Determining whether IM can be generated by the existing non-linear elements in the model is important because previous work on dichoptic flicker IM has considered a variety of simple models of dichoptic flicker summation and has favored models involving either a non-linear combination of linear monocular inputs (Baitch and Levi, Vis Research, 1988) or a non-linear combination of rectified (non-linear) monocular inputs (Regan and Regan, Canadian J Neurol Sci, 1989). In either case, the last stage of binocular combination is non-linear, rather than linear. The authors' model is different - it has a stage of divisive binocular interaction and this "quasi-monocular" stage feeds a linear binocular combination stage.

There is a second opportunity to test the proposed model that the authors could take advantage of. In the initial review, two of the reviewers were curious about what is predicted for counter-phase inputs to the two eyes. The authors indicate that the class of models they are using could be extended to cover this case. As it turns out, this experiment has been done for dichoptic full-field flicker (Sherrington, BrJPsychiatr, 1904); van der Tweel and Estevez, Ophthalmologica, 1974; Odom and Chao, IntJNeurosci, 1995; Cavonius, QJExpPsych, 1979; Levi et al., BJO, 1982). More importantly, the predictions of several binocular combination models for anti-phase inter-ocular flicker stimulation have been tested for both the VEP and psychophysics (Odom and Chao, Int J Neurosci). Varying the relative phase of the two eyes inputs from in phase to antiphase, Odom and Chao observed that the 2nd harmonic response went to a minimum at 90 deg of interocular phase. This will happen because a 2nd order nonlinearity in the monocular path will double the phase shift of the second harmonic, putting the two eyes' 2nd harmonic response out of phase when the interocular phase is 90 deg. Summing these inputs thus leads to cancellation at 90 deg, rather than 180 deg of interocular phase. Does the authors' model predict this behavior with typical parameters used in the modeling? In the end, to account for details of both VEP and psychophysical data, Odom and Chao favored a two-path model with one path comprising non-linear monocular inputs being combined linearly and a second path combining linear monocular inputs at a non-linear binocular stage. A similar set of results and models has been developed for inter-ocular presentation of gratings (Zemon et al., PNAS, 1995).

The Odom/Chao/Zemon VEP and psychophysical data are directly relevant to the authors' work and need to be taken into account in sufficient detail so that we can judge the consistency of the proposed framework with their data and the similarities and differences in the model predictions for dichoptic flicker combination. These models are also relevant to the generation of IM, a concern raised above.

Reviewer #2 (Public Review):

The manuscript by Dacheux et al. reported homozygous deleterious variants of ZMYND12 in four unrelated men with asthenoteratozoospermia. Based on the immunofluorescence assays in human sperm cells, it was shown that ZMYND12 deficiency altered the localization of DNAH1, DNALI1, WDR66 and TTC29 (four of the known key proteins involved in sperm flagellar formation). Trypanosoma brucei and mouse models were further employed for mechanistic studies, which revealed that ZMYND12 is part of the same axonemal complex as TTC29 and DNAH1. Their findings are solid, and this manuscript will be very informative for clinicians and basic researchers in the field of human infertility.

Reviewer #2 (Public Review):

The authors found that the age-related reduction in the serum CNP concentration was highly correlated with decreased oocyte quality. Treatment with exogenous CNP promoted follicle growth and ovulation in aged mice and enhanced meiotic competency and fertilization ability. The cytoplasmic maturation of aged oocytes was thoroughly improved by CNP treatment. CNP treatment also ameliorated DNA damage and apoptosis caused by ROS accumulation in aged oocytes. CNP reversed the defective phenotypes in aged oocytes by alleviating oxidative damage and suppressing excessive PINK1/Parkin-mediated mitophagy. CNP functioned as a cAMP/PKA pathway modulator to decrease PINK1 stability and inhibit Parkin recruitment. CNP may be used to improve the overall success rates of clinically assisted reproduction in older women.

The author has modified the text and the level of the article has been improved. Additional experiments will further enhance the credibility of the article.

1）The control also needs to be pre-cultured as that in CNP treatment.

2）The mechanism is done 6 days later after CNP treatment. It is hard to know whether it is direct or indirect.

Reviewer #2 (Public Review):

The manuscript by Genzoni et al. provides evidence that trophic eggs laid by the queen in the ant Pogonomyrmex rugosis have an inhibitory effect on queen development. The authors also compare a number of features of trophic eggs, including protein, DNA, RNA, and miRNA content, to reproductive eggs. To support their argument that trophic eggs have an inhibitory effect on queen development, the authors show that trophic eggs have a lower content of protein, triglycerides, glycogen, and glucose than reproductive eggs, and that their miRNA distributions are different relative to reproductive eggs. Although the finding of an inhibitory influence of trophic eggs on queen development is indeed arresting, the egg cross-fostering experiment that supports this finding can be effectively boiled down to a single figure (Figure 6). The rest of the data are supplementary and correlative in nature (and can be combined), especially the miRNA differences shown between trophic and reproductive eggs. This means that the authors have not yet identified the mechanism through which the inhibitory effect on queen development is occurring. To this reviewer, this finding is more appropriate as a short report and not a research article. A full research article would be warranted if the authors had identified the mechanism underlying the inhibitory effect on queen development. Furthermore, the article is written poorly and lacks much background information necessary for the general reader to properly evaluate the robustness of the conclusions and to appreciate the significance of the findings.

Reviewer #2 (Public Review):

The manuscript by Nishikawa et al. addresses time-dependent changes in the electron transfer energetics in the photosynthetic reaction center from Blastochloris viridis, whose time-dependent structural changes upon light illumination were recently demonstrated by time-resolved serial femtosecond crystallography (SFX) using X-ray free-electron laser (XFEL) (Dods et al., Nature, 2021). Based on the redox potential Em values of bacteriopheophytin in the electron transfer active branch (BL) by solving the linear Poisson-Boltzmann equation, the authors found that Em(HL) values in the charge-separated 5-ps structure obtained by XFEL are not clearly changed, suggesting that the P+HL- state is not stabilized owing to protein reorganization. Furthermore, chlorin ring deformation upon HL- formation, which was expected from their QM/MM calculation, is not recognized in the 5-ps XFEL structure. Then the authors concluded that the structural changes in the XFEL structures are not related to the actual time course of charge separation. They argued that their calculated changes in Em and chlorin ring deformations using the XEFL structures may reflect the experimental errors rather than the real structural changes; they mentioned this problem is due to the fact that the XFEL structures were obtained at not high resolutions (mostly at 2.8 Å). I consider that their systematic calculations may suggest a useful theoretical interpretation of the XFEL study.

Comments on latest version:

The authors have satisfied my concerns. I consider that their present manuscript is more attractive and informative for readers.

Reviewer #2 (Public Review):

The manuscript by Nishikawa et al. addresses time-dependent changes in the electron transfer energetics in the photosynthetic reaction center from Blastochloris viridis, whose time-dependent structural changes upon light illumination were recently demonstrated by time-resolved serial femtosecond crystallography (SFX) using X-ray free-electron laser (XFEL) (Dods et al., Nature, 2021). Based on the redox potential Em values of bacteriopheophytin in the electron transfer active branch (BL) by solving the linear Poisson-Boltzmann equation, the authors found that Em(HL) values in the charge-separated 5-ps structure obtained by XFEL are not clearly changed, suggesting that the P+HL- state is not stabilized owing to protein reorganization. Furthermore, chlorin ring deformation upon HL- formation, which was expected from their QM/MM calculation, is not recognized in the 5-ps XFEL structure. Then the authors concluded that the structural changes in the XFEL structures are not related to the actual time course of charge separation. They argued that their calculated changes in Em and chlorin ring deformations using the XEFL structures may reflect the experimental errors rather than the real structural changes; they mentioned this problem is due to the fact that the XFEL structures were obtained at not high resolutions (mostly at 2.8 Å). I consider that their systematic calculations may suggest a useful theoretical interpretation of the XFEL study. However, the present manuscript insists as a whole negatively that the experimental errors may hamper to provide the actual structural changes relevant to the electron transfer events.

Reviewer #2 (Public Review):

Summary:<br /> The authors tried to identify new genes involved in melanin metabolism and its spatial distribution in the silkworm Bombyx mori. They identified the gene Bm-mamo as playing a role in caterpillar pigmentation. By functional genetic and in silico approaches, they identified putative target genes of the Bm-mamo protein. They showed that numerous cuticular proteins are regulated by Bm-mamo during larval development.

Strengths:<br /> -preliminary data about the role of cuticular proteins to pattern the localization of pigments<br /> - timely question<br /> - challenging question because it requires the development of future genetic and cell biology tools at the nanoscale

Weaknesses:<br /> - statistical sampling limited<br /> - the discussion would gain in being shorter and refocused on a few points, especially the link between cuticular proteins and pigmentation. The article would be better if the last evolutionary-themed section of the discussion is removed.

A recent paper has been published on the same gene in Bombyx mori (https://www.sciencedirect.com/science/article/abs/pii/S0965174823000760) in August 2023. The authors must discuss and refer to this published paper through the present manuscript.

Reviewer #2 (Public Review):

Summary:<br /> The authors report the successful retrieval of mitogenomes from extinct Pleistocene megafauna (woolly Mammoth and woolly rhino) from recent sediment cores from two close Siberian lakes. The cores are too recent to represent real time points of these two extinct species (known to have been extinct for several thousands of years) and therefore, the most plausible interpretation is that permafrost thawing and similar physical processes in the lakes have made surface old ancient DNA, maybe from nearby, deep-buried carcasses.

Strengths:<br /> The pattern of postmortem damage at the end of the Mammoth DNA reads as well as the length distribution (reported in Figure 1) is expected for authentic ancient DNA extracts (besides the phylogenetic evidence). These results pose a question, in my view, on the general reliability of sedimentary DNA in similar contexts, especially in the absence of direct radiocarbon dating of associated remains and in the absence of an understanding of the local geo-physical dynamics. At the same time, the evidence reported here suggests that, at least in Siberian lakes, the sediments can preserve a rich ancient DNA record that it is worth surveying.

Weaknesses:<br /> Although admittedly the work can represent two cases of environments with singular thermal conditions and geodynamics, it opens also the possibility of studying more lake sediments for trying to understand if these findings can be generalized.

Reviewer #2 (Public Review):

Neininger-Castro et al report on their original study entitled "Independent regulation of Z-lines and M-lines during sarcomere assembly in cardiac myocytes revealed by the automatic image analysis software sarcApp", In this study, the research team developed two software, yoU-Net and sarcApp, that provide new binarization and sarcomere quantification methods. The authors further utilized human induced pluripotent stem cell-derived cardiomyocytes (hiCMs) as their model to verify their software by staining multiple sarcomeric components with and without the treatment of Blebbistatin, a known myosin II activity inhibitor. With the treatment of different Blebbistatin concentrations, the morphology of sarcomeric proteins was disturbed. These disrupted sarcomeric structures were further quantified using sarcApp and the quantification data supported the phenotype. The authors further investigated the roles of muscle myosins in sarcomere assembly by knocking down MYH6, MYH7, or MYOM in hiCMs. The knockdown of these genes did not affect Z-line assembly yet the knockdown of MYOM affected M-line assembly. The authors demonstrated that different muscle myosins participate in sarcomere assembly in different manners.

Reviewer #2 (Public Review):

Summary:<br /> The authors' main goal was to evaluate how both behavioral responses to odor, and their early sensory representations are modified by repeated exposure to odor, asking whether the process of adaptation is equivalent to reducing the concentration of an odor. They open with behavioral experiments that actually establish that repeated odor presentation increases the likelihood of evoking a behavioral response in their experimental subjects - locusts. They then examine neural activity patterns at the second layer of the olfactory circuit. At the population level, repeated odor exposure reduces total spike counts, but at the level of individual cells there seems to be no consistent guiding principle that describes the adaptation-related changes, and therefore no single mechanism could be identified.

Both population vector analysis and pattern correlation analysis indicate that odor intensity information is preserved through the adaptation process. They make the closely related point that responses to an odor in the adapted state are distinct from responses to lower concentration of the same odor. These analyses are appropriate, but the point could be strengthened by explicitly using some type of classification analysis to quantify the adaptation effects. e.g. a confusion matrix might show if there is a gradual shift in odor representations, or whether there are trials where representations change abruptly.

Strengths:<br /> One strength is that the work has both behavioral read-out of odor perception and electrophysiological characterization of the sensory inputs and how both change over repeated stimulus presentations. It is particularly interesting that behavioral responses increase while neuronal responses generally decrease. Although the behavioral effect could occur fully downstream of the sensory responses the authors measure, at least those sensory responses retain the core features needed to drive behavior despite being highly adapted.

Weaknesses:<br /> Ultimately no clear conceptual framework arises to understand how PN responses change during adaptation. Neither the mechanism (vesicle depletion versus changes in lateral inhibition) nor even a qualitative description of those changes. Perhaps this is because much of the analysis is focused on the entire population response, while perhaps different mechanisms operate on different cells making it difficult to understand things at the single PN level.

From the x-axis scale in Fig 2e,f it appeared to me that they do not observe many strong PN responses to these stimuli, everything being < 10 spikes/sec. So perhaps a clearer effect would be observed if they managed to find the stronger responding PNs than captured in this dataset.

DOI: 10.1371/journal.pbio.3002223

Resource: RRID:ZFIN_ZDB-ALT-060221-2

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-060221-2

What is this?

DOI: 10.1371/journal.pbio.3002223

Resource: (ZFIN Cat# ZDB-ALT-081027-2,RRID:ZFIN_ZDB-ALT-081027-2)

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-081027-2

What is this?

Reviewer #2 (Public Review):

In this study, the authors seek to answer two main questions: 1) Whether interfering with lactate availability in hepatocytes through depletion of hepatocyte specific MCT-1 depletion would reduce steatosis, and 2) Whether MCT-1 in stellate cells promote fibrogenesis. While the first question is based on the observation that haploinsufficiency of MCT-1 makes mice resistant to steatosis, the rationale behind how MCT-1 could impact fibrogenesis in stellate cells is not clear. A more detailed discussion regarding how lactate availability would regulate two different processes in two different cell types would be helpful. The authors employ several mouse models and in vitro systems to show that MCT1 inhibition in hepatic stellate cells reduces the expression of COL-1. The significance of the findings is moderately impacted due to the following considerations:

a) Fibrosis in human NAFLD is a significant problem as a predictor of liver related mortality and is associated with type 1 and type 3 collagen. However, the reduction in COL1 in stellate cells did not amount to a reduction in liver fibrosis even in cell specific KO (in Fig 7E, there is no indication of whether Sirius red staining was different between HSC KO and control mice- the authors mention a downward trend in the text). The authors postulate that type 1 COL may not be the more predominant form of fibrosis in the model. This does not seem likely, since the same ob/ob mouse model was used to determine that fibrosis was enhanced with hepatocyte specific MCT-1 KO and decreased with Chol MCT-1KO. Measurements of different types of collagens in their model and the effect of MCT-1 on different types could be more informative. In particular, although collagens are the structural building blocks for hepatic fibrosis, fibrosis can also be controlled by matrix remodeling factors such as Timp1, Serpine 1, PAI-1 and Lox.

b) The authors use multiple animal models including cell specific KO to conclude that stellate cell MCT-1 inhibition decreases COL-1. However, the mechanisms behind this reduced expression of COL-1 are not discussed or explored, making it descriptive.

c) Different types of diets are used in this study which could impact lactate availability. Choline deficiency diets are reported to cause weight loss, and importantly have none of the metabolic features of human NASH. Therefore, their utility is doubtful, especially for this study which proposes to investigate if metabolic dysregulation and substrate availability could be a tool for therapy.

d) Hepatocyte specific MCT-1 KO mice seem to have increased COL-1 production, despite no noticeable difference in hepatocyte steatosis. The reasons for this are not discussed. Fibrosis in NASH is thought to be from stellate cell activation secondary to signals from hepatocellular damage. There is no evidence that there was a difference in either of these parameters in the mouse models used.

e) The authors report that serum lactate levels did not rise after MCT-1 silencing, but the reasons behind this are unclear. There is insufficient data about lactate production and utilization in this model, which would be useful to interpret data regarding steatosis and fibrosis development. For example, does the MCT-1 KO prevent hepatocyte and stellate cell net import or export of lactate? What is the downstream metabolic consequence in terms of pyruvate, acetylCoA and the NAD/NADH levels. Does the KO have downstream effects on mitochondrial TCA cycling?

f) MCT-1 protein expression is measured only in the in vitro assay. Similar quantitation through western blot is not shown in the animal models.

Reviewer #2 (Public Review):

The authors present new data of endocranial surface details from the early Homo specimen KNM-ER 3732 and discuss the evolution of brain surface features that might be related to the evolution of language in the hominin lineage.

Comments and issues raised by the reviewers have been addressed adequately. I am sure that this contribution will revive discussion about these issues.

Reviewer #2 (Public Review):

In this work, the authors investigated the pectoralis work loop and the function of the supracoracoideus muscle in the down stroke during slow flight in doves. The aim of this study was to determine how aerodynamic force is generated, using simultaneous high-speed measurements of the wings' kinematics, aerodynamics, and activation and strain of pectoralis muscles during slow flight. The measurements show a reduction in the angle of attack during mid-downstroke, which induces a peak power factor and facilitates the tensioning of the supracoracoideus tendon with pectoralis power, which then can be released in the up-stroke. By combining the data with a muscle mechanics model, the timely tuning of elastic storage in the supracoracoideus tendon was examined and showed an improvement of the pectoralis work loop shape factor. Finally, other bird species were integrated into the model for a comparative investigation.

The major strength of the methods is the simultaneous application of four high-speed techniques - to quantify kinematics, aerodynamics and muscle activation and strain - as well as the implementation of the time-resolved data into a muscle mechanics model. With a thorough analysis which supports the conclusions convincingly, the authors achieved their goal of reaching an improved understanding of the interplay of the pectoralis and supracoracoideus muscles during slow flight and the resulting energetic benefits.

Reviewer #2 (Public Review):

Summary:

The manuscript by Knecht et al entitled "Non-cognate immunity proteins provide broader defenses against interbacterial effectors in microbial communities" aims at characterizing a new type VI secretion system (T6SS) effector immunity pair using genetic and biochemical studies primarily focused on Proteus mirabilis and metagenomic analysis of human-derived data focused on Rothia and Prevotella sequences. The authors provide evidence that RdnE and RdnI of Proteus constitute an E-I pair and that the effector likely degrades nucleic acids. Further, they provide evidence that expression of non-cognate immunity derived from diverse species can provide protection against RdnE intoxication. Overall, this general line of investigation is underdeveloped in the T6SS field and conceptually appropriate for a broad audience journal. The paper is well-written and, aside from a few cases, well-cited. As detailed below however, there are several aspects of this paper where the evidence provided is somewhat insufficient to support the claims. Further, there are now at least two examples in the literature of non-cognate immunity providing protection against intoxication, one of which is not cited here (Bosch et al PMID 37345922 - the other being Ting et al 2018). In general therefore I think that the motivating concept here in this paper of overturning the predominant model of interbacterial effector-immunity cognate interactions is oversold and should be dialed back.

Strengths:

One of the major strengths of this paper is the combination of diverse techniques including competition assays, biochemistry, and metagenomics surveys. The metagenomic analysis in particular has great potential for understanding T6SS biology in natural communities. Finally, it is clear that much new biology remains to be discovered in the realm of T6SS effectors and immunity.

Weaknesses:

The authors have not formally shown that RdnE is delivered by the T6SS. Is it the case that there are not available genetics tools for gene deletion for the BB2000 strain? If there are genetic tools available, standard assays to demonstrate T6SS-dependency would be to interrogate function via inactivation of the T6SS (e.g. by deleting tssC).

For swarm cross-phyla competition assays (Figure 4), at what level compared to cognate immunity are the non-cognate immunity proteins being expressed? This is unclear from the methods and Figure 4 legend and should be elaborated upon. Presumably these non-cognate immunity proteins are being overexpressed. Expression level and effector-to-immunity protein stoichiometry likely matters for interpretation of function, both in vitro as well as in relevant settings in nature. It is important to assess if native expression levels of non-cognate cross-phyla immunity (e.g. Rothia and Prevotella) protect similarly as the endogenously produced cognate immunity. This experiment could be performed in several ways, for example by deleting the RdnE-I pair and complementing back the Rothia or Prevotella RdnI at the same chromosomal locus, then performing the swarm assay. Alternatively, if there are inducible expression systems available for Proteus, examination of protection under varying levels of immunity induction could be an alternate way to address this question. Western blot analysis comparing cognate to non-cognate immunity protein levels expressed in Proteus could also be important. If the authors were interested in deriving physical binding constants between E and various cognate and non-cognate I (e.g. through isothermal titration calorimetry) that would be a strong set of data to support the claims made. The co-IP data presented in supplemental Figure 6 are nice but are from E. coli cells overexpressing each protein and do not fully address the question of in vivo (in Proteus) native expression.

Lines 321-324, the authors infer differences between E and I in terms of read recruitment (greater abundance of I) to indicate the presence of orphan immunity genes in metagenomic samples (Figure 5A-D). It seems equally or perhaps more likely that there is substantial sequence divergence in E compared to the reference sequence. In fact, metagenomes analyzed were required only to have "half of the bases on reference E-I sequence receiving coverage". Variation in coverage again could reflect divergent sequence dipping below 90% identity cutoff. I recommend performing metagenomic assemblies on these samples to assess and curate the E-I sequences present in each sample and then recalculating coverage based on the exact inferred sequences from each sample.

A description of gene-level read recruitment in the methods section relating to metagenomic analysis is lacking and should be provided.

Reviewer #2 (Public Review):

The work by Bærentsen et al., entitled "Structural basis for regulation of a tripartite toxin-antitoxin system by dual phosphorylation" deals with the structural aspects of the control of the hipBST TA operon, the role of auto-phosphorylation in the activation and neutralisation of the enzyme and the direct effects of HipS and HipB in neutralisation. This is a follow-up to the Vang Nielsen et al., and Gerdes et al., papers from the same authors on this very unique TA module, that brings forth a thorough and well written dissection of an unusually complex regulatory system.

This is a much improved manuscript, the paper is more focused and the message is now clear.

Reviewer #2 (Public Review):

Tian et al. perform a meta-analysis of 113 genome-wide origin profile datasets in humans to assess the reproducibility of experimental techniques and shared genomics features of origins. Techniques to map DNA replication sites have quickly evolved over the last decade, yet little is known about how these methods fare against each other (pros and cons), nor how consistent their maps are. The authors show that high-confidence origins recapitulate several known features of origins (e.g., correspondence with open chromatin, overlap with transcriptional promoters, CTCF binding sites). However, surprisingly, they find little overlap between ORC/MCM binding sites and origin locations.

Overall, this meta-analysis provides the field with a good assessment of the current state of experimental techniques and their reproducibility, but I am worried about: (a) whether we've learned any new biology from this analysis; (b) how binding sites and origin locations can be so mismatched, in light of numerous studies that suggest otherwise; and (c) some methodological details described below.

Major comments:

-- Line 26: "0.27% were reproducibly detected by four techniques" -- what does this mean? Does the fragment need to be detected by ALL FOUR techniques to be deemed reproducible? And what if the technique detected the fragment is only 1 of N experiments conducted; does that count as "detected"? Later in Methods, the authors (line 512) say, "shared origins ... occur in sufficient number of samples" but what does *sufficient* mean? Then on line 522, they use a threshold of "20" samples, which seems arbitrary to me. How are these parameters set, and how robust are the conclusions to these settings? An alternative to setting these (arbitrary) thresholds and discretizing the data is to analyze the data continuously; i.e., associate with each fragment a continuous confidence score.

-- Line 20: "50,000 origins" vs "7.5M 300bp chromosomal fragments" -- how do these two numbers relate? How many 300bp fragments would be expected given that there are ~50,000 origins? (i.e., how many fragments are there per origin, on average)? This is an important number to report because it gives some sense of how many of these fragments are likely nonsense/noise. The authors might consider eliminating those fragments significantly above the expected number, since their inclusion may muddle biological interpretation.

-- Line 143: I'm not terribly convinced by the PCA clustering analysis, since the variance explained by the first 2 PCs is only ~25%. A more robust analysis of whether origins cluster by cell type, year etc is to simply compute the distribution of pairwise correlations of origin profiles within the same group (cell type, year) vs the correlation distribution between groups. Relatedly, the authors should explain what an "origin profile" is (line 141). Is the matrix (to which PCA is applied) of size 7.5M x 113, with a "1" in the (i,j) position if the ith fragment was detected in the jth dataset?

-- It's not clear to me what new biology (genomic features) has been learned from this meta-analysis. All the major genomic features analyzed have already been found to be associated with origin sites. For example, the correspondence with TSS has been reported before:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320713/<br /> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547456/

So what new biology has been discovered from this meta-analysis?

-- Line 250: The most surprising finding is that there is little overlap between ORC/MCM binding sites and origin locations. The authors speculate that the overlap between ORC1 and ORC2 could be low because they come from different cell types. Equally concerning is the lack of overlap with MCM. If true, these are potentially major discoveries that butts heads with numerous other studies that have suggested otherwise. More needs to be done to convince the reader that such a mis-match is true. Some ideas are below:

Idea 1) One explanation given is that the ORC1 and ORC2 data come from different cell types. But there must be a dataset where both are mapped in the same cell type. Can the authors check the overlap here? In Fig S4A, I would expect the circles to not only strongly overlap but to also be of roughly the same size, since both ORC's are required in the complex. So something seems off here.

Idea 2) Another explanation given is that origins fire stochastically. One way to quantify the role of stochasticity is to quantify the overlap of origin locations performed by the same lab, in the same year, in the same experiment, in the same cell type -- i.e., across replicates -- and then compute the overlap of mapped origins. This would quantify how much mis-match is truly due to stochasticity, and how much may be due to other factors.

Idea 3) A third explanation is that MCMs are loaded further from origin sites in human than in yeast. Is there any evidence of this? How far away does the evidence suggest, and what if this distance is used to define proximity?

Idea 4) How many individual datasets (i.e., those collected and published together) also demonstrate the feature that ORC/MCM binding locations do not correlate with origins? If there are few, then indeed, the integrative analysis performed here is consistent. But if there are many, then why would individual datasets reveal one thing, but integrative analysis reveal something else?

Idea 5) What if you were much more restrictive when defining "high-confidence" origins / binding sites. Does the overlap between origins and binding sites go up with increasing restriction?

Overall, I have the sense that these experimental techniques may be producing a lot of junk. If true, this would be useful for the field to know! But if not, and there are indeed "unexplored mechanisms of origin specification" that would be exciting. But I'm not convinced yet.

-- It would be nice in the Discussion for the authors to comment about the trade-offs of different techniques; what are their pros and cons, which should be used when, which should be avoided altogether, and why? This would be a valuable prescription for the field.

Reviewer #2 (Public Review):

Summary:<br /> The authors provide new evidence for motion-induced sound camouflage and can link the hunting approach to hunting success (detailing the adaptation and inferring a fitness consequence).

Strengths:<br /> Strong evidence by combining high-resolution accelerometer data with a ground-truthed data set on prey provisioning at nest boxes. A good set of co-variates to control for some of the noise in the data provides some additional insights into owl hunting attempts.

Weaknesses:<br /> There is a disconnect between the hypotheses tested and the results presented, and insufficient detail is provided on the statistical approach. R2 values of the presented models are very small compared to the significance of the effect presented. Without more detail, it is impossible to assess the strength of the evidence. The authors seem to overcome persisting challenges associated with the validation and calibration of accelerometer data by ground-truthing on-board measures with direct observations in captivity, but here the methods are not described any further and sample sizes (2 owls - how many different loggers were deployed?) might be too small to achieve robust behavioural classifications.

Reviewer #2 (Public Review):

This is a well-written manuscript about a strong comparative study of diversity of facial movements in three macaque species to test arguments about social complexity influencing communicative complexity.

Reviewer #2 (Public Review):

The ATPase protein machine cohesin shapes the genome by loop extrusion and holds sister chromatids together by topological entrapment. When executing these functions, cohesin is tightly regulated by multiple cofactors, such as Scc2/Nipbl, Pds5, Wapl, and Eco1/Esco1/2, and it undergoes dynamic conformational changes with ATP binding and hydrolysis. The mechanisms by which cohesin extrudes DNA loops and medicates siter-chromatid cohesion are still not understood. A major reason for the lack of understanding of cohesin dynamics and regulation is the failure to capture the structures of intact cohesin in different nucleotide-bound states and in complex with various regulators. So far only the ATP state cohesin bound to NIPBL and DNA have been experimentally determined.

In this manuscript, Nasmyth et al. made use of the powerful protein structure prediction tool, AlphaFold2 (AF), to predict the models of tens of cohesin subcomplexes from different species. The results provide important insight into how the Smc3-Scc1 DNA exiting gate is opened, how Pds5 and Wapl maintain the opened gate, how Pds5 and Scc3/SA recruit different cofactors, how Eco1 and Sororin antagonize Wapl, and how Scc2/Nipbl interacts with Scc3/SA. The models are for the most part consistent with published mutations in these proteins that affect cohesin's functions in vitro and in vivo and raise testable hypotheses of cohesin dynamics and regulation. This study also serves as an example of how to use AF to build models of protein complexes that involve the docking of flexible regions to globular domains.

Major points

(1)The revised manuscript is still too long and would be difficult for readers to read. While the authors have made some efforts to streamline their presentations and remove excessive speculations and models of minor importance, the changes are not enough.<br /> (2) AF has been accurate in predicting both the fold and sidechain conformations of globular domains. It is less accurate in predicting structural regions with conformational flexibility. Comparisons of predicted and determined structures of large protein complexes have shown considerable differences, particularly with respect to regions lacking tertiary fold. The authors should be more cautious in interpreting some of their models, particularly when the predicted models are inconsistent with determined structures and published biochemical data. For example, human WAPL-C in isolation does not interact with the SA-SCC1 complex while the N-terminal region of WAPL does.<br /> (3) The predicted SA/Scc3-Pds5-Scc1-WaplC quaternary complex is fascinating. Can the authors provide some experimental evidence to support the formation of this quaternary complex or at least the formation of the SA/Scc3-Pds5-WaplC ternary complex? In vitro pulldown or gel filtration can be used to test their predictions. The authors have decided not the test their models experimentally.

Reviewer #2 (Public Review):

The present study by Ye et al. characterizes some of the major effects of ferroptotic stress on tooth morphogenesis.

The strengths of this study are its innovative nature and beautiful histology. Mechanistic data are convincing Overall, the study is well done.

Reviewer #2 (Public Review):

In this manuscript, Touray et al investigate the mechanisms by which PIP5Pase and RAP1 control VSG expression in T. brucei and demonstrate an important role for this enzyme in a signalling pathway that likely plays a role in antigenic variation in T. brucei. While these data do not definitively show a role for this pathway in antigenic variation, the data are critical for establishing this pathway as a potential way the parasite could control antigenic variation and thus represent a fundamental discovery.

The methods used in the study are generally well-controlled. The authors provide evidence that RAP1 binds to PI(3,4,5)P3 through its N-terminus and that this binding regulates RAP1 binding to VSG expression sites, which in turn regulates VSG silencing. Overall their results support the conclusions made in the manuscript. Readers should take into consideration that the epitope tags on RAP1 could alter its function, however.

There are a few small caveats that are worth noting. First, the analysis of VSG derepression and switching in Figure 1 relies on a genome which does not contain minichromosomal (MC) VSG sequences. This means that MC VSGs could theoretically be mis-assigned as coming from another genomic location in the absence of an MC reference. As the origin of the VSGs in these clones isn't a major point in the paper, I do not think this is a major concern, but I would not over-interpret the particular details of switching outcomes in these experiments.

Another aspect of this work that is perhaps important, but not discussed much by the authors, is the fact that signalling is extremely poorly understood in T. brucei. In Figure 1B, the RNA-seq data show many genes upregulated after expression of the Mut PIP5Pase (not just VSGs). The authors rightly avoid claiming that this pathway is exclusive to VSGs, but I wonder if these data could provide insight into the other biological processes that might be controlled by this signaling pathway in T. brucei.

Overall, this is an excellent study which represents an important step forward in understanding how antigenic variation is controlled in T. brucei. The possibility that this process could be controlled via a signalling pathway has been speculated for a long time, and this study provides the first mechanistic evidence for that possibility.

Reviewer #2 (Public Review):

This paper aims at establishing the role of WRN-interacting protein 1 (WRNIP1) and its UBZ domain (an N-terminal ubiquitin-binding zinc finger domain) on genome instability caused by mild inhibition of DNA synthesis by aphidicolin. The authors used human MRC5 fibroblasts investigated with standard methods in the field. The results clearly showed that WRNIP1 silencing and UBZ-mutation (D37A) increased DNA damage, chromosome aberrations, and transcription-replication conflicts caused by aphidicolin.

The conclusions of the paper are overall well supported by results, however, aspects of some data analyses would need to be clarified and/or extended.

1 The methods (immunofluorescence microscopy and dot-blots) to determine R-loop levels can lack sensitivity and specificity. In particular, since the S9.6 antibody can bind to other structures besides heteroduplex, dot-blot analyses only grossly assess R-loop levels in cellular samples of purified nucleic acids, which are constituted by many different types of DNA/RNA structures.

2 Experimental plan has analyzed the impact of WRNIP1 lack or mutations at steady-state conditions. Thus, the possible role of WRNIP1 at an early step of the mechanism would require some sort of kinetics analysis of the molecular process, therefore not at steady-state conditions. The findings of a co-localization of R-loops and WRNIP1 have been obtained with the S9.6 antibody, which recognizes DNA-RNA heteroduplexes. Since WRNIP1 is known to be recruited at stalled forks and DNA cleavage sites, it is not surprising that WRNIP1 is very close to heteroduplexes, abundant structures at replication forks and cleavage sites. Similar interpretations may also be valid for Rad51/S9.6 co-localization findings.

3 Determination of DNA damage, chromosome aberration, and co-localization data are reported as means of measurements with appropriate statistics. However, the fold-change values relative to corresponding untreated samples are not reported. In some instances, it seems that WRNIP1 silencing or mutations actually reduce or do not affect aphidicolin effects. That leaves open the interpretation of specific results.

Reviewer #2 (Public Review):

Summary:

Spikol et al performed a technical tour de force by combining numerous novel tools and approaches to investigate for the first time the connectivity and motor functions of nucleus incertus subset of neurons genetically defined by the expression of specific markers in the larval zebrafish brain.

Strengths:

By using expression of the specific markers relaxin 3 and gsc2, the authors generated novel knock-in transgenic lines enabling them to investigate the connectivity, recruitment and roles of these neurons in locomotion. Their work should enable numerous subsequent studies in zebrafish & inspire new paths of investigations in other animal models.

Weaknesses:

More precision is required for the anatomical data and further analysis is needed to describe the recruitment and role in spontaneous exploration of the rln3- and gsc2- expressing neurons.

Reviewer #2 (Public Review):

Peterson et al., perform a series of behavioral experiments to study the repertoire and variance of Mongolian gerbil vocalizations across social groups (families). A key strength of the study is the use of a behavioral paradigm which allows for long term audio recordings under naturalistic conditions. This experimental set-up results in the identification of additional vocalization types. In combination with state of the art methods for vocalization analysis, the authors demonstrate that the distribution of sound types and the transitions between these sound types across three gerbil families is different. This is a highly compelling finding which suggests that individual families may develop distinct vocal repertoires. One potential limitation of the study lies in the cluster analysis used for identifying distinct vocalization types. The authors use a Gaussian Mixed Model (GMM) trained on variational auto Encoder derived latent representation of vocalizations to classify recorded sounds into clusters. Through the analysis the authors identify 70 distinct clusters and demonstrate a differential usage of these sound clusters across families. While the authors acknowledge the inherent challenges in cluster analysis and provide additional analyses (i.e. maximum mean discrepancy, MMD), additional analysis would increase the strength of the conclusions. In particular, analysis with different cluster sizes would be valuable. An additional limitation of the study is that due to the methodology that is used, the authors can not provide any information about the bioacoustic features that contribute to differences in sound types across families which limits interpretations about how the animals may perceive and react to these sounds in an ethologically relevant manner.

The conclusions of this paper are well supported by data, but certain parts of the data analysis should be expanded and more fully explained.

• Can the authors comment on the potential biological significance of the 70 sound clusters? Does each cluster represent a single sound type? How many vocal clusters can be attributed to a single individual? Similarly, can the authors comment on the intra-individual and inter-individual variability of the sound types within and across families?<br /> • As a main conclusion of the paper rests on the different distribution of sound clusters across families, it is important to validate the robustness of these differences across different cluster parameters. Specifically, the authors state that "we selected 70 clusters as the most parsimonious fit". Could the authors provide more details about how this was fit? Specifically, could the authors expand upon what is meant by "prior domain knowledge about the number of vocal types...". If the authors chose a range of cluster values (i.e. 10, 30, 50, 90) does the significance of the results still hold?<br /> • While VAEs are powerful tools for analyzing complex datasets in this case they are restricted to analysis of spectrogram images. Have the authors identified any acoustic differences (i.e. in pitch, frequency, and other sound components) across families?

Reviewer #2 (Public Review):

In this work the IGluSnFR3 sensor, recently developed by Marvin et al (2023) is mutated position S72, which was previously reported to switch the specificity from Glu to Asp. They made 3 mutations at this position, selected a S72P mutant, then made a second mutation at S27 to generate an Asp-specific version of the sensor. This was then characterized thoroughly and used on some test experiments, where it was shown to detect and allow visualization of aspartate concentration changes over time. It is an incremental advance on the iGluSnFR3 study, where 2 predictable mutations are used to generate a sensor that works on a close analog of Glu, Asp. It is shown to have utility and will be useful in the field of Asp-mediated biological effects.

Reviewer #2 (Public Review):

Making state-of-the-art (super-resolution) microscopy widely available has been the subject of many publications in recent years as correctly referenced in the manuscript. By advocating the ideas of open-microscopy and trying to replace expensive, scientific-grade components such as lasers, cameras, objectives, and stages with cost-effective alternatives, interested researchers nowadays have a number of different frameworks to choose from. In the iteration of the theme presented here, the authors used the existing modular UC2 framework, which consists of 3D printable building blocks, and combined a cheapish laser, detector and x,y,(z) stage with expensive filters/dichroics and a very expensive high-end objective (>15k Euros). This particular choice raises a first technical question, to which extent a standard NA 1.3 oil immersion objective available for <1k would compare to the chosen NA 1.49 one.

The choice of using the UC2 framework has the advantage, that the individual building blocks can be 3D printed, although it should be mentioned that the authors used injection-molded blocks that will have a limited availability if not offered commercially by a third party. The strength of the manuscript is the tight integration of the hardware and the software (namely the implementations of imSwitch as a GUI to control data acquisition, OS SMLM algorithms for fast sub-pixel localisation and access to Napari).

The presented experimental data is convincing, demonstrating (1) extended live cell imaging both using bright-field and fluorescence in the incubator, (2) single-particle tracking of quantum dots, and (3) and STORM measurements in cells stained against tubulin.

In the following I will raise two aspects that currently limit the clarity and the potential impact of the manuscript.

First, the manuscript would benefit from further refinement. Elements in Figure 1d/e are not described properly. Figure 2c is not described in the caption. GPI-GFP is not introduced. MMS (moment scaling spectrum) could benefit from a one sentence description of what it actually is. In Figure 6, the size of the STORM and wide-field field of views are vastly different, the distances between the peaks on the tubuili are given in micrometers rather than nanometers. (more in the section on recommendations for the author)

Second, and this is the main criticism at this point, is that although all the information and data is openly available, it seems very difficult to actually build the setup due to a lack of proper documentation (as of early July 2023).<br /> 1. The bill of materials (https://github.com/openUC2/UC2-STORM-and-Fluorescence#bill-of-material) should provide a link to the commercially available items. Some items are named in German. Maybe split the BoM in commercially available and 3D printable parts (I first missed the option to scroll horizontally).<br /> 2. The links to the XY and Z stage refer to the general overview site of the UC2 project (https://github.com/openUC2/) requiring a deep dive to find the actual information.<br /> 3. Detailed building instructions are unfortunately missing. How to assemble the cubes (pCad files showing exploded views, for example)? Trouble shooting?<br /> 4. Some of the hardware details (e.g. which laser was being used, lenses, etc) should be mentioned in the manuscript (or SI)

I fully understand that providing such level of detail is very time consuming, but I hope that the authors will be able to address these shortcomings.

Reviewer #2 (Public Review):

This study compares the activity of neural populations in the primary and non-primary auditory cortex of ferrets while the animals actively behaved or passively listened to a sound discrimination task. Using a variety of methods, the authors convincingly show differential effects of task engagement on population neural activity in primary vs non-primary auditory cortex; notably that in the primary auditory cortex, task-engagement (1) improves discriminability for both task-relevant and non-task relevant dimensions, and (2) improves the alignment between covariability and sound discrimination axes; whereas in the non-primary auditory cortex, task-engagement (1) improves discriminability for only task-relevant dimensions, and (2) does not affect the alignment between covariability and sound discrimination axes. They additionally show that task-engagement changes in gain can account for the selectivity noted in the discriminability of non-primary auditory neurons. They also admirably attempt to isolate task-engagement from arousal fluctuations, by using fluctuations in pupil size as a proxy for physiological arousal. This is a well-carried out study with thoughtful analyses which in large part achieves its aims to evaluate how task-engagement changes neural activity across multiple auditory regions. As with all work, there are several caveats or areas for future study/analysis. First, the sounds used here (tones, and narrow-band noise) are relatively simple sounds; previous work suggests that exactly what activity is observed within each region (e.g., sensory only, decision-related, etc) may depend in part upon what stimuli are used. Therefore, while the current study adds importantly to the literature, future work may consider the use of more varied stimuli. Second, the animals here were engaged in a behavioral task; but apart from an initial calculation of behavioral d', the task performance (and its effect on neural activity) is largely unaddressed.

Reviewer #2 (Public Review):

Summary:<br /> Through a series of four experiments, Yuan, Wang and Jiang examined pupil size responses to emotion signals in point-light motion stimuli. Experiment 1 examined upright happy, sad and neutral point-light biological motion (BM) walkers. The happy BM induced a significantly larger pupil response than the neutral, whereas the sad BM evoked a significantly smaller pupil size than the neutral BM. Experiment 2 examined inverted BM walkers. Experiment 3 examined BM stimuli with acceleration removed. No significant effects of emotion were found in neither Experiment 2 nor Experiment 3. Experiment 4 examined scrambled BM stimuli, in which local motion features were preserved while the global configuration was disrupted. Interestingly, the scrambled happy and sad BM led to significantly greater pupil size than the scrambled neutral BM at a relatively early time, while no significant difference between the scrambled happy and sad BM was found. Thus, the authors argue that these results suggest multi-level processing of emotions in life motion signals.

Strengths:<br /> The experiments were carefully designed and well-executed, with point-light stimuli that eliminate many potential confounding effects of low-level visual features such as luminance, contrast, and spatial frequency.

Weaknesses:<br /> Correlation results with limited sample size should be interpreted with extra caution.

It would be helpful to add discussions as a context to compare the current results with pupil size reactions to emotion signals in picture stimuli.

Overall, I think this is a well-written paper with solid experimental results that support the claim of the authors, i.e., the human visual system may process emotional information in biological motion at multiple levels. Given the key role of emotion processing in normal social cognition, the results will be of interest not only to basic scientists who study visual perception, but also to clinical researchers who work with patients of social cognitive disorders. In addition, this paper suggests that examining pupil size responses could be a very useful methodological tool to study brain mechanisms underlying emotion processing.

Reviewer #2 (Public Review):

This short manuscript by Zhu et al. describes an investigation into the role of gamma protocadherins in synaptic connectivity in the mouse cerebral cortex. First, the authors conduct a single-cell RNA-seq survey of postnatal day 11 mouse cortical neurons, using an adapted 10X Genomics method to capture the 5' sequences that are necessary to identify individual gamma protocadherin isoforms (all 22 transcripts share the same three 3' "constant" exons, so standard 3'-biased methods can't distinguish them). This method adaptation is an advance for examining individual gamma transcripts, and it is helpful to publish the method, the characterization of which is improved in this revised manuscript. The results largely confirm what was known from other approaches, which is that a few of the 19 A and B subtype gamma protocadherins are expressed in an apparently stochastic and combinatorial fashion in each cortical neuron, while the 3 C subtype genes are expressed ubiquitously. Second, using elegant paired electrophysiological recordings, the authors show that in gamma protocadherin cortical slices, the likelihood of two neurons on layers 2/3 being synaptically connected is increased. That suggests that gamma protocadherins generally inhibit synaptic connectivity in the cortex; again, this has been reported previously using morphological assays, but it is important to see it confirmed here with physiology. Finally, the authors use an impressive sequential in utero electroporation method to provide evidence that the degree of isoform matching between two neurons negatively regulates their reciprocal synaptic connectivity. These are difficult experiments to do, and while some caveats remain, the main result is consistent. Strengths include the impressive methodology and improved demonstration of the previously-reported finding that gamma protocadherins work via homophilic matching to put a brake on synapse formation in the cortex. Weaknesses include the writing, which even in the revision fails to completely put the new results in context with prior work, which together has largely shown similar results; a still-incomplete characterization of a new alpha protocadherin KO mouse (a minor point but it should still be addressed); and a lack of demonstration of protein levels in electroporated brains. Because of the unique organization and expression pattern of the gamma protocadherins, it is unlikely that these results will be directly applicable to the broader understanding of the role of cell adhesion molecules in synapse development. However, the methodology, which is now better described, should be applicable more broadly and the improved demonstration of the role of gamma protocadherin's negative role in cortical synaptogenesis is helpful.

Reviewer #2 (Public Review):

Summary:<br /> In this work, Liang et al. investigate whether an abstract social space is neurally represented by a grid-like code. They trained participants to 'navigate' around a two-dimensional space of social agents characterized by the traits of warmth and competence, then measured neural activity as participants imagined navigating through this space. The primary neural analysis consisted of three procedures: 1) identifying brain regions exhibiting the hexagonal modulation characteristic of a grid-like code, 2) estimating the orientation of each region's grid, and 3) testing whether the strength of the univariate neural signal increases when a participant is navigating in a direction aligned with the grid, compared to a direction that is misaligned with the grid.<br /> From these analyses, the authors find the clearest evidence of a grid-like code in the prefrontal cortex and weaker evidence in the entorhinal cortex.

Strengths:<br /> The work demonstrates the existence of a grid-like neural code for a socially-relevant task, providing evidence that such coding schemes may be relevant for a variety of two-dimensional task spaces.

Weaknesses:<br /> In various parts of this manuscript, the authors appear to use a variety of terms to refer to the (ostensibly) same neural regions: prefrontal cortex, frontal pole, ventromedial prefrontal cortex (vmPFC), and orbitofrontal cortex (OFC). It would be useful for the authors to use more consistent terminology to avoid confusing readers.

Claims about a grid code in the entorhinal cortex are not well-supported by the analyses presented. The whole-brain analysis does not suggest that the entorhinal cortex exhibits hexagonal modulation; the strength of the entorhinal BOLD signal does not track the putative alignment of the grid code there; multivariate analyses do not reveal any evidence of a grid-like representational geometry.

On a conceptual level, it is not entirely clear how this work advances our understanding of grid-like encoding of two-dimensional abstract spaces, or of social cognition. The study design borrows heavily from Constantinescu et al. 2016, which is itself not an inherent weakness, but the Constantinescu et al. study already suggests that grid codes are likely to underlie two-dimensional spaces, no matter how abstract or arbitrary. If there were a hypothesis that there is something unique about how grid codes operate in the social domain, that would help motivate the search for social grid codes specifically, but no such theory is provided. The authors do note that warmth and competence likely have ecological importance as social traits, but other past studies have used slightly different social dimensions without any apparent loss of generality (e.g., Park et al. 2021). There are some (seemingly) exploratory analyses examining how individual difference measures like social anxiety and avoidance might affect the brain and behavior in this study, but a strong theoretical basis for examining these particular measures is lacking.

I found it difficult to understand the analyses examining whether behavior (i.e., reaction times) and individual difference measures (i.e., social anxiety and avoidance) can be predicted by the hexagonal modulation strength in some region X, conditional on region X having a similar estimated grid alignment with some other region Y. It is possible that I have misunderstood the authors' logic and/or methodology, but I do not feel comfortable commenting on the correctness or implications of this approach given the information provided in the current version of this manuscript.

It was puzzling to see passing references to multivariate analyses using representational similarity analysis (RSA) in the main text, given that RSA is only used in analyses presented in the supplementary material.

References:<br /> Constantinescu, A. O., O'Reilly, J. X., & Behrens, T. E. (2016). Organizing conceptual knowledge in humans with a gridlike code. Science, 352(6292), 1464-1468.

Park, S. A., Miller, D. S., & Boorman, E. D. (2021). Inferences on a multidimensional social hierarchy use a grid-like code. Nature Neuroscience, 24(9), 1292-1301.

Reviewer #2 (Public Review):

This manuscript by Xue et al. describes the effects of a long noncoding RNA, lncDACH1, on the localization of Nav channel expression, the magnitude of INa, and arrhythmia susceptibility in the mouse heart. Because lncDACH1 was previously reported to bind and disrupt membrane expression of dystrophin, which in turn is required for proper Nav1.5 localization, much of the findings are inferred through the lens of dystrophin alterations.

The results report that cardiomyocyte-specific transgenic overexpression of lncDACH1 reduces INa in isolated cardiomyocytes; measurements in whole heart show a corresponding reduction in conduction velocity and enhanced susceptibility to arrhythmia. The effect on INa was confirmed in isolated WT mouse cardiomyocytes infected with a lncDACH1 adenoviral construct. Importantly, reducing lncDACH1 expression via either a cardiomyocyte-specific knockout or using shRNA had the opposite effect: INa was increased in isolated cells, as was conduction velocity in heart. Experiments were also conducted with a fragment of lnDACH1 identified by its conservation with other mammalian species. Overexpression of this fragment resulted in reduced INa and greater proarrhythmic behavior. Alteration of expression was confirmed by qPCR.

The mechanism by which lnDACH1 exerts its effects on INa was explored by measuring protein levels from cell fractions and immunofluorescence localization in cells. In general, overexpression was reported to reduce Nav1.5 and dystrophin levels and knockout or knockdown increased them.

Reviewer #2 (Public Review):

This manuscript illustrates the power of "combined" research, incorporating a range of tools, both old and new to answer a question. This thorough approach identifies a novel target in a well-established signalling pathway and characterises a new player in Drosophila CNS development.

Largely, the experiments are carried out with precision, meeting the aims of the project, and setting new targets for future research in the field. It was particularly refreshing to see the use of multi-omics data integration and Targeted DamID (TaDa) findings to triage scRNA-seq data. Some of the TaDa methodology was unorthodox (and should be justifed/caveats mentioned in the main text), however, this does not affect the main finding of the study.

Their discovery of Spar as a neuropeptide precursor downstream of Alk is novel, as well as its ability to regulate activity and circadian clock function in the fly. Spar was just one of the downstream factors identified from this study, therefore, the potential impact goes beyond this one Alk downstream effector.

Reviewer #2 (Public Review):

The manuscript by Kaneko set out to understand the mechanisms underlying cell proliferation in hepatocytes lacking Shp2 signals. To do this, the authors focused on CD133 as the proliferating clusters of cells in the Shp2 knockout (SKO) livers are CD133 expressing. After excluding the contribution of progenitors that are CD133 to this cell population, the authors focused on the intrinsic regulation of CD133 by Met/Shp2 regulated Ras/Erk parthway and showed upregulation of CD133 to be a compensatory signal to overcome loss of Ras/Erk signal and suggested Wnt10a in the regulation of CD133 signal. The study then focused on the observed filament localization of CD133 in the CD133+ cluster of cells. The study went on to identify the CD133+ vesicles that contain primarily mRNA vs. microRNA like other EVs. Specifically, the authors identified several mRNA species that encode IEGs, indicating a potential role for these CD133+ vesicles in cell proliferation signal transmission to neighboring cells via delivery of the IEG mRNAs as cargos. Finally, they showed that the induction of CD133 (and by derivative, the CD133+ vesicles) are necessary for maintaining cell proliferation in the cell cluster with high proliferation capacities in the SKO livers; and in intestinal crypt organoids treated with Met inhibitors to block Ras/ERk signal. In the revised manuscript, the authors more definitively identified the CD133+ vesicles. The authors also provided additional experimental evidence demonstrating the role of these CD133+ vesicles in cell-cell communication. The functional significance of CD133 on this cell-cell communication was further demonstrated with genetic knockout studies.

Reviewer #2 (Public Review):

Summary:

The preprint by Pang, Deluca, et al. investigates the molecular events occurring during germline stem cell (GSC) differentiation into an oocyte. The study highlights several critical observations:

1. Gene Expression and Chromatin State: GSCs exhibit an open chromatin state and express a large number of genes. However, during differentiation, the number of genes expressed decreases.<br /> 2. Gene Clustering and Chromatin Domains: Genes promoting GSC fate are found in clusters close to centric heterochromatin domains.<br /> 3. Epigenetic Marks: The transition from GSC fate to oocyte/nurse cell fate is marked by an increase in H3K27me3 and H3K9me3 on regions, including centric heterochromatin.<br /> 4. Metabolic Rewiring: Genes related to metabolism undergo changes during this fate transition, indicating metabolic rewiring.

Strengths:<br /> The conclusions are strongly supported by a substantial amount of data. Multiple complementary methods are employed, such as increased H3K9me3 heterochromatin and reporter assays, to validate the increase of H3K9me3 during meiosis.

The wealth of data presented will be valuable to the scientific community, providing further insights into critical molecular events during GSC differentiation.

The study uncovers new biology, notably the proximity of stem cell genes to centric heterochromatin and its regulation.

Key observations include the low H3K9me3 levels on transposons in GSCs, which warrant further investigation.

Weaknesses:<br /> To make the paper more accessible to a broader audience, the authors can use fewer jargon terms. In particular, the abbreviations used for staging can be confusing.

Some sections in the results contain extensive discussion that may be better suited for the discussion section. For example, see page 9.

Reviewer #2 (Public Review):

Please note that I am not a structural biologist and cannot critically evaluate the details of figures 1 to 3; my review focuses on the cell biology experiments in figures 4 and 5.

Paine and colleagues investigated structural requirements for the interaction between the ESCRT-III subunit IST1 and the protease CAPN7. This is a continuation of previous work by the same group (Wenzel et al., eLife 2022), which showed that Capn7 is recruited to the midbody by Ist1 and that Capn7 promotes both normal abscission and NoCut abscission checkpoint function. In this article, the structural determinants of the Ist1-Capn7 interaction are characterised in more detail, focusing on the structure of Capn7 MIT domains and their binding to Ist1. Notably, point mutations in Capn7 MIT domains known to mediate binding to Ist1 and midbody recruitment are shown here to be required for abscission functions, as expected from the authors' previous paper. Furthermore, the report shows that a Capn7 point mutant lacking proteolytic activity behaves as a loss-of-function in abscission assays, despite showing normal midbody localisation. These are important results that will help in future studies to understand how the Capn7 protease regulates abscission mechanistically.

The report is clearly written and the results support the main conclusions. Some technical limitations and alternative interpretations of the data should be discussed in the text, as outlined below.

1. It is not always clearly stated how the results presented in this report relate to those in the Wenzel paper. For example, the finding that Ist1 recruits Capn7 to midbodies (p. 6 and figure 4) was first shown in the Wenzel paper. The novelty here is not that Capn7 MIT mutants fail to localise to midbodies, but that they phenocopy the previously described knockdown of Capn7, failing to support normal abscission and NoCut function (fig. 5). This supports and extends the findings of Wenzel et al. It is important to make this explicit and explain the conceptual advances shown here more clearly.<br /> 2. The NoCut checkpoint can be triggered by chromatin bridges, DNA replication stress, and nuclear basket defects, but only basket defects are tested here. Therefore, it is not clear if NoCut is still functional in Capn7-defective cells after replication stress and/or with chromatin bridges. Ideally, this should be tested experimentally, or alternatively discussed in the text, especially since the molecular details of how NoCut is engaged under different conditions remain unclear. For example, "abscission checkpoint bodies" proposed to control abscission timing form in response to nuclear basket defects and aphidicolin treatment, but not in the presence of chromatin bridges (Strohacker et al., eLife 2021).<br /> 3. The current data suggest that Capn7 is a regulator of abscission timing, but in my opinion do not quite establish this, for two main reasons. First, abscission timing is not directly measured in this study. Time-lapse imaging would be required to rule out alternative interpretations of the data in figure 5. For example, a delay in an earlier cell cycle stage could in principle lead to a decrease in the overall fraction of midbody-stage cells. Second, the absence of the midbody is not necessarily a marker of complete abscission. Indeed, midbody disassembly is associated with the completion of abscission in unchallenged HeLa cells, but not in cells with chromatin bridges (Steigemann et al, Cell 2009). Midbodies remain a useful marker for pre-abscission cells, but the absence of midbodies should not be immediately interpreted as completion of abscission without further assays. Formally, a direct measurement of abscission timing would require imaging of the plasma membrane, for example using time-lapse phase-contrast microscopy (Fremont et al., 2016 Nat Comm). These limitations should be mentioned in the text.<br /> 4. IST1 plays a role in nuclear envelope sealing by recruiting the co-factor Spastin (Vietri et al., Nature 2015), a known IST1 co-factor also confirmed in the previous interactome screen (Wenzel et al. 2022). CAPN7 could have a role in maintaining nuclear integrity upon the KD of Nup153 and Nup50 (Mackay et al. 2010) instead of/in addition to its proposed role in delaying abscission as part of the NoCut checkpoint at the midbody. I don't think the authors can differentiate between these two possibilities, and it would be interesting to consider their possible implications on how the "NoCut" checkpoint is triggered.<br /> 5. Figure 5 should include images of representative cells, highlighting midbody-positive and multinucleated cells. Without images, it is not possible to evaluate the quality of these data.

Reviewer #2 (Public Review):

The cortical hem is one of the main signaling centers in the vertebrate forebrain, regulating neurogenesis of the medial pallium and the generation of Cajal-Retzius neurons. The authors examine how this signaling center is formed and functions. Previously, transcription factors playing instructive roles in the development of the cortical hem have been identified, but a master regulator had not been found so far. The authors build on their previous work studying the transcription factor Lmx1a which is one of the earliest and most specific cortical hem markers.

By combining loss- and gain-of-function studies, RNA sequencing, histology, and analysis of downstream factors, the authors rigorously show Lmx1a is required for the expression of signaling molecules in the hem, the proliferation and functionality of dentate gyrus neurons, the cell cycle exit and differentiation (and also migration) of cajal-retzius cells and this by activating different downstream regulators.

They use golden standard experiments in the field such as BrdU-Ki67 cell-cycle exit measurements, RNA sequencing, and patch clamping; combined with state-of-the-art techniques such as RNAscope and laser capture microdissection. These convincingly show that Lmx1a regulates the proliferation of dentate gyrus progenitor cells and a malformation of the transhilar scaffold. The authors also claim a migration deficit for dentate gyrus progenitors, but they do not consider apoptosis or show direct evidence for migration abnormalities.<br /> In the hem, the authors report normal proliferation and apoptosis in the Lmx1a mutants, but aberrant cell-cycle-exit, from which the authors conclude a problem in differentiation. However, this could be a cell cycle progression problem too (stuck in a certain cell cycle phase?), as the RNAseq data suggest. The authors should acknowledge this possibility.

The RNAseq dataset provides candidate downstream regulators of the observed phenotypes and the authors test the functionality of Wnt3a, Tbr2, and Cdkn1a, showing they are involved in distinct processes.<br /> Strikingly, Wnt3a is not significantly downregulated in the RNAseq data in the Lmx1a mutant, but quantification of in situ hybridization signal (which is less robust) did reveal a significant difference. Is this a splice variant issue? A timing issue or specificity of the RNAscope probe? The authors should look into this more carefully.

To study the role of Cdkn1a, the authors performed rescue experiments using in utero electroporation, which is a standard in the field. However, they argued before that "CR cell migration and DG morphogenesis are complex processes that require precise expression levels of key genes" when studying downstream factors Wnt3a and Tbr2. Why is this no longer an issue studying Cdkn1a?<br /> To study cell-cycle exit in this model, the authors quantified GFP and Ki67. Since electroporation not only targets the progenitor cells (see e.g. Govindan et al. 2018, Nature protocols), the authors should confirm these results with a BrdU/Ki67 quantification as in previous experiments, or confirm electroporation only targeted progenitor cells in their model.

Lastly, the authors ectopically expressed Lmx1a and convincingly show its ability to generate a hem-like structure. Could the authors elaborate on the necessity for a medial signature? Can the hem be ectopically induced in the lateral pallium?

Reviewer #2 (Public Review):

In the past few years, single-cell transcriptomics analysis has uncovered cellular states associated with disease in experimental models and humans, revealing previously unrecognized disease-associated macrophage states. In particular, a macrophage state characterized by high expression of SPP1 (encoding osteopontin), and by a specific gene expression signature including the expression of TREM2, has been observed in various pathologies and given various names depending on the context e.g. TREM2hi macrophages, lipid-associated macrophages (LAM), disease-associated microglia (DAM), Scar-associated macrophages (SAM), etc... However, a focused investigation and comparison of SPP1+ macrophages across disease contexts were lacking. Here, the authors aimed to systematically analyze SPP1+ macrophages in the context of tissue fibrosis, and integrated single-cell RNA-seq data of >200,000 human macrophages in 6 organs in health and tissue fibrosis.

Beyond confirming the presence of SPP1+ macrophages with a conserved gene expression module (TREM2, CD9, GPNMB, etc...) across tissues and their association with fibrosis, the authors identified a previously unknown cell subset within SPP1+ macrophages, that was enriched for the expression of genes involved in remodeling of the extracellular matrix, which they termed SPP1+ matrisome-associated macrophages (SPP1+MAM+). The authors further used computational tools to compare these SPP1+MAM+ macrophages to previously described SPP1+ macrophage states (LAM, DAM, SAM), investigate the differentiation and activation trajectory of SPP1+MAM+ macrophages, and identify potential transcriptional regulators involved in their differentiation. Finally, the authors show that SPP1+MAM+ macrophages are associated with ageing in both humans and mice.

Overall, the conclusions of the authors are well supported by the data. The authors made excellent use of available computational tools, and the figures are clear and informative. The methods are well-described and appropriately used. In particular, the authors made a nice effort in explaining and justifying some key decisions in their scRNA-seq data analysis workflow, including a data-driven approach to decisions in the clustering analysis.

The author's findings are of broad interest to the fields of tissue inflammation, fibrosis, macrophage biology, and immunology, and their report constitutes a valuable resource, and a basis for further investigations of macrophage differentiation mechanisms in tissue fibrosis, and how macrophages could be targeted to alleviate pathological tissue fibrosis.

Reviewer #2 (Public Review):

In this study, the authors pursue a line of inquiry related to the impacts of cholesterol depletion on macrophage gene expression. The authors find that depletion of cholesterol with either statins or methyl-cyclodextrin induces robust gene expression changes, including changes to JMJD3 expression, an epigenetic regulator.

The authors then seek to dissect the mechanistic determinants of the regulation of JMJD3 in macrophages converging on a metabolic regulation hypothesis that requires mitochondrial activity.

A strength of the paper is the use of multiple macrophage cell models and multiple tools for perturbation to improve the rigor of their conclusions. A weakness of the paper is that it relies heavily on chemical approaches without ever using genetic tools to confirm that their conclusions can be supported using an alternative approach, and when perturbing metabolic pathways as described here, it is difficult to understand how the entire cell state has changed. In fact, the unique focus on JMJD3 without utilizing a control set of genes to show that the impacts of these metabolic perturbations are specific to JMJD3 makes it hard to understand if this is a truly specific pathway for JMJD3 or a general cellular health change.

The authors make an interesting claim in the early part of their manuscript about the potential for statins to regulate the epigenome which they show; however, in the present presentation, it is unclear if this is related to the JMJD3 effect or a separate form of regulation.

This work has the potential to contribute to an improved understanding of the impact of statins on immune function.

Reviewer #2 (Public Review):

For most organs including lung produced by blastocyst complementation, certain cells including the blood vessels are still derived from host tissues, making them unfit for transplantation. To address this issue, Miura et al. explored the origin and the program of whole lung epithelium and mesenchyme, and identified the crucial Foxa2 lineage for lung organogenesis by using lineage tracing mice and human iPSC derived lung differentiation. They found that Foxa2 lineage cells contribute to both lung epithelium and mesenchyme formation, which suggest targeting Fox2 lineage cells could create an empty developmental niche for blastocyst complementation in mice. They further deplete Fgfr2 gene in Foxa2 lineage cells to induce the lung agenesis phenotype in mice, and donor mouse iPSCs injected into Fgfr2 mutant blastocysts occupied the empty niche and formed the missing lung.

Strengths:

To fill our knowledge gap of the origin of all lung cell types, especially pulmonary mesenchyme and endothelium, the authors investigated the lineage hierarchy of specified lung precursors in gastrulating mesendoderm. Using mouse lineage trancing and human iPSC derived lung differentiation, they clarified the msendoderm gene Expression pattern and progression, and compared the contributions of Pdgfra and Foxa2 lineage cells during lung development. They further demonstrate that the defective Foxa2 lineage in critically important for efficient lung complementation, which provide insight for next generation lung transplant therapies.

Weakness:

1. Several lineage tracing experiment lack rigorous quantification, the authors using "partially labels" or "labels a part of" in the text to describe their finding and conclusion, which make the evidence less solid.

2. The ideal lung for transplant should be functional for gas exchange, the lung complementation was only analyzed at E17.5 and E14.5, these two stages were too early to determine the function of the lungs generated via CBC.

3. Immune cells contribute large proportion in the lung, and are critical for lung transplant, the chimerism analysis of immune cells is missing in this study.

Reviewer #2 (Public Review):

In this study, Yang et al. have shown that SIRT2 plays an adverse effect on the heart in response to injury. Further, the groups showed that the deletion of Sirt2 is protective through stabilization and increased nuclear translocation of NRF2, which leads to increased expression of antioxidant genes. They also show that pharmacological inhibition of SIRT2 protects the heart against the development of cardiac hypertrophy. Although this is an interesting finding, at the same time, this study contradicts the previous findings where Xiaoqiang Tang et al. https://doi.org/10.1161/CIRCULATIONAHA.117.028728Circulation. 2017;136:2051-2067) have shown that SITR2 as a cardioprotective deacetylase and Sirt2 knockout is markedly exaggerated cardiac hypertrophy and fibrosis. Although Yang et al. in the discussion part have mentioned that this could be due to mice background. In my opinion, this is not satisfactory. It is important for the scientific community to come to a conclusion about the function of SIRT2 in the heart. The author could use the Global germ-line Sirt2-KO mice (C57BL/6J background; stock no. 012772) TAC model to show the function of SITR2 in cardiac hypertrophy.

The strength of this article is the mechanism where the author showed that SIRT2 function is through the increased NRF2 deacetylation and its degradation and eventual reduction in the levels of antioxidant genes.

Reviewer #2 (Public Review):

The work reports a minor modification in the protocol for Prp formation in vitro. Using this the authors evaluate the role of Syntaxin 6 in modulating prion formation in vitro and the toxicity of the amyloid fibrils in cell culture models. The authors show that while prions/amyloids formed by PrP are non-toxic, mixed aggregates formed by Stx6/PrP are toxic; they claim that this is due to the toxic aggregation intermediates that accumulate more in the presence of Stx6. However, the basis of enhanced toxicity of Stx6/PrP mixed aggregates is not clear and doesn't seem to be physiologically relevant; there is no evidence that Stx6 and PrP forms mixed aggregates in vivo. Which is the toxic component of the Stx6/Prp co-aggregate? Is it the Stx6 component or the Prp component? Additionally, the authors do not have mechanistic explanation for the effect of Stx6 on PrP prion formation

Reviewer #2 (Public Review):

This manuscript describes the structural differences between the prototypical TPC1 channel from Arabidopsis thaliana and its ortholog in Vicia faba that are responsible for divergent channel sensitivity to luminal Ca2+ and in turn make the Vf vacuoles hyperexcitable. It is speculated that these differences might be responsible for species-specific adaptation of these plant species to their environments. Luminal Ca2+ inhibits both of these TPCs (to a different extent) by slowing down the voltage activation and shifting the voltage dependence towards positive potentials. Previously, authors demonstrated that site 3 residues (E605, D606 and D607) in AtTPC1 sense inhibitory luminal Ca2+ that is allosterically coupled to the voltage sensor.

The main strength of the current manuscript is that authors cloned the VfTPC1 channel, and then using a combination of mutagenesis and electrophysiology to demonstrate that only two of the above three residues at site 3 (that are acidic in At but neutral in Vf) are primarily responsible for the luminal Ca2+-sensitivity differences observed between these two species (At and Vf). These modifications make Vf vacuoles hyperexcitable relative to At. The experimental data is robust, and the primary conclusions are mostly justified.

Although the results are fascinating, they are not entirely surprising based on the data presented in a previous publication from the same group. The exact significance of these findings with respect to species-specific plant physiology is not clear, yet this presents a promising and fertile ground for future research.

DOI: 10.7554/eLife.83974

Resource: ZFIN_ZDB-GENO-070501-2

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-GENO-070501-2

What is this?

Reviewer #2 (Public Review):

Summary:<br /> The authors sought to identify the impact skin viscoelasticity has on neural signalling of contact forces that are representative of those experienced during normal tactile behaviour. The evidence presented in the analyses indicates there is a clear effect of viscoelasticity on the imposed skin movements from a force-controlled stimulus. Both skin mechanics and evoked afferent firing were affected based on prior stimulation, which has not previously been thoroughly explored. This study outlines that viscoelastic effects have an important impact on encoding in the tactile system, which should be considered in the design and interpretation of future studies. Viscoelasticity was shown to affect the mechanical skin deflections and stresses/strains imposed by previous and current interaction force, and also the resultant neuronal signalling. The result of this was an impaired coding of contact forces based on previous stimulation. The authors may be able to strengthen their findings, by using the existing data to further explore the link between skin mechanics and neural signalling, giving a clearer picture than demonstrating shared variability. This is not a critical addition, but I believe would strengthen the work and make it more generally applicable.

Strengths:<br /> -Elegant design of the study. Direct measurements have been made from the tactile sensory neurons to give detailed information on touch encoding. Experiments have been well designed and the forces/displacements have been thoroughly controlled and measured to give accurate measurements of global skin mechanics during a set of controlled mechanical stimuli.<br /> -Analytical techniques used. Analysis of fundamental information coding and information representation in the sensory afferents reveals dynamic coding properties to develop putative models of the neural representation of force. This advanced analysis method has been applied to a large dataset to study neural encoding of force, the temporal dynamics of this, and the variability in this.

Weaknesses:<br /> -Lack of exploration of the variation in neural responses. Although there is a viscoelastic effect that produces variability in the stimulus effects based on prior stimulation, it is a shame that the variability in neural firing and force-induced skin displacements have been presented, and are similarly variable, but there has been no investigation of a link between the two. I believe with these data the authors can go beyond demonstrating shared variability. The force per se is clearly not faithfully represented in the neural signal, being masked by stimulation history, and it is of interest if the underlying resultant contact mechanics are.

Validity of conclusions:<br /> The authors have succeeded in demonstrating skin viscoelasticity has an impact on skin contact mechanics with a given force and that this impacts the resultant neural coding of force. Their study has been well-designed and the results support their conclusions. The importance and scope of the work is adequately outlined for readers to interpret the results and significance.

Impact:<br /> This study will have important implications for future studies performing tactile stimulation and evaluating tactile feedback during motor control tasks. In detailed studies of tactile function, it illustrates the necessity to measure skin contact dynamics to properly understand the effects of a force stimulus on the skin and mechanoreceptors.

Reviewer #2 (Public Review):

Summary:<br /> CD8+ QFL T cells recognize a peptide, FYAEATPML (FL9), presented on Erap1-deficient cells. QFL T cells are present at a high frequency in the spleen of naïve mice. They express an antigen-experienced phenotype, and about 80% express an invariant TCRα chain Vα3.2Jα21.

Here, Guan and colleagues report that QFL T cells are present not only in the spleen but also in the intestinal epithelium, where they display several phenotypic and functional peculiarities. The establishment of spleen and gut Vα3.2+ QFL T cells is TAP-dependent, and their phenotype is regulated by the presence/absence of Qa-1b and Erap1. Maintenance of gut Vα3.2+ QFL T cells depends on the gut microbiota and is associated with colonization by Pediococcus pentosaceus.

Strengths:<br /> This article contains in-depth studies of a peculiar and interesting subset of unconventional CD8 T cells, based partly on generating two novel TCR-transgenic models.

The authors discovered a clear relation between the gut microbiome and the maintenance of gut QFL T cells. One notable observation is that monocolonization of the gut with Pediococcus pentosaceus is sufficient to sustain gut QFL T cells.

Weaknesses:<br /> In the absence of immunopeptidomic analyses, the presence or absence of the FL9 peptide on various cell types is inferred based on indirect evidence.

Analyses of the homology between the FL9 and bacterial peptides were limited to two amino acid residues (P4 and P6).

The potential function of QFL T cells remains elusive.

Reviewer #2 (Public Review):

The manuscript "IQCH regulates spermatogenesis by interacting with CaM to promote RNA-binding proteins' expression" by Ruan et al. identified a homozygous variant affecting the splicing of IQCH in two infertile men from a Chinese family. The authors also generated an IQCH knockout mouse model to confirm the abnormal sperm phenotypes associated with IQCH deficiency. Further molecular biological assays supported the important role and mechanism of IQCH in spermatogenesis. This manuscript is informative for clinical and basic research on male infertility.

Reviewer #2 (Public Review):

The manuscript by Chambert et al. describes a thorough and careful characterization of inositol pyrophosphate isomers and the PHO pathways in different genetic backgrounds in S. cerevisiae. The paper ultimately arrives at a proposed model in which the inositol pyrophosphate 1,5-IP8 signals phosphate abundance to SPX-domain containing proteins. To arrive at their conclusion, the authors rely heavily on CE-MS analysis of inositol pyrophosphates in different yeast strains, and monitoring inositol pyrophosphate depletion over time in response to phosphate starvation. This analysis is complemented by different reporter systems of PHO pathway activation, such as Pho4 translocation and Pho81 expression.

The experiments are well-designed and the results interpreted with care. With their findings, the authors demonstrate convincingly, that a previous study by O'Shea and co-workers (reference 15 and 16) had been misleading. Lee et al. claimed that the PHO pathway in S. cerevisiae is triggered by an increase in 1-IP7. This claim has been debated heavily in the community, and several groups were not able to reproduce this putative increase of inositol pyrophosphates (references 6, 11, 18). The confusion regarding these discrepancies has been resolved by the current study and is of significant importance to the community.

Reviewer #2 (Public Review):

Summary:

Planctomycetes encompass a group of bacteria with unique biological traits, the compartmentalized cells make them appear to be organisms in between prokaryotes and eukaryotes. However, only a few of the Planctomycetes bacteria are cultured thus far, and this hampers insight into the biological traits of these evolutionarily important organisms.

This work reports the methodology details of how to isolate the deep-sea bacteria that could be recalcitrant to laboratory cultivation, and further reveals the distinct characteristics of the new species of a deep-sea Planctomycetes bacterium, such as the chronic phage release without breaking the host and promote the host and related bacteria in nitrogen utilization. Therefore, the finding of this work is of importance in extending our knowledge of bacteria.

Strengths:

Through the combination of microscopic, physiological, genomics, and molecular biological approaches, this reports the isolation and comprehensive investigation of the first anaerobic representative of the deep-sea Planctomycetes bacterium, in particular in that of the budding division, and release phage without lysis of the cells. Most of the results and conclusions are supported by the experimental evidence.

Weaknesses:

1. While EMP glycolysis is predicted to be involved in energy conservation, no experimental evidence indicated any sugar utilization by the bacterium.<br /> 2. "anaerobic representative" is indicated in the Title, the contrary, TCA in energy metabolism is predicted by the bacterium.<br /> 3. The possible mechanisms of the chronic phage release without breaking the host are not discussed.

Reviewer #2 (Public Review):

Summary:

The authors develop a computational approach-avoidance-conflict (AAC) task, designed to overcome the limitations of existing offer based AAC tasks. The task incorporated likelihoods of receiving rewards/ punishments that would be learned by the participants to ensure computational validity and estimated model parameters related to reward/punishment and task induced anxiety. Two independent samples of online participants were tested. In both samples participants who experienced greater task induced anxiety avoided choices associated with greater probability of punishment. Computational modelling revealed that this effect was explained by greater individual sensitivities to punishment relative to rewards.

Strengths:

Large internet-based samples, with discovery sample (n = 369), pre-registered replication sample (n = 629) and test-retest sub group (n = 57). Extensive compliance measures (e.g. audio checks) seek to improve adherence.

There is a great need for RL tasks that model threatening outcomes rather than simply loss of reward. The main model parameters show strong effects and the additional indices with task based anxiety are a useful extension. Associations were broadly replicated across samples. Fair to excellent reliability of model parameters is encouraging and badly needed for behavioral tasks of threat sensitivity.

The task seems to have lower approach bias than some other AAC tasks in the literature.

Weaknesses:

The negative reliability of punishment learning rate is concerning as this is an important outcome.

The Kendall's tau values underlying task induced anxiety and safety reference/ various indices are very weak (all < 0.1), as are the mediation effects (all beta < 0.01). The interaction with P(punishment|conflict) does explain some of this.

The inclusion of only one level of reward (and punishment) limits the ecological validity of the sensitivity indices.

Appraisal and impact:

Overall this is a very strong paper, describing a novel task that could help move the field of RL forward to take account of threat processing more fully. The large sample size with discovery, replication and test-retest gives confidence in the findings. The task has good ecological validity and associations with task-based anxiety and clinical self-report demonstrate clinical relevance. Test-retest of the punishment learning parameter is the only real concern. Overall this task provides an exciting new probe of reward/threat that could be used in mechanistic disease models.

Additional context:

The sex differences between the samples are interesting as effects of sex are commonly found in AAC tasks. It would be interesting to look at the main model comparison with sex included as a covariate.

Reviewer #2 (Public Review):

The study is performed with old tool Spamo (12 year ago), source data from Encode (2010-2012), even peak caller tool version MACS is old ~ 2013. De novo motif search tool is old too (new one STREME is not mentioned). Any composite element search tool published for the recent 12 years are not cited, there are some issues in data analysis in presentation. Almost all references are from about 8-10 year ago (the most recent date is 2019)

The title is misleading<br /> Instead of<br /> A new pipeline SPICE identifies novel JUN-IKZF1 composite elements<br /> It should be written as<br /> Application of SpaMo tool identifies novel JUN-IKZF1 composite elements<br /> It reflects the pipeline better but honestly shows that the novelty is missed.

The study was performed on too old data from ENCODE, authors mentioned 343 Encode ChIP-Seq libraries, but authors even did not care even about to set for each library the name of target TF (Figure 1E, Figure S2, Table 2).

Reviewer #2 (Public Review):

Most farming is done by subtracting or adding what people want based in nature. However, in nature, crops interact with various objects, and mostly we are unaware of their effects. In order to increase agricultural productivity, finding useful objects is very important. However, in an uncontrolled environment, it coexists with so many biological objects that it is very inefficient to verify them all experimentally. It is therefore necessary to develop an effective screening method to identify external environmental factors that can increase crop productivity. This study identified factors presumed to be important to crop growth based on metabarcoding analysis, field sampling, and non-linear analysis/information theory, and conducted a mesocosm experiment to verify them experimentally. In conclusion, the object proposed by the author did not increase rice yield, but rather rice growth rate.

The authors responded to my general concerns and all of my specific comments. The manuscript has significantly improved. The flow of aims and approaches is more understandable. Extra supplementary material -especially the visual ones, is useful.

I agree with the other reviewers that the study needs more data and evidence. However, this study aims to introduce ecological concepts and advanced statistical methods to the field. Also, most time series analyses require absolute abundance data, but the manuscript provides solutions for the sequencing data.

Reviewer #2 (Public Review):

In this study, Maillie et al. have carried out a set of multiscale molecular dynamics simulations to investigate the interactions between the viral membrane and four broadly neutralizing antibodies that target the membrane proximal exposed region (MPER) of the HIV-1 envelope trimer. The simulation recapitulated in several cases the binding sites of lipid head groups that were observed experimentally by X-ray crystallography, as well as some new binding sites. These binding sites were further validated using a structural bioinformatics approach. Finally, steered molecular dynamics was used to measure the binding strength between the membrane and variants of the 4E10 and PGZL1 antibodies.

The conclusions from the paper are mostly well supported by the simulations, however, they remain very descriptive and the key findings should be better described and validated. In particular:

It has been shown that the lipid composition of HIV membrane is rich in cholesterol [1], which accounts for almost 50% molar ratio. The authors use a very different composition and should therefore provide a reference. It has been shown for 4E10 that the change in lipid composition affects dynamics of the binding. The robustness of the results to changes of the lipid composition should also be reported.

The real advantage of the multiscale approach (coarse grained (CG) simulation followed by a back-mapped all atom simulation) remains unclear. In most cases, the binding mode in the CG simulations seem to be an artifact.

The results reported in this study should be better compared to available experimental data. For example how does the approach angle compare to cryo-EM structure of the bnAbs engaging with the MPER region, e.g. [2-3]? How do these results from this study compare to previous molecular dynamics studies, e.g.[4-5]?

References<br /> 1. Brügger, Britta, et al. "The HIV lipidome: a raft with an unusual composition." Proceedings of the National Academy of Sciences 103.8 (2006): 2641-2646.<br /> 2. Rantalainen, Kimmo, et al. "HIV-1 envelope and MPER antibody structures in lipid assemblies." Cell Reports 31.4 (2020).<br /> 3. Yang, Shuang, et al. "Dynamic HIV-1 spike motion creates vulnerability for its membrane-bound tripod to antibody attack." Nature Communications 13.1 (2022): 6393.<br /> 4. Carravilla, Pablo, et al. "The bilayer collective properties govern the interaction of an HIV-1 antibody with the viral membrane." Biophysical Journal 118.1 (2020): 44-56.<br /> 5. Pinto, Dora, et al. "Structural basis for broad HIV-1 neutralization by the MPER-specific human broadly neutralizing antibody LN01." Cell host & microbe 26.5 (2019): 623-637.

Reviewer #2 (Public Review):

The exact dynamics of responses to volatiles from herbivore-attacked neighbouring plants have been little studied so far. Also, we still lack evidence of whether herbivore-induced plant volatiles (HIPVs) induce or prime plant defences of neighbours. The authors investigated the volatile emission patterns of receiver plants that respond to the volatile emission of neighbouring sender plants which are fed upon by herbivorous caterpillars. They applied a very elegant approach (more rigorous than the current state-of-the-art) to monitor temporal response patterns of neighbouring plants to HIPVs by measuring volatile emissions of senders and receivers, senders only and receivers only. Different terpenoids were produced within 2 h of such exposure in receiver plants, but not during the dark phase. Once the light turned on again, large amounts of terpenoids were released from the receiver plants. This may indicate a delayed terpene burst, but terpenoids may also be induced by the sudden change in light. A potential caveat exists with respect to the exact timing and the day-night cycle. The timing may be critical, i.e. at which time-point after onset of light herbivores were placed on the plants and how long the terpene emission lasted before the light was turned off. If the rhythm or a potential internal clock matters, then this information should also be highly relevant. Moreover, light on/off is a rather arbitrary treatment that is practical for experiments in the laboratory but which is not a very realistic setting. Particularly with regard to terpene emission, the sudden turning on of light instead of a smooth and continuous change to lighter conditions may trigger emission responses that are not found in nature. As one contrasting control, the authors also studied the time-delay in volatile emission when plants were just kept under continuous light (just for the experiment or continuously?). Here they also found a delayed terpenoid production, but this seemed to be lower compared to the plants exposed to the day-night-cycle. Another helpful control would be to start the herbivory treatment in the evening hours and leave the light on. If then again plants only release volatiles after a 17 h delay, the response is indeed independent of the diurnal clock of the plant.

Interestingly, internal terpene pools of one of the leaves tested here remained more comparable between night and day, indicating that their pools stay higher in plants exposed to HIPVs. In contrast, terpene synthases were only induced during the light-phase, not in the dark-phase. Moreover, jasmonates were only significantly induced 22 h after the onset of the volatile exposure and thus parallel with the burst of terpene release.

An additional experiment exposing plants to the green leaf volatile (glv) (Z)-3-hexenyl acetate revealed that plants can be primed by this glv, leading to a stronger terpene burst. The results are discussed with nice logic and considering potential ecological consequences. Some data are not discussed, e.g. the jasmonate and gene induction pattern.

Overall, this study provides intriguing insights into the potential interplay between priming and induction, which may co-occur, enhancing (indirect and direct) plant defence. Follow-up studies are suggested that may provide additional evidence.

Reviewer #2 (Public Review):

The manuscript titled "Calcineurin Inhibition Enhances Caenorhabditis elegans Lifespan by Defecation Defects-Mediated Calorie Restriction and Nuclear Hormone Signaling" by Priyanka Das, Alejandro Aballay, and Jogender Singh reveals that inhibiting calcineurin, a conserved protein phosphatase, in C. elegans affects the defecation motor program (DMP), leading to intestinal bloating and increased susceptibility to bacterial infection. This intestinal bloating mimics calorie restriction, ultimately resulting in an enhanced lifespan. The research identifies the involvement of HLH-30 and NHR-8 proteins in this lifespan enhancement, providing new insights into the role of calcineurin in C. elegans DMP and mechanisms for longevity.

The authors present novel findings on the role of calcineurin in regulating the defecation motor program in C. elegans and how its inhibition can lead to lifespan enhancement. The evidence provided is solid with multiple experiments supporting the main claims.

Strengths:<br /> The manuscript's strength lies in the authors' use of genetic and biochemical techniques to investigate the role of calcineurin in regulating the DMP, innate immunity, and lifespan in C. elegans. Moreover, the authors' findings provide a new mechanism for calcineurin inhibition-mediated longevity extension, which could have significant implications for understanding the molecular basis of aging and developing interventions to promote healthy aging.

1. The study uncovers a new role for calcineurin in the regulation of C. elegans DMP and a potential novel pathway for enhancing lifespan via calorie restriction involving calcineurin, HLH-30, and NHR-8 in C. elegans.<br /> 2. Multiple signaling pathways involved in lifespan enhancement were investigated with fairly strong experimental evidence supporting their claims.

Weaknesses:<br /> The manuscript's weaknesses include the lack of mechanistic details regarding how calcineurin inhibition leads to defects in the DMP and induces calorie restriction-like effects on lifespan.

The exact site of calcineurin action, i.e., whether in the intestine or enteric muscles (Lee et al., 2005), and the possible molecular mechanisms linking calcineurin inhibition, DMP defects, and lifespan were not adequately explored. Although characterization of the full mechanism is probably beyond the scope of this paper, given the relative simplicity and advantages of using C. elegans as a model organism for this study, some degree of rigor is expected with additional straightforward control experiments as listed below:

The authors state that tax-6 knockdown animals had drastically reduced expulsion events (Figure 2G), leading to irregular DMP (Lines 144-145), but did not describe the nature of DMP irregularity. For example, did the reduced expulsion events still occur with regular intervals but longer cycle lengths? Or was the rhythmicity completely abolished? The former would suggest the intestine clock is still intact, and the latter would indicate that calcineurin is required for the clock to function. Therefore, ethograms of DMP in both wild-type and tax-6 mutant animals are warranted to be included in the manuscript. Along the same line, besides the cycle length, the three separable motor steps (aBoc, pBoc, EMC) are easily measurable, with each step indicating where the program goes wrong, hence the site of action, which is precisely the beauty of studying C. elegans DMP. Unfortunately, the authors did not use this opportunity to characterize the exact behavior phenotypes of the tax-6 mutant to guide future investigations. Furthermore, it is interesting that about 64% of tax-6 (p675) animals had normal DMP. The authors attributed this to p675 being a weak allele. It would be informative to further examine tax-6 RNAi as in other experiments or to make a tax-6 null mutant with CRISPR. In addition, in one of the cited papers (Lee et al., 2005), the exact calcineurin loss-of-function strain tax-6(p675) was shown to have normal defecation, including normal EMC, while the gain-of-function mutant of calcineurin tax-6(jh107) had abnormal EMC steps. It wasn't clear from Lee et al., 2005, if the reported "normal defecation" was only referring to the expulsion step or also included the cycle length. Nevertheless, this potential contradiction and calcineurin gain-of-function mutant is highly relevant to the current study and should be further explored as a follow-up to previously reported results. For some of the key experiments, such as tax-6's effects on susceptibility to PA14, DMP, intestinal bloating, and lifespan, additional controls, as the norm of C. elegans studies, including second allele and rescue experiments, would strengthen the authors' claims and conclusions.

The second weakness of this manuscript is the data presentation for all survival rate curves. The authors stated that three independent experiments or biological replicates were performed for each group but only showed one "representative" curve for each plot. Without seeing all individual datasets or the averaged data with error bars, there is no way to evaluate the variability and consistency of the survival rate reported in this study.

Overall, the authors' claims and conclusions are justified by their data, but further experiments are needed to confirm their findings and establish the detailed mechanisms underlying the observed effects of calcineurin inhibition on the DMP, calorie restriction, and lifespan in C. elegans.

Reviewer #2 (Public Review):

Summary:<br /> Laham et al. investigate how the projection from adult-born granule cells into CA2 affects the retrieval of social memories at various developmental points. They use chemogenetic manipulations and electrophysiological recordings to test how this projection affects hippocampal network properties during behavior. I find the study to be very interesting, the results are important for our understanding of how social memories of different natures (remote or immediate) are encoded and supported by the hippocampal circuitry. I have some points that I added below that I think could help clarify the conclusions:<br /> - My major concern with the manuscript was that making the transitions between the different experiments for each result section is not very smooth. Maybe they can discuss a bit in a summary conclusion sentence at the end of each result section why the next set of experiments is the most logical step.<br /> - In line 113, the authors say that "the DG is known to influence hippocampal theta-gamma coupling and SWRs". Another recent study Fernandez-Ruiz et al. 2021, examined how various gamma frequencies in the dentate gyrus modulate hippocampal dynamics.<br /> - Having no single cells in the electrophysiological recordings makes it difficult to interpret the ephys part. Perhaps having a discussion on this would help interpret the results. If more SWRs are produced from the CA2 region (perhaps aided by projections from abGC), more CA2 cells that respond to social stimuli (Oliva et al. 2020) would reactivate the memories, therefore making them consolidate faster/stronger. On the other hand, the projections from abGC that the authors see, also target a great deal of PV+ interneurons, which have been shown to pace the SWRs frequency (Stark et al 2014, Gan et al 2017), which further suggests that this projection could be involved in SWRs modulation.<br /> - The authors should cite and discuss Shuo et al., 2022 (A hypothalamic novelty signal modulates hippocampal memory).<br /> - I think the authors forgot to refer to Fig 3a-f, maybe around lines 163-168.<br /> - Are the SWRs counted only during interaction time or throughout the whole behavior session for each condition?<br /> - Figure 3t shows a shift in the preferred gamma phase within theta cycles as a result of abGC projections to CA2 ablation with CNO, especially during Mother CNO condition. I think this result is worth mentioning in the text.<br /> - Figure 3u in the legend mention "scale bars = 200um", what does this refer to?<br /> - What exactly is calculated as SWR average integral? Is it a cumulative rate? Please clarify.<br /> - Alexander et al 2017, "CA2 neuronal activity controls hippocampal oscillations and social behavior", examined some of the CA2 effects in the hippocampal network after CNO silencing, and the authors should cite it.

Strengths:<br /> Behavioral experiments after abGC projections to CA2 are compelling as they show clearly distinct behavioral readout.

Weaknesses:<br /> Electrophysiological experiments are difficult to interpret without additional quantifications (single-cell responses during interactions etc.)

Reviewer #2 (Public Review):

Summary:<br /> This study uses transcriptome sequence from a dioecious plant to compare evolutionary rates between genes with male- and female-biased expression and distinguish between relaxed selection and positive selection as causes for more rapid evolution. These questions have been explored in animals and algae, but few studies have investigated this in dioecious angiosperms, and none have so far identified faster rates of evolution in male-biased genes (though see Hough et al. 2014 https://doi.org/10.1073/pnas.1319227111).

Strengths:<br /> The methods are appropriate to the questions asked. Both the sample size and the depth of sequencing are sufficient, and the methods used to estimate evolutionary rates and the strength of selection are appropriate. The data presented are consistent with faster evolution of genes with male-biased expression, due to both positive and relaxed selection.

This is a useful contribution to understanding the effect of sex-biased expression in genetic evolution in plants. It demonstrates the range of variation in evolutionary rates and selective mechanisms, and provides further context to connect these patterns to potential explanatory factors in plant diversity such as the age of sex chromosomes and the developmental trajectories of male and female flowers.

Weaknesses:<br /> The presence of sex chromosomes is a potential confounding factor, since there are different evolutionary expectations for X-linked, Y-linked, and autosomal genes. Attempting to distinguish transcripts on the sex chromosomes from autosomal transcripts could provide additional insight into the relative contributions of positive and relaxed selection.

Reviewer #2 (Public Review):

The authors employed the Mendelian Randomization method to analyze the association between type 2 diabetes (T2D) and fracture using the UK Biobank data. They found that "genetically predicted T2D was associated with higher BMD and lower risk of fracture". Additionally, they identified 10 loci that were associated with both T2D and fracture risk, with the SNP rs4580892 showing the highest signal. While the negative relationship between T2D and fracture has been previously observed, the discovery of these 10 loci adds an intriguing dimension to the findings, although the clinical implications remain uncertain.

Reviewer #2 (Public Review):

In this manuscript, the authors use newly-generated, large-scale transcriptomic data along with histological data to attempt to dissect the mechanisms by which individuals with Tibetan ancestry are able to mitigate the negative effects of high elevation on birth weight. They present detailed analyses of the transcriptomic data and find significant sex differences in the placenta transcriptome.

I have significant concerns about the conclusions that are presented. The analyses also lack the information necessary to evaluate their reliability.

The experimental design does not include a low elevation comparison and thus cannot be used to answer questions about how ancestry influences hypoxia responses and thus birthweight at high elevations. Importantly, because the placenta tissues (and trophoblasts specifically) are quickly evolving, there are a priori good reasons to expect to find population differences irrespective of adaptive evolution that might contribute to fetal growth protection. There are also significant details missing in the analyses that are necessary to substantiate and replicate the analyses presented.

Although the datasets are ultimately valuable as reference sets, the absence of low elevation comparisons for Tibetans and Han Chinese individuals undermines the ability of the authors to assess whether differences observed between populations are linked to hypoxia responses or variation in the outcomes of interest (i.e., hypoxia-dependent fetal growth restriction).

The authors attempt to tackle this phenotypic association by looking for correlations between gene networks (WGCNA) and individual genes with birthweight and other measurements collected at birth. I have some reservations about this approach with only two groups (i.e., missing the lowland comparison), but it is further problematic that the authors do not present data demonstrating that there are differences in birthweight or any other traits between the populations in the samples they collected.

Throughout, I thus find conclusions about the adaptive value and hypoxia-responses made by the authors to be unsubstantiated and/or the data to be inadequate. There are also a gratuitous number of speculative statements about mechanisms by which differential gene expression leads to the protection of birthweight that are not evaluated and thus cannot be substantiated by the data presented.

As currently presented and discussed, these results thus can only be used to evaluate population differences and tissue-specific variation therein.

There is also some important methodological information missing that makes it difficult or impossible to assess the quality of the underlying data and/or reproduce the analyses, further limiting the potential impact of these data:<br /> 1. Transcriptome data processing and analyses: RNA quality information is not mentioned (i.e., RIN). What # of reads are mapped to annotated regions? How many genes were expressed in each tissue (important for contextualizing the # of DE genes reported - are these a significant proportion of expressed genes or just a small subset?).<br /> 2. The methods suggest that DE analyses were run using data that were normalized prior to reading them into DESeq2. DESeq2 has an internal normalization process and should not be used on data that was already normalized. Please clarify how and when normalization was performed.<br /> 3. For enrichment analyses, the background gene set (all expressed genes? all genes in the genome? or only genes expressed in the tissue of interest?) has deterministic effects on the outcomes. The background sets are not specified for any analyses.<br /> 4. In the WGCNA analysis, P-values for correlations of modules with phenotype data (birthweight etc.) should be corrected for multiple testing (i.e., running the module correlation for each outcome variables) and p.adjust used to evaluate associations to limit false positives given the large number of correlations being run.<br /> 6. The plots for umbilical histological data (Fig 5 C) contain more than 5 points, but the use of replicate sections is not specified. If replicate sections were used, the authors should control for non-independence of replicate sections in their analyses (i.e., random effects model).

On more minor notes:<br /> There is significant and relevant published data on sex differences and hypoxia in rodents (see Cuffe et al 2014, "Mid- to late-term hypoxia in the mouse alters placental morphology, glucocorticoid regulatory pathways, and nutrient transporters in a sex-specific manner" and review by Siragher and Sferuzzi-Perro 2021, "Placental hypoxia: What have we learnt from small animal models?"), and historical work reporting sex differences in placental traits associated with high elevation adaptation in Andeans (series of publications by Moira Jackson in the late 1980s, reviewed in Wilsterman and Cheviron 2021, "Fetal growth, high altitude, and evolutionary adaptation: A new perspective").

Reviewer #2 (Public Review):

In this manuscript, Xie and colleagues investigate the contribution of osteocytes to bone metastasis of non-small cell lung carcinoma (NSCLC) using a combination of clinical samples and in vitro and in vivo data. They find that metastatic NSCLC cells exhibit lower levels of the proliferation marker Ki-67 when located in areas adjacent to the bone surface in both NSCLC patients and an intraosseous animal model of NSCLC. Using in vitro approaches, they show that osteocyte-like cells inhibit the proliferation of NSCLC cells through the secretion of small extracellular vesicles (sEVs). They identify miR-99b-3p as a component of sEVs and demonstrate that miR-99b3p inhibits the proliferation of NSCLC cells by targeting the transcription factor MDM2. Interestingly, the data also shows that mechanical stimulation of osteocytes enhances the inhibitory effect of osteocytes on NSCLC cell proliferation via increasing sEVs release. By performing different in vivo studies, the authors show that tibial loading and moderate exercise (treadmill running), before and after tumor cell inoculation, suppress tumor progression in bone and protect bone mass. Intriguingly, the moderate exercise regime shows additive/synergistic effects with the co-administration of anti-resorptive therapy. These data add to the growing evidence pointing towards osteocytes as important cells of the tumor microenvironment capable of influencing the progression of tumors in bone.

The conclusions of the paper, however, are not well supported by the data, and some critical aspects of image analysis and data analysis need to be clarified and extended.

1) The histological images are analyzed in a qualitative manner, with no description of the methodology used. In bone metastases, cancer cells are frequently mixed with bone marrow cells. The lack of cell markers to identify NSCLC cells versus bone marrow cells makes the interpretation of the imaging data difficult. The authors rely on KI-67 as a marker of proliferation. Yet, it is intriguing that some osteocytes, non-proliferating cells by definition, are often positive for this marker, which questions the specificity of the staining. To make these results more solid, the authors should have provided the proper immunostaining controls to check for specificity and use additional markers of proliferation.

2) Adding control groups to fully assess the impact of the in vivo interventions (tibial loading, moderate exercise, anti-resorptive therapy) on bone mass would be needed. The authors should have used naive mice or analyzed the bones from the non-injected contralateral legs. Further, validating the in vivo work with other osteocyte-like cells or primary osteocytes would have strengthened the results.

3) The data on miRNA99b-3p on NSCLC in Supplementary Figure 3 is not convincing. The positive cells are difficult to see and most of the osteocyte lack nuclei. Better data, in humans and the mouse model, would have helped to confirm that osteocytes produce miRNA99b-3p.

4) The conclusions of the paper are not fully supported by the data provided. Osteocytes, as well as other bone cells, can respond to mechanical stimulation and thus could virtually be responsible for the protective effects of mechanical loading or moderate exercise. In vivo experiments demonstrating a direct role of osteocytes-produced miRNA99b-3p are needed to support the notion that osteocytes maintain tumor dormancy in NSCLC bone metastasis. Further, the authors solely rely on Ki-67 as a marker of dormancy. Completing this analysis with an assessment of a dormant gene expression signature or in vivo studies assessing tumor dormancy directly would be needed to confirm this notion.

Reviewer #2 (Public Review):

Summary:

This work presents a previously undescribed neuroanatomical and neurophysiological analog between mammals and songbirds. Juvenile zebra finches learn to sing by memorizing an adult song and then, through practice, converging to a close copy of the stored template. Previous work identified pathways emanating from the avian auditory cortical regions (AIV) and basal ganglia that, through ventral pallium (VP), and the subthalamic nucleus, innervate the finches' ventral tegmental area (VTA). As in mammals, the dopaminergic projections of the VTA onto the avian striatopallidal nucleus, area X, deliver a prediction error signal. This signal encodes a surprisingly better or worse performance of the ongoing song and therefore allows the birds to improve.

In mammals, lateral Habenula (LHb) neurons contribute to learning by signaling disappointing trial outcomes or aversive stimuli. Using viral tract tracing Roesner et al. identify projections from the zebra finch VP and AIV to the LHb as well as from the LHb to the VTA. The authors use functional mapping to show that the VP activates the LHb and that the LHb suppresses the Area X-projecting VTA neurons. Then, the authors show that lesioning the LHb in juvenile finches does not prevent them from copying their tutor's song but still leads to worse performance than controls due to the production of highly abnormal vocalizations, peppered in both lone and female-directed songs. In contrast, lesioning the LHb in adult finches has no effect on the song. Together, these findings suggest that the LHb may be part of a song evaluation system and may participate in learning by signaling vocalizations that deviate from the desired tutor template.

The LHb is an evolutionarily conserved structure that connects the forebrain and midbrain with the epithalamus in vertebrates. By identifying the LHb as a component in song learning, the authors lay the grounds for a trove of new research into the various emotional, biophysical, memory, and sensory processes that contribute to learning within and through the LHb. Most conclusions of this paper are well supported by data, but some conceptual and analytic aspects require framing with respect to methodological limitations.

Strengths:<br /> The use of both anatomical tracing and functional circuit mapping is a uniquely-powerful approach to addressing the main line of inquiry in this work. Specifically, collision testing and antidromic identification allow identifying LHb-->VTA and VTA-->X projecting neurons and therefore testing the response of these specific learning-related projections to stimulation in VP and LHb (respectively).

The evaluation of abnormal vocalizations using a variational autoencoder (VAE) is a particularly strong approach that is immune to observer biases. By training this artificial neural network model with sham or pre-lesion animals, the authors clearly distinguish abnormal syllables because of their significantly poorer reconstruction through the VAE. This approach allowed the authors to provide strong quantitative support to the effect of LHb lesion in juvenile finches on their adult song.

Weaknesses:<br /> The lesions in juveniles, as the authors discuss, were histologically examined at the end of the song development, months after their creation. The authors mention not being able to rule out damage to the medial part of the Hb. But the effect of the lesions could perhaps be mediated by damage to other brain regions, such as DLM, or passing fibers (when using electrolytic lesions).

Additionally, the effect on learning could also be mediated indirectly. In mammals, the outputs of the LHb target dopaminergic regions, serotonergic regions, and a cholinergic region. In birds, the LHb may also have a diverse impact on neuromodulators and therefore an impact on behavior states and on sleep. Disrupted behavior states may lead to poorer or less frequent practice and indirectly to abnormal results that do not stem from erroneous performance evaluation.

Reviewer #2 (Public Review):

In this study the authors sought to investigate how the metabolic state of iNKT cells impacts their potential pathological role in allergic asthma. The authors used two mouse models, OVA and HDM-induced asthma, and assessed genes in glycolysis, TCA, B-oxidation and FAS. They found that acetyl-coA-carboxylase 1 (ACC1) was highly expressed by lung iNKT cells and that ACC1 deficient mice failed to develop OVA-induced and HDM-induced asthma. Importantly, when they performed bone marrow chimera studies, when mice that lacked iNKT cells were given ACC1 deficient iNKT cells, the mice did not develop asthma, in contrast to mice given wildtype NKT cells. In addition, these observed effects were specific to NKT cells, not classic CD4 T cells. Mechanistically, iNKT cell that lack AAC1 had decreased expression of fatty acid-binding proteins (FABPs) and peroxisome proliferator-activated receptor (PPAR)γ, but increased glycolytic capacity and increased cell death. Moreover, the authors were able to reverse the phenotype with the addition of a PPARg agonist. When the authors examined iNKT cells in patient samples, they observed higher levels of ACC1 and PPARG levels, compared to healthy donors and non-allergic-asthma patients.

Reviewer #2 (Public Review):

Summary:<br /> Sotiropoulou et al. present an interesting study of an incredibly rare premature aging disease (De Barsy syndrome), examining both the underlying mechanisms at play behind the condition as well as how that biology may have a larger role in understanding features of normal aging, and in particular, human skin aging. The authors link one of the underlying genetic defects in De Barsy syndrome (PYCR1 mutations) to its phenotypic manifestations and then extrapolate those findings to present more preliminary data to suggest that a loss of PYCR1 may be a biomarker of normal human skin aging.

Strengths:<br /> - The study is important as De Barsy syndrome is challenging to study given its rarity, thus making it an understudied condition. Here the authors combine both human patient samples and murine models to offer a nice contribution to further understanding the pathophysiology of this disease.

- The authors are able to link some of the observed features in De Barsy syndrome preliminarily to more common aging models and processes (senescence, human skin dermal aging). They nicely show that the loss of Pycr1 in mice can provoke thinning of the dermis of mice while not affecting the epidermis. Furthermore, they present compelling data to suggest that Pycr2 may be compensating in mice (while not in humans) and this may contribute to the differences in lifespan observed between the mice and humans.

- Should these results be further verified, this could suggest that further study of Pycr1 and Pycr2 biology may offer new insights into aging and senescence in other tissues.

Weaknesses:<br /> - Some of the data appears preliminary and seems like it needs further analysis as described further below in my suggestions for the authors:

1) While the authors report that there is no difference in the lifespan of the Pycr1-KO mice, can they report whether there was overall weight loss or any size differences between the mice? This is helpful particularly when comparing the dermal thickness as well as considering how the global loss of Pycr1 may affect overall systemic health.

2) In Figure S2E, the comparison "pairs" seems somewhat arbitrarily chosen and it seems from the quantifications of these pairs that depending upon which young sample you compare to which old sample, you may end up with differing results. I think the more appropriate way to make this quantitative comparison would be to average the young samples and average the old samples and then compare them and perform statistics. This seems critical to really assess whether PYCR1 loss would be a consistent marker of human skin dermal aging. Additionally, it would be helpful to also look at Pycr2 expression in the normal young versus old dermis to see if the reported difference in Pycr1 is really something specific for Pycr1 and not something more general.

3) Are the labels mixed up in Figures 1J and 1K or am I reading it incorrectly? From what I can see the graph is showing that the dermal thickness and collagen intensity is higher in the Pycr1-/- mice. Similarly, the authors state that there is "significantly less collagen fiber staining", although in Figure S1G neither the quantification of collagen I or collagen III are shown to be significant. These discrepancies need to be discussed or corrected.

4) Can the authors speculate further on why Pycr2 is also diminished in human patients (while it clearly remains present in the mice).

5) Can the authors comment on whether other canonical senescence features are seen in De Barsy syndrome (p16 positivity, senescence associated secretory phenotype, etc.)? Along these lines, there is an abundance of publicly available RNA-seq datasets from various forms of senescent cells. It would be interesting to examine these and see whether there is any loss of expression of PYCR1 or PYCR2 in these data, or is the loss of PYCR1 only seen at the protein level?

Reviewer #2 (Public Review):

Milovic, Duong, and Barbour investigate the inflammatory response of three species of small mammals (P. leucopus, M. musculus, and R. norvegicus) to endotoxin lipopolysaccharide (LPS) injection via genome-wide transcriptomics from blood samples. Understanding the inflammation response of P. leucopus is of importance as they are a reservoir for several pathogens. The study is a thorough, controlled, well researched analysis that will be valuable for designing and interpreting future studies. The authors discuss the limitations of the data and the potential directions. Clearly P. leucopus respond differently to the LPS exposure which is very interesting and opens the door for numerous other comparative studies.

The conclusions of the manuscript are thoughtful and mostly supported by the data, but there are a couple of points for clarification.

1) How were the number of animals for each experiment selected? Was a power analysis conducted?

2) The authors conducted a cursory evaluation of sex differences of P. leucopus and reported no difference in response except for Il6 and Il10 expression being higher in the males than the females in the exposed group. The data was not presented in the manuscript. Nor was sex considered for the other two species. A further discussion of the role that sex could play and future studies would be appreciated.

3) The ratio of Nos2 and Arg1 copies for LPS treated and control P. leucopus and M.musculus in Table 3 show that in P. leucopus there is not a significant difference but in M.musculus there is an increase in Nos2 copies with LPS treatment. The authors then used a targeted RNA-seq analysis to show that in P. leucopus the number of Arg1 reads after LPS treatment is significantly higher than the controls. These results are over oversimplified in the text as an inverse relationship for Nos2/Arg1 in the two species.

Reviewer #2 (Public Review):

Summary:

This work addresses the question of how human macrophages restrict intracellular replication of Salmonella.

Strengths:

Through a series of genetic knockouts and using specific inhibitors, Egan et al. demonstrated that the inflammasome components caspase-1, caspase-4, gasdermin D (GSDMD), and the final lytic death effector ninjurin-1 (NINJ1) are required for control of Salmonella replication in human macrophages. Interestingly, caspase-1 proved crucial in restricting Salmonella early during infection, whereas caspase-4 was essential in the later stages of infection. Furthermore, using a chloroquine resistance assay and state-of-the-art microscopy, the authors found that NAIP receptor and caspase-1 mostly regulate replication of cytosolic bacteria, with smaller, yet significant, impact on the vacuolar bacteria.

The finding that inflammasomes are critical in the restriction of replication of intracellular Salmonella in human macrophages contrasts with the published minimal role of inflammasomes in restriction of replication of intracellular Salmonella in murine macrophages. These findings demonstrate yet another example of interspecies and intercellular differences in regulation of bacterial infections by the immune system.

Weaknesses: none.

Reviewer #2 (Public Review):

Summary: The authors have previously demonstrated that the E3 ligase PDLIM2 inhibits NF-kB and STAT3 and is epigenetically repressed in human lung cancers (Sun et al. Nat. Comm. 2019 10: 5324); therefore, PDLIM2 is a tumor suppressor in lung cancer. In this manuscript, they follow up on their previous findings and show that expression of PDLIM2 is downregulated in human lung cancers by both genetic deletion and promoter methylation. They further describe a novel approach to restore the expression of PDLIM2 in mouse lung tumors by systemically administering PDLIM2 plasmids encapsulated in nanoparticles (termed "nanoPDLIM2"). The nanoPDLIM2 approach was shown to exhibit efficacy with low toxicity in a urethane-induced mouse lung cancer model. The authors further demonstrated the synergy of nanoPDLIM2 with chemotherapy and PD-1 blockade immunotherapy. The combination therapy of nanoPDLIM2, chemotherapy, and immunotherapy proved most effective with complete tumor remission in 60% of mice. Mechanistically, nanoPDLIM2 upregulated MHC-I expression, enhanced CD4/CD8 T cell activation and tumor infiltration, and suppressed MDR1 induction and nuclear expression of STAT3, RelA and prosurvival genes in tumors. Overall, this study is important because it reinforces the critical roles of PDLIM2 in suppressing lung cancer, and also identifies a potential approach to restoring PDLIM2 expression in lung tumors. The experiments were well executed; the data are convincing and support the conclusions made by the authors.

Reviewer #2 (Public Review):

In a study by Shen et al., the authors investigated YAP/TAZ target genes that play a role in the formation of processing bodies (P-bodies). P-bodies are membraneless cytoplasmic granules that contain translationally repressed mRNAs and components of mRNA turnover. GO enrichment analysis of the RNA-Seq data of colorectal cancer cells (HCT116) after YAP/TAZ knockdown showed that the downregulated genes were enriched in P-body resident proteins. Overexpression, knockdown, and ChIP-qPCR analyses showed that SAMD4A, PNRC1, AJUBA, and WTIP are YAP-TEAD target genes that also play a role in P-body biogenesis. Using P-body markers such as DDX6 and DCP1A, the authors showed that the knockdown of YAP in the HCT116 cell line causes a reduction in the number of P-bodies. Similarly, overexpression of constitutively active YAP (YAP 5SA) increased the P-body number. The YAP-TEAD target genes SAMD4A and AJUBA positively regulate P-body formation, because lowering their expression levels using siRNA reduces the number of P-bodies. The other YAP target gene, PNRC1, is a negative regulator of P-body biogenesis and consistently YAP suppresses its expression through the recruitment of the NuRD complex. YAP target genes that modulate P-body formation play prominent roles in oncogenesis. PNRC1 suppression is key to YAP-mediated proliferation, colony formation, and tumorigenesis in HCT116 xenografts. Similarly, SAMD4 and AJUBA knockdown abrogated cell viability. In summary, this study demonstrated that SAMD4, AJUBA, WTIP, and PNRC1 are bona fide YAP-TEAD target genes that play a role in P-body formation, which is also linked to the oncogenesis of colon cancer cells.

Major Strengths:

The majority of the experiments were appropriately planned so that the generated data could support the conclusions drawn by the authors. The phenotype observed with YAP/TAZ knockdown correlated inversely with YAP5SA overexpression, which is complementary. Where possible, the authors also used point mutations that selectively disrupt protein-protein interactions, such as YAP S94A and PNRC1 W300A. The CRC cell line HCT116 was used throughout the study; additionally, data from other cancer cell lines were used to support the generality of the findings.

Weaknesses:

The authors did not elucidate the mechanistic link between P-body formation and oncogenesis; therefore, it is unclear why an increase in the number of P-bodies is pro-tumorigenic. AJUBA and SAMD4 may have housekeeping functions and reduce the proliferation of YAP-independent cell lines. Figure 6 - Figure Supplement 4 shows a reduction in cell viability and migration in control HCT116 cell lines upon AJUBA/SAMD4 knockdown. Therefore, it is unclear whether their tumor suppressive role is YAP-dependent. The authors extrapolated and suggested that their findings could be exploited therapeutically, without providing much detail. How do they plan to stimulate the expression of PNRC1? It is not necessary for every scientific finding to lead to a therapeutic benefit; therefore, they can tone down such statements if therapeutic exploitation is not realistic. The authors elucidated a mechanism for PNRC1 repression and one wonders why no attempts were made to understand the mechanism of activation of SAMD4, AJUBA, and WTIP expression.

Reviewer #2 (Public Review):

Summary:<br /> ​In the present study, Gardeux et al provide a web-based tool for curated association mapping results from DRP studies. The tool lets users view association results for phenotypes and compare mean phenotype ~ phenotype correlations between studies. In the manuscript, the authors provide several example utilities associated with this new resource, including pan-study summary statistics for sex, traits, and loci. They highlight cross-trait correlations by comparing studies focused on longevity with phenotypes such as oxphos and activity.

Strengths:<br /> -Considerable efforts were dedicated toward curating the many DRG studies provided.<br /> -Available tools to query large DRP studies are sparse and so new tools present appeal

Weaknesses:<br /> The creation of a tool to query these studies for a more detailed understanding of physiologic outcomes seems underdeveloped. These could be improved by enabling usages such as more comprehensive queries of meta-analyses, molecular information to investigate given genes or pathways, and links to other information such as in mouse rat or human associations.

Reviewer #2 (Public Review):

Summary: In this study of the mouse homolog of acidic mammalian chitinase, the overall goal is to provide a mechanistic explanation for the unusual observation of two pH optima for the enzyme. The study includes biochemical assays to establish kinetic parameters at different solution pH, structural studies of enzyme/substrate complexes, and theoretical analysis of amino acid side chain pKas and molecular dynamics.

Strengths: The biochemical assays are rigorous and nicely complemented by the structural and computational analysis. The mechanistic proposal that results from the study is well rationalized by the observations in the study.

Weaknesses: The overall significance of the work could be made more clear. Additional details could be provided about the limitations of prior biochemical studies of mAMC that warranted the kinetic analysis. The mouse enzyme seems unique in terms of its behavior at high and low pH, so it remains unclear how the work will enhance broader understanding of this enzyme class. It was also not clear can the findings be used for therapeutic purposes, as detailed in the abstract, if the human enzyme works differently.

Reviewer #2 (Public Review):

This submission is about the role of Dbp5/Gle1 in tRNA export. The manuscript provides data showing that Dbp5/Gle1 are involved in tRNA export from the nucleus which is an essential process critical to translation. The authors provide data that largely supports conclusions, however, there are some pieces of data that are misinterpreted. (Figure 1A and B look the same; in Fig 1E, the DAPI staining is abnormal; in Fig 4 the bands can't be seen.)

Additionally, the methods used are fairly standard so the article does not contain any new technical achievements.

Reviewer #2 (Public Review):

The authors presented a well-written manuscript describing the comparison of active-learning methods with state-of-art methods for several datasets of pharmaceutical interest. This is a very important topic since active learning is similar to a cyclic drug design campaign such as testing compounds followed by designing new ones which could be used to further tests and a new design cycle and so on. The experimental design is comprehensive and adequate for proposed comparisons. However, I would expect to see a comparison regarding other regression metrics and considering the applicability domain of models which are two essential topics for the drug design modelers community.

Reviewer #2 (Public Review):

A limitation in using SNPs to understand recent histories of genomes is their low mutation frequency. Tellier et al. explore the possibility of adding hypermutable markers to SNP based methods for better resolution over short time frames. In particular, they hypothesize that epimutations (CG methylation and demethylation) could provide a useful marker for this purpose. Individual CGs in Arabidopsis tends to be either close to 100% methylated or close to 0%, and are inherited stably enough across generations that they can be treated as genetic markers. Small regions containing multiple CGs can also be treated as genetic markers based on their cumulative methylation level. In this manuscript, Tellier et al develop computational methods to use CG methylation as a hypermutable genetic marker and test them on theoretical and real data sets. They do this both for individual CGs and small regions. My review is limited to the simple question of whether using CG methylation for this purpose makes sense at a conceptual level, not at the level of evaluating specific details of the methods. I have a small concern in that it is not clear that CG methylation measurements are nearly as binary in other plants and other eukaryotes as they are in Arabidopsis. However, I see no reason why the concept of this work is not conceptually sound. Especially in the future as new sequencing technologies provide both base calling and methylating calling capabilities, using CG methylation in addition to SNPs could become a useful and feasible tool for population genetics in situations where SNPs are insufficient.

Reviewer #2 (Public Review):

In this study, Lewis et al seek to further define the role of ROM1. ROM1 is a tetraspanin protein that oligomerizes with another tetraspanin, PRPH2, to shape the rims of the membrane discs that comprise the light-sensitive outer segment of vertebrate photoreceptors. ROM1 knockout mice and several PRPH2 mutant mice are reexamined. The conclusion reached is that ROM1 is redundant to PRPH2 in regulating the size of newly forming discs, although excess PRPH2 is required to compensate for the loss of ROM1.

This replicates earlier findings while adding rigor using a mass spectrometry-based approach to quantitate the ratio of ROM1 and PRPH2 to rhodopsin (the protein packed in the body of the disc membranes) and careful analysis of tannic acid labeled newly forming discs using transmission electron microscopy.

In ROM1 knockout mice PRPH2 expression was found to be increased so that the level of PRPH2 in those mice matches the combined amount of PRPH2 and ROM1 in wildtype mice. Despite this, there are defects in disc formation that are resolved when the ROM1 knockout is crossed to a PRPH2 overexpressing line. A weakness of the study is that the molar ratios between ROM1, PRPH2 and rhodopsin were not measured in the PRPH2 overexpressing mice. This would have allowed the authors to be more precise in their conclusion that a 'sufficient' excess of PRPH2 can compensate for defects in ROM1.

Reviewer #2 (Public Review):

In this study, the authors examined how the maintenance of mitochondrial-associated endoplasmic reticulum membranes (MAM) is critical for the prevention of muscle atrophy under microgravity conditions. They observed, a reduction in MAM in myotubes placed in a microgravity condition; in addition, MFN2-deficient human iPS cells showed a decrease in the number of MAM, similar to in myotubes differentiated under microgravity conditions, in addition to the activation of the Notch signaling pathway. The authors, moreover, observed that treatment with the gamma-secretase inhibitor with DAPT preserved the atrophic phenotype of differentiated myotubes in microgravity and improve the regenerative capacity of Mfn2-deficient muscle stem cells in dystrophic mice.

The entire study was well conducted, bringing an interesting analysis in vitro and in vivo of aging conditions. In my opinion, it is necessary to improve the analysis of both genes and proteins to better support the conclusions

The study can contribute to a better understanding of one of the major problems of aging, such as muscle atrophy and inhibition of muscle regeneration, emphasizing the importance of the NOTCH pathway in these pathological situations. The work will be of interest to all scientists working on aging.

Reviewer #2 (Public Review):

Summary:

Cell-free DNA (cfDNA) are short DNA fragments released into the circulation when cells die. Plasma cfDNA level is thought to reflect the degree of cell-death or tissue injury. Indeed, plasma cfDNA is a reliable diagnostic biomarker for multiple diseases, providing insights into disease severity and outcomes. In this manuscript, Dr. Sender and colleagues address a fundamental question: What fraction of DNA released from cell death is detectable as plasma cfDNA? The authors use public data to estimate the amount of DNA produced from dying cells. They also utilize public data to estimate plasma cfDNA levels. Their calculations showed that <10% of DNA released is detectable as plasma cfDNA, the fraction of detectable cfDNA varying by tissue sources. The study demonstrates new and fundamental principles that could improve disease diagnosis and treatment via cfDNA.

Strengths:

1. The experimental approach is resource-mindful taking advantage of publicly available data to estimate the fraction of detectable cfDNA in physiological states. The authors did not assess if the fraction of detectable cfDNA changes in disease conditions. Nonetheless, their pioneering study lays the foundation and provides the methods needed for a similar assessment in disease states.<br /> 2. The findings of this study potentially explain discrepancies in measured versus expected tissue-specific cfDNA from some tissues. For example, the gastrointestinal tract is subject to high cell turnover and release of DNA. Yet, only a small fraction of that DNA ends up in plasma as gastrointestinal cfDNA.<br /> 3. The study proposes potential mechanisms that could account for the low fraction of detectable cfDNA in plasma relative to DNA released. This includes intracellular or tissue machinery that could "chew up" DNA released from dying cells, allowing only a small fraction to escape into plasma as cfDNA. Could this explain why the gastrointestinal track with an elaborate phagosome machinery contributes a small fraction of plasma cfDNA? Given the role of cfDNA as damage-associated molecular pattern in some diseases, targeting such a machinery may provide novel therapeutic opportunities.

Weaknesses: In vitro and in vivo studies are needed to validate these findings and define tissue machinery that contribute to cfDNA production. The validation studies should address the following limitations of the study design: -

1. Align the cohorts to estimate DNA production and plasma cfDNA levels. Cellular turnover rate and plasma cfDNA levels vary with age, sex, circadian clock, and other factors (Madsen AT et al, EBioMedicine, 2019). This study estimated DNA production using data abstracted from a homogenous group of healthy control males (Sender & Milo, Nat Med 2021). On the other hand, plasma cfDNA levels were obtained from datasets of more diverse cohort of healthy males and females with a wide range of ages (Loyfer et al. Nature, 2023 and Moss et al., Nat Commun, 2018).<br /> 2. "cfDNA fragments are not created equal". Recent studies demonstrate that cfDNA composition vary with disease state. For example, cfDNA GC content, fraction of short fragments, and composition of some genomic elements increase in heart transplant rejection compared to no-rejection state (Agbor-Enoh, Circulation, 2021). The genomic location and disease state may therefore be important factors to consider in these analyses.<br /> 3. Alternative sources of DNA production should be considered. Aside from cell death, DNA can be released from cells via active secretion. This and other additional sources of DNA should be considered in future studies. The distinct characteristics of mitochondrial DNA to genomic DNA should also be considered.

Reviewer #2 (Public Review):

Summary:<br /> This study seeks to advance our knowledge of how vitamin D may be protective in allergic airway disease in both adult and neonatal mouse models. The rationale and starting point are important human clinical, genetic/bioinformatic data, with a proposed role for vitamin D regulation of 2 human chromosomal loci (Chr17q12-21.1 and Chr17q21.2) linked to the risk of immune-mediated/inflammatory disease. The authors have made significant contributions to this work specifically in airway disease/asthma. They link these data to propose a role for vitamin D in regulating IL-2 in Th2 cells implicating genes associated with these loci in this process.

Strengths:<br /> Here the authors draw together evidence from multiple lines of investigation to propose that amongst murine CD4+ T cell populations, Th2 cells express high levels of VDR, and that vitamin D regulates many of the genes on the chromosomal loci identified to be of interest, in these cells. The bottom line is the proposal that vitamin D, via Ikfz3/Aiolos, suppresses IL-2 signalling and reduces IL-2 signalling in Th2 cells. This is a novel concept and whilst the availability of IL-2 and the control of IL-2 signalling is generally thought to play a role in the capacity of vitamin D to modulate both effector and especially regulatory T cell populations, this study provides new data.

Weaknesses:<br /> Overall, this is a highly complicated paper with numerous strands of investigation, methodologies etc. It is not "easy" reading to follow the logic between each series of experiments and also frequently fine detail of many of the experimental systems used (too numerous to list), which will likely frustrate immunologists interested in this. There is already extensive scientific literature on many aspects of the work presented, much of which is not acknowledged and largely ignored. For example, reports on the effects of vitamin D on Th2 cells are highly contradictory, especially in vitro, even though most studies agree that in vivo effects are largely protective. Similarly, other reports on adult and neonatal models of vitamin D and modulation of allergic airway disease are not referenced. In summary, the data presentation is unwieldy, with numerous supplementary additions, which makes the data difficult to evaluate and the central message lost. Whilst there are novel data of interest to the vitamin D and wider community, this manuscript would benefit from editing to make it much more readily accessible to the reader.

Wider impact: Strategies to target the IL-2 pathway have long been considered and there is a wealth of knowledge here in autoimmune disease, transplantation, GvHD etc - with some great messages pertinent to the current study. This includes the use of IL-2, including low dose IL-2 to boost Treg but not effector T cell populations, to engineered molecules to target IL-2/IL-2R.

Reviewer #2 (Public Review):

Summary:<br /> The present study explores the molecular function of LRRC23 in male fertility, specifically in the context of the regulation of spermiogenesis. The author initiates the investigation by identifying LRRC23 mutations as a potential cause of male sterility based on observations made in closely related individuals affected by asthenozoospermia (ASZ). To further investigate the function of LRRC23 in spermatogenesis, mutant mice expressing truncated LRRC23 proteins are created, aligning with the identified mutation site. Consequently, the findings confirm the deleterious effects of LRRC23 mutations on sperm motility in these mice while concurrently observing no significant abnormalities in the overall flagella structure. Furthermore, the study reveals LRRC23's interaction with the RS head protein RSPH9 and its active involvement in the assembly of the axonemal RS. Notably, LRRC23 mutations result in the loss of the RS3 head structure and disruption of the RS2-RS3 junction structure. Therefore, the author claimed that LRRC23 is an indispensable component of the RS3 head structure and suggests that mutations in LRRC23 underlie sterility in mice.

Strengths:<br /> The key contribution of this article lies in confirming LRRC23's involvement in assembling the RS3 head structure in sperm flagella. This finding represents a significant advancement in understanding the complex architecture of the RS3 structural complex, building upon previous studies. Moreover, the article's topic is interesting and originates from clinical research, which holds significant implications for potential clinical applications.

Weaknesses:<br /> 1. While the author generated mutant mice expressing truncated LRRC23 proteins, the expression of these truncated proteins was not detected in sperm. This implies that, in terms of sperm structure, the mutant LRRC23 protein behaves similarly to the complete knockout of the LRRC23 protein, which has been previously reported and characterized (Zhang et al., 2021).

2. This reviewer questions the proposal that LRRC23 is an integral component of RS3, as the results indicate not only the loss of the RS3 head structure but also an incomplete RS2-RS3 junction structure. In addition, the interaction of LRRC23 with RSPH9 alone does not fully explain its involvement solely in RS3 assembly. Additional evidence is required to examine the influence of LRRC23 on the RS2-RS3 junction.

3. The article does not explore how these mutations affect the flagella structure in human sperm, which needs further study. Expanding the study to include human sperm structure would undoubtedly enhance the quality of the article.

Reviewer #2 (Public Review):

This study investigates the excitability of neurons in the peri-infarct cortex during recovery from ischemic stroke. The excitability of neurons in the peri-infarct cortex during stroke recovery has produced contradictory findings: some studies suggest hyper-excitability to direct-brain stimulation, while others indicate diminished responsiveness to physical stimuli. However, most studies have used anesthetized animals, which can disrupt cortical activity and functional connectivity. The present study used two-photon Ca2+ imaging after focal photothrombotic stroke to examine neural activity patterns in awake mice. The authors found reduced neuronal spiking in the peri-infarct cortex that was strongly correlated with motor performance deficits. Additionally, the authors found disruptions in neural activation, functional connectivity, and assembly architecture in the immediate peri-infarct region but not in the distal cortex regions.

The findings of this study are very important as they show that there is no measurable change in terms of neuronal activation and reorganization in distal regions of remapped cortical response areas after stroke.

However, cortical response areas are calculated using a threshold of 95% peak activity within a trial. The threshold is presumably used to discriminate between the sensory-evoked response and collateral activation / less "relevant" response (noise). Since the peak intensity is lower after stroke, the "response" area is larger - lower main signal results in less noise exclusion. Predictably, areas that show a higher response before stroke than after are excluded from the response area before the stroke and included after.

We suggest a reinterpretation of the findings: much of the non-remapped areas are included when using a within-trial threshold as a criterion, and the absence of increased neuronal activation and reorganization is evidence for this claim. The take-home message of this study should be that we need a much better criterion for what we consider remapping.

Reviewer #2 (Public Review):

Summary:<br /> This is an interesting and impressive study that provides a rare opportunity to learn about brain-behaviour links of learning systems at a relatively early stage of development.

Strengths:<br /> The main strengths are that the authors followed a relatively large group of children over 2 years and used a reinforcement learning task aimed at assessing learning that depends on both the striatum and the hippocampus. The authors also included a thorough overview of the computational models and the choices they made. I think this paper would be of considerable interest and contributes to knowledge about how learning and memory systems change with development.

Weaknesses:<br /> There were a few things that I thought would be helpful to clarify. First, what exactly are the anatomical regions included in the striatum here? Second, it was mentioned that for the reduced dataset, object recognition memory focused on "sure" ratings. This seems like the appropriate way to do it, but it was not clear whether this was also the case for the full analyses in the main text. Third, the children's fitted parameters were far from optimal; is it known whether adults would be closer to optimal on the task?

The main thing I would find helpful is to better integrate the differences between the main results reported and the many additional results reported in the supplement, for example from the reduced dataset when excluding non-learners. I found it a bit challenging to keep track of all the differences with all the analyses and parameters. It might be helpful to report some results in tables side-by-side in the two different samples. And if relevant, discuss the differences or their implication in the Discussion. For example, if the patterns change when excluding the poor learners, in particular for the associations between delayed feedback and hippocampal volume, and those participants were also those less well fit by the value-based model, is that something to be concerned about and does that affect any interpretations? What was not clear to me is whether excluding the poor learners at one extreme simply weakens the general pattern, or whether there is a more qualitative difference between learners and non-learners. The discussion points to the relevance of deficits in hippocampal-dependent learning for psychopathology and understanding such a distinction may be relevant.

Reviewer #2 (Public Review):

This study examined the role of CRF neurons in the BNST in both phasic and sustained fear in males and females. The authors first established a differential fear paradigm whereby shocks were consistently paired with tones (Full) or only paired with tones 50% of the time (Part), or controls who were exposed to only tones with no shocks. Recall tests established that both Full and Part conditioned male and female mice froze to the tones, with no difference between the paradigms. Additional studies using the NSF and startle test, established that neither fear paradigm produced behavioral changes in the NSF test, suggesting that these fear paradigms do not result in an increase in anxiety-like behavior. Part fear conditioning, but not Full, did enhance startle responses in males but not females, suggesting that this fear paradigm did produce sustained increases in hypervigilance in males exclusively. Photometry studies found that while undifferentiated BNST neurons all responded to shock itself, only Full conditioning in males lead to a progressive enhancement of the magnitude of this response. BNST neurons in males, but not females, were also responsive to tone onset in both fear paradigms, but only in Full fear did the magnitude of this response increase across training. Knockdown of CRF from the BNST had no effect on fear learning in males or females, nor any effect in males on fear recall in either paradigm, but in females enhanced both baseline and tone-induced freezing only in Part fear group. When looking at anxiety following fear training, it was found in males that CRF knockdown modulated anxiety in Part fear trained animals and amplified startle in Fully trained males but had no effect in either test in females. Using 1P imaging, it was found that CRF neurons in the BNST generally decline in activity across both conditioning and recall trials, with some subtle sex differences emerging in the Part fear trained animals in that in females BNST CRF neurons were inhibited after both shock and omission trials but in males this only occurred after shock and not omission trials. In recall trials, CRF BNST neuron activity remained higher in Part conditioned mice relative to Full conditioned mice.

Overall, this is a very detailed and complex study that incorporates both differing fear training paradigms and males and females, as well as a suite of both state of the art imaging techniques and gene knockdown approaches to isolate the role and contributions of CRF neurons in the BNST to these behavioral phenomena. The strengths of this study come from the thorough approach that the authors have taken, which in turn helped to elucidate nuanced and sex specific roles of these neurons in the BNST to differing aspects of phasic and sustained fear. More so, the methods employed provide a strong degree of cellular resolution for CRF neurons in the BNST. In general, the conclusions appropriately follow the data, although the authors do tend to minimize some of the inconsistencies across studies (discussed in more depth below), which could be better addressed through discussion of these in greater depth. As such, the primary weakness of this manuscript comes largely from the discussion and interpretation of mixed findings without a level of detail and nuance that reflects the complexity, and somewhat inconsistency, across the studies. These points are detailed below:

-Given the focus on CRF neurons in the BNST, it is unclear why the photometry studies were performed in undifferentiated BNST neurons as opposed to CRF neurons specifically (although this is addressed, to some degree, subsequently with the 1P studies in CRF neurons directly). This does limit the continuity of the data from the photometry studies to the subsequent knockdown and 1P imaging studies. The authors should address the rationale for this approach so it is clear why they have moved from broader to more refined approaches.

-The CRF KD studies are interesting, but it remains speculative as to whether these effects are mediated locally in the BNST or due to CRF signaling at downstream targets. As the literature on local pharmacological manipulation of CRF signaling within the BNST seems to be largely performed in males, the addition of pharmacological studies here would benefit this to help to resolve if these changes are indeed mediated by local impairments in CRF release within the BNST or not. While it is not essential to add these experiments, the manuscript would benefit from a more clear description of what pharmacological studies could be performed to resolve this issue.

-While I can appreciate the authors perspective, I think it is more appropriate to state that startle correlates with anxiety as opposed to outright stating that startle IS anxiety. Anxiety by definition is a behavioral cluster involving many outputs, of which avoidance behavior is key. Startle, like autonomic activation, correlates with anxiety but is not the same thing as a behavioral state of anxiety (particularly when the startle response dissociates from behavior in the NSF test, which more directly tests avoidance and apprehension). Throughout the manuscript the use of anxiety or vigilance to describe startle becomes interchangeable, but then the authors also dissociate these two, such as in the first paragraph of the discussion when stating that the Part fear paradigm produces hypervigilance in males without influencing fear or anxiety-like behaviors. The manuscript would benefit from harmonization of the language used to operationally define these behaviors and my recommendation would be to remain consistent with the description that startle represents hypervigilance and not anxiety, per se.

-The interpretation of the anxiety data following CRF KD is somewhat confusing. First, while the authors found no effect of fear training on behavior in the NSF test in the initial studies, now they do, however somewhat contradictory to what one would expect they found that Full fear trained males had reduced latency to feed (indicative of an anxiolytic response), which was unaltered by CRF KD, but in Part fear (which appeared to have no effect on its own in the NSF test), KD of CRF in these animals produced an anxiolytic effect. Given that the Part fear group was no different from control here it is difficult to interpret these data as now CRF KD does reduce latency to feed in this group, suggesting that removal of CRF now somehow conveys an anxiolytic response for Part fear animals. In the discussion the authors refer to this outcome as CRF KD "normalizing" the behavior in the NSF test of Part fear conditioned animals as now it parallels what is seen after Full fear, but given that the Part fear animals with GFP were no different then controls (and neither of these fear training paradigms produced any effect in the NSF test in the first arm of studies), it seems inappropriate to refer to this as "normalization" as it is unclear how this is now normalized. Given the complexity of these behavioral data, some greater depth in the discussion is required to put these data in context and describe the nuance of these outcomes, in particular a discussion of possible experimental factors between the initial behavioral studies and those in the CRF KD arm that could explain the discrepancy in the NSF test would be good (such as the inclusion of surgery, or other factors that may have differed between these experiments). These behavioral outcomes are even more complex given that the opposite effect was found in startle whereby CRF KD amplified startle in Full trained animals. As such, this portion of the discussion requires some reworking to more adequately address the complexity of these behavioral findings.

Reviewer #2 (Public Review):

In this manuscript, González-Segarra et al. investigated how ISNs regulate sugar and water ingestion in Drosophila. In their previous paper, authors have shown that inhibiting neurotransmission in ISNs has opposite effects on sugar and water ingestion. In this manuscript, the authors first identified the effector molecules released by ISNs. Their RNAi screen found that, surprisingly, ISNs use ilp3 as a neuromodulator. Next, using light and electron microscopy, they investigated the downstream neural circuits ISNs connect with to regulate water or sugar ingestion. These analyses identified a new group of neurons named Bilateral T-shaped neurons (BiT) as the main output of ISNs, and several other peptidergic neurons as downstream effectors of ISNs. While BiT activity regulated both sugar and water ingestion, BiT downstream neurons, such as CCHa2R, only impacted water ingestion. These results suggested that ISNs might interact with distinct neural circuits to control sugar or water ingestion. The authors also investigated other ISN downstream neurons, such as ilp2 and CCAP, and revealed that their activity also contributes to ingestive behaviors in flies.

Major strengths:<br /> 1. This manuscript presents a comprehensive investigation of the downstream neurons connected to ISNs.

2. The authors have identified and characterized a diverse set of peptidergic neurons that regulate ingestive behaviors in the fly brain.

Weaknesses:<br /> 1. Only one RNAi hairpin is used to knock down Ilp3 in ISNs? There is a concern about off-targeting effects without the presence of another hairpin or mutant data. Do ilp3 mutants also have similar defects in sugar/water ingestion compared to ISN ilp3 knockdown?

2. Throughout the paper, authors use either voltage or calcium sensors without explaining why they choose to use either method to determine the functional connectivity between neurons.

3. How these diverse sets of peptidergic neurons interact to regulate ingestive behaviors is unclear and requires further investigation.

Reviewer #2 (Public Review):

Summary:

This manuscript by Kroon, Grunewald, Marrink and coworkers present the development of Vermouth library for coarse grain assignment and parameterization and an updated version of python script, the Martinize2 program, to build Martini coarse grained (CG) models, primarily for protein systems.

Strengths:

In contrast to many mature and widely used tools to build all-atom (AA) models, there are few well-accepted programs for CG model constructions and parameterization. The research reported in this manuscript is among the ongoing efforts to build such tools for Martini CG modeling, with a clear goal of high-throughput simulations of complex biomolecular systems and, ultimately, whole-cell simulations. Thus, this manuscript targets a practical problem in computational biophysics. The authors see such an effort to unify operations like CG mapping, parameterization, etc. as a vital step from the software engineering perspective.

Weaknesses:

However, the manuscript in this shape is unclear in the scientific novelty and appears incremental upon existing methods and tools. The only "validation" (more like an example application) is to create Martini models with two protein structure sets (I-TASSER and AlphaFold). The success rate in building the models was only 73%, while the significant failure is due to incomplete AA coordinates. This suggests a dependence on the input AA models, which makes the results less attractive for high-throughput applications (for example, preparation/creation of the AA models can become the bottleneck). There seems to be an improvement in considering the protonation state and chemical modification, but convincing validation is still needed. Besides, limitations in the existing Martini models remain (like the restricted dynamics due to the elastic network, the electrostatic interactions or polarizability).

Reviewer #2 (Public Review):

In this manuscript, authors firstly investigated the role of a transcriptional factor BATF in hepatic lipid metabolism both in vivo and in vitro. By using a AAV transfection to overexpress BATF in liver, the mice with overexpression of BATF resisted the high fat diets induced obesity and attenuated the hepatic steatosis. Mechanically, the PD1 mediated its effect on lipid accumulation in hepatocyte and IL-27 mediated its effect on adiposity reduction in vivo.

Strengths:

1) This work found the transcription factor BATF was positive to reduce hepatic lipid accumulation and offered a potential target to treat NAFLD.<br /> 2) PD1 antibody is always used to treat cancer, authors here have developed its new function in metabolic disease. PD1 antibody could help mice to combat obesity and hepatic steatosis induced by high fat diets.<br /> 3) Overexpression of BATF in the liver not only decreased the lipid accumulation in the liver but also reduced the fat mass. IL-27 secretion in the liver was enhanced to affect the adipose tissue. The cross talk in liver and adipose tissue was also validated in this paper.

Reviewer #2 (Public Review):

The manuscript by G. Viejo et al. describes a new open-source toolbox called Pynapple, for data analysis of electrophysiological recordings, calcium imaging, and behavioral data. It is an object-oriented python package, consisting of 5 main object types: timestamps (Ts), timestamped data (Tsd), TsGroup, TsdFrame, and IntervalSet. Each object has a set of built-in core methods and import tools for common data acquisition systems and pipelines.

Pynapple is a low-level package that uses NWB as a file format, and further allows for other more advanced toolsets to build upon it. One of these is called Pynacollada which is a toolset for data analysis of electrophysiological, imaging, and behavioral data.

Pynapple and Pynacollada have the potential to become very valuable and foundational tools for the analysis of neurophysiological data. NWB still has a steep learning curve and Pynapple offers a user-friendly toolset that can also serve as a wrapper for NWB.

Reviewer #2 (Public Review):

In this study, Lamire et al. use a calcium imaging approach, behavioural tests, and pharmacological manipulations to identify the molecular mechanisms behind visual habituation. They show a valuable drug screen paradigm to assess the impact of pharmacological compounds on the behaviour of larval zebrafish.

The pharmacological screen identifies an expected suppression of habituation by GABA receptor antagonists. More interestingly, it identifies potentially new contributions of melatonin receptor agonists, and oestrogen receptor agonists to habituation, as they seem to increase the rate of habituation.

The volumetric calcium imaging of habituation to dark flashes is valuable, but the mix of responses to visual cues that are not relevant to the dark flash escape, such as the slow increase back to baseline luminosity, lowers the clarity of the results. The link between the calcium imaging results and free-swimming behaviour is not especially convincing, however, that is a common issue of head-restrained imaging with larval zebrafish. The identification of a cluster of neurons with potentiating responses, which could drive the habituation is intriguing, but more characterizations of these neurons would be needed to fully understand their function in habituation. The pharmacological manipulation of the habituation circuits mapped in the first part does not arrive at any satisfying conclusion, which is acknowledged by the authors.

Overall, the authors did identify interesting new molecular pathways that may be involved in habituation to dark flashes. Their screening approach, while not novel, will be a powerful way to interrogate other behavioural profiles. The authors identified circuit loci apparently involved in habituation to dark flashes, and the potentiation and no adaptation clusters have not been previously observed and are interesting targets for future work. This work suggests that the circuits and mechanisms underlying habituation are likely more complex than anticipated. The data will be useful to guide follow-up experiments by the community on the new pathway candidates that this screen has uncovered, including behaviours beyond dark flash habituation.

Reviewer #3 (Public Review):

Moutard, Laura, et al. investigated the gene expression and functional aspects of Leydig cells in a cryopreservation/long-term culture system. The authors found that critical genetic markers for Leydig cells were diminished when compared to the in-vivo testis. The testis also showed less androgen production and androgen responsiveness. Although they did not produce normal testosterone concentrations in basal media conditions, the cultured testis still remained highly responsive to gonadotrophin exposure, exhibiting a large increase in androgen production. Even after the hCG-dependent increase in testosterone, genetic markers of Leydig cells remained low, which means there is still a missing factor in the culture media that facilitates proper Leydig cell differentiation. Optimizing this testis culture protocol to help maintain proper Leydig cell differentiation could be useful for future human testis biopsy cultures, which will help preserve fertility and child cancer patients.

Overall, the authors addressed most comments and questions from the previous review. The additional data regarding the necrotic area is helpful for interpreting the quality of the cultures.

The authors did not conduct a multiple comparison tests although there are multiple comparisons conducted on for a single dependent variable (Fig 2J, Fig 3F, among many others), however, the addition of this multiple comparison is unlikely to change the conclusions of the paper or the figure and, thus is a minor technical detail in this case.

Reviewer #2 (Public Review):

Summary:<br /> Ma et al. employed a myeloid progenitor/microglia differentiation protocol to produce human-induced pluripotent stem cell (hiPSC)-derived microglia in order to examine the potential of microglial cell replacement as a treatment for retinal disorders. They characterized the iPSC-derived microglia by gene expression and in vitro assay analysis. By evaluating xenografted microglia in the partly microglia-depleted retina, the function of the microglia was further assessed.

Strengths:

Overall, the study and the data are convincing, and xenografted microglia were also tested in a RPE injury paradigm.

Weaknesses:

Gene expression analysis of mature microglia cells should be better interpreted and it would be beneficial to compare the iPSC-derived microglia gene set to a human microglial cell line (for example, HMC3) instead of myeloid progenitor cells.<br /> The way that the manuscript has been written, unfortunately, is not optimal. I recommend that the entire manuscript be edited and proofread in English. The text contains spelling and grammar mistakes, and the manuscript is inconsistent in several parts. The manuscript should also be revised for a scientific paper format.

Reviewer #2 (Public Review):

Understanding disease conditions often yields valuable insights into the physiological regulation of biological functions, as well as potential therapeutic approaches. In previous investigations, the author's research group identified abnormal expression of brain-derived neurotrophic factor (BDNF) in the hypothalamus of a mouse model exhibiting Smith-Magenis syndrome (SMS), which is caused by heterozygous mutations of the Rai1 gene. Human SMS is associated with distinct facial characteristics, sleep disturbances, behavioral issues, and intellectual disabilities, often accompanied by obesity. Conditional knockout (cKO) of the Bdnf gene from the paraventricular hypothalamus (PVH) in mice led to hyperphagic obesity, while overexpression of the Bdnf gene in the PVH of Rai1 heterozygous mice restored the SMS-like obese phenotype. Based on these preceding findings, the authors of the present study discovered that homozygous Rai1 cKO restricted to Bdnf-expressing cells, or Rai1 gene knockdown solely in Bdnf-positive neurons in the PVH, induced obesity along with intricate alterations in adipose tissue composition, energy expenditure, locomotion, feeding patterns, and glucose tolerance, some of which varied between sexes. Additionally, the authors demonstrated that a brain-penetrating drug capable of activating the TrkB pathway, a downstream signaling pathway of BDNF, partially alleviated the SMS-like obesity phenotype in female mice with Rai1 heterozygous mutations. Although the specific (neural) cell type responsible for this TrkB signaling remains an open question, the present study unequivocally highlights the importance of Rai1 gene function in PVH Bdnf neurons for the obesity phenotype, providing valuable insights into potential therapeutic strategies for managing obesity associated with SMS.

In the proteomic analysis (Fig. 1), the authors elucidated that multiple phospho-protein signaling pathways, including Akt and mTOR pathways, exhibited significant attenuation in the SMS model mice. Of significance, the manifestation of haploinsufficiency of the Rai1 gene exclusively within the BDNF+ cells demonstrated negligible impact on body weight (Fig. 2-supple 3D), despite observing a reduction in BDNF levels in the heterozygous Rai1 mutant (Fig. 1A). Conversely, the homozygous Rai1 cKO in the BDNF+ cells prominently displayed an obesity phenotype, suggesting substantial dissimilarities in the gene expression profiles between Rai1 heterozygous and homozygous conditions within the BDNF+ cell population. It would be advantageous to precisely identify the responsible differentially expressed genes, possibly including Bdnf itself, in the homozygous cKO model. The observed reduction in the excitability of PVH BDNF+ cells (Fig. 3) is presumably attributed to aberrant gene expression other than Bdnf itself, which may serve as a prospective target for gene expression analysis. Notably, the Rai1 homozygous cKO mice in BDNF+ cells exhibited some sexual dimorphisms in feeding and energy expenditures, as evidenced by Fig. 2 and related figures. Exploring the potential relevance of these sexual differences to human SMS cases and investigating the underlying cellular/molecular mechanisms in the future would provide valuable insights.

Although the CRISPR-mediated knockdown of the Rai1 gene (Fig. 4) appears to be highly effective, given the broad transduction of AAV serotype 9, it may be helpful to exclude the possibility of other brain regions adjacent to the PVH, such as the DMH or VMH, being affected by this viral procedure. If the PVH-specificity is established, the majority of Rai1 cKO effects in Bdnf+ cells are primarily attributed to PVH-Bdnf+ cells based on the similarity of phenotypes observed. With regards to the apparent rescue of the body weight phenotype in Rai1 heterozygous mutants using a selective TrkB activator, the specific biological processes, and neurons responsible for this effect remain unclear to this reviewer. Elucidating these aspects would be significant when considering potential applications to human SMS cases.

Overall, the present study represents a valuable addition to the authors' series of high-quality molecular genetic investigations into the in vivo functions of the Rai1 gene. This reviewer particularly commends their diligent efforts to enhance our comprehension of SMS and contribute to the future development of more effective therapies for this syndrome.

Reviewer #2 (Public Review):

The authors provide convincing evidence that optogenetic stimulation of ChR2-expressing motor neurons implanted in muscles effectively restores innervation of severely affected skeletal muscles in the aggressive SOD1 mouse model of ALS, and conclude that this method can be applied to selectively control the function of implicated muscles. This was supported by convincing data presented in the paper.

This is an interesting paper providing new/improved optogenetic methods to restore or improve muscle strength in ALS. In general, it is of high significance in both the techniques and concept, and the paper was well written. The evidence supporting the conclusions is convincing, with rigorous muscle tension physiological analysis, and nerve and muscle histology and image analysis. The work will be of broad interest to medical biologists on muscle disorders.

One weak point is that proper control experiments were not clearly presented - these could be shown in the paper. For example, one control experiment with only YFP but no ChR2 expression with optogenetic stimulation should be performed, following similar procedures and analysis applied to the ChR2-transduced animals.

Reviewer #2 (Public Review):

Summary: Two early Cambrian taxa of linguliform brachiopods are assigned to the family Eoobolidae. The taxa exhibit a columnar shell structure and the phylogenetic implications of this shell structure in relation to other early Cambrian families are discussed.

Strengths: Interesting idea regarding the evolution of shell structure.

Weaknesses: The early record of shell structures of linguliform brachiopods is incomplete and partly contradictory. The authors maintain silence regarding contradictory information throughout the article to the extent that information is cited wrongly.<br /> The structure and language of the article need reworking in my opinion, the systematic part can be in the appendix but the main results and the results relevant for the discussion should be in the main article. A critical revision of the family Eoobolidae and Lingulellotretidae including a revision of the type species of Eoobolus and Lingulellotreta is needed.

Reviewer #2 (Public Review):

I have read with great interest the manuscript entitled "The optimal clutch size revisited: separating individual quality from the costs of reproduction" by LA Winder and colleagues. The paper consists in a meta-analysis comparing survival rates from studies providing clutch sizes of species that are unmanipulated and from studies where the clutch sizes are manipulated, in order to better understand the effects of differences in individual quality and of the costs of reproduction. I find the idea of the manuscript very interesting. However, I am not sure the methodology used allows to reach the conclusions provided by the authors (mainly that there is no cost of reproduction, and that the entire variation in clutch size among individuals of a population is driven by "individual quality").

I write that I am not sure, because in its current form, the manuscript does not contain a single equation, making it impossible to assess. It would need at least a set of mathematical descriptions for the statistical analysis and for the mechanistic model that the authors infer from it.<br /> The texts mixes concepts of individual vs population statistics, of within individual vs among-individuals measures, of allocation trade-offs and fitness trade-offs, etc ....which means it would also require a glossary of the definitions the authors use for these various terms, in order to be evaluated.

This problem is emphasised by the following sentence to be found in the discussion "The effect of birds having naturally larger clutches was significantly opposite to the result of increasing clutch size through brood manipulation". The "effect" is defined as the survival rate (see Fig 1). While it is relatively easy to intuitively understand what the "effect" is for the unmanipulated studies: the sensitivity of survival to clutch size at the population level, this should be mentioned and detailed in a formula. Moreover, the concept of effect size is not at all obvious for the manipulated ones (effect of the manipulation? or survival rate whatever the manipulation (then how could it measure a trade-off ?)? at the population level? at the individual level ?) despite a whole appendix dedicated to it. This absolutely needs to be described properly in the manuscript.

Despite the lack of information about the underlying mechanistic model tested and the statistical model used, my impression is still that the interpretation in the introduction and discussion is not granted by the outputs of the figures and tables. Let's use a model similar to that of (van Noordwijk and de Jong, 1986): imagine that the mechanism at the population level is<br /> a.c_(i,q)+b.s_(i,q)=E_q<br /> Where c_(i,q) are s_(i,q) are respectively the clutch size for individual i which is of quality q, and E_q is the level of "energy" that an individual of quality q has available during the given time-step (and a and b are constants turning the clutch size and survival rate into energy cost of reproduction and energy cost of survival, and there are both quite "high" so that an extra egg (c_(i,q) is increased by 1) at the current time-step, decreases s_(i,q) markedly (E_q is independent of the number of eggs produced), that is, we have strong individual costs of reproduction). Imagine now that the variance of c_(i,q) (when the population is not manipulated) among individuals of the same quality group, is very small (and therefore the variance of s_(i,q) is very small also) and that the expectation of both are proportional to E_q. Then, in the unmanipulated population, the variance in clutch size is mainly due to the variance in quality. And therefore, the larger the clutch size c_(i,q) the higher E_q, and the higher the survival s_(i,q).<br /> In the manipulated populations however, because of the large a and b, an artificial increase in clutch size, for a given E_q, will lead to a lower survival s_(i,q). And the "effect size" at the population level may vary according to a,b and the variances mentioned above. In other words, the costs of reproduction may be strong, but be hidden by the data, when there is variance in quality; however there are actually strong costs of reproduction (so strong actually that they are deterministic and that the probability to survive is a direct function of the number of eggs produced)

Moreover, it seems to me that the costs of reproduction are a concept closely related to generation time. Looking beyond the individual allocative (and other individual components of the trade-off) cost of reproduction and towards a populational negative relationship between survival and reproduction, we have to consider the intra-population slow fast continuum (some types of individuals survive more and reproduce less (are slower) than other (which are faster)). This continuum is associated with a metric: the generation time. Some individuals will produce more eggs and survive less in a given time-period because this time-period corresponds to a higher ratio of their generation time (Gaillard and Yoccoz, 2003; Gaillard et al., 2005). It seems therefore important to me, to control for generation time and in general to account for the time-step used for each population studied when analysing costs of reproduction. The data used in this manuscript is not just clutch size and survival rates, but clutch size per year (or another time step) and annual (or other) survival rates.

Finally, it is important to relate any study of the costs of reproduction in a context of individual heterogeneity (in quality for instance), to the general problem of the detection of effects of individual differences on survival (see, e.g., Fay et al., 2021). Without an understanding of the very particular statistical behaviour of survival, associated to an event that by definition occurs only once per life history trajectory (by contrast to many other traits, even demographic, where the corresponding event (production of eggs for reproduction, for example) can be measured several times for a given individual during its life history trajectory).

References:<br /> Fay, R. et al. (2021) 'Quantifying fixed individual heterogeneity in demographic parameters: Performance of correlated random effects for Bernoulli variables', Methods in Ecology and Evolution, 2021(August), pp. 1-14. doi: 10.1111/2041-210x.13728.<br /> Gaillard, J.-M. et al. (2005) 'Generation time: a reliable metric to measure life-history variation among mammalian populations.', The American naturalist, 166(1), pp. 119-123; discussion 124-128. doi: 10.1086/430330.<br /> Gaillard, J.-M. and Yoccoz, N. G. (2003) 'Temporal Variation in Survival of Mammals: a Case of Environmental Canalization?', Ecology, 84(12), pp. 3294-3306. doi: 10.1890/02-0409.<br /> van Noordwijk, A. J. and de Jong, G. (1986) 'Acquisition and Allocation of Resources: Their Influence on Variation in Life History Tactics', American Naturalist, p. 137. doi: 10.1086/284547.

Reviewer #2 (Public Review):

To reveals cellular and molecular heterogeneity in enthesis, the authors established a single-cell temporal atlas during development. This study provides a transcriptional resource for further investigation of fibrocartilage development.

Reviewer #2 (Recommendations for the authors):

1. As known, Fei Fang et al. have established single-cell transcriptomes of mouse supraspinatus tendon enthesis cells (Cell Stem Cell, 2022). It is suggested that the authors introduced Fei Fang et al.'s work in Introduction and emphasize the significant novelty compared with Fei Fang et al.'s work.<br /> 2. In Fig1, the authors highlighted P7 was a critical stage for enthesis differentiation. But this section was less associated with the following content. The authors should link these results with the scRNASeq data. Is there any time-dependent change/signaling in scRNASeq data at this critical time point?<br /> 3. In the H&E staining of Fig1a, the tendon structure was separated and random. It is suggested that the authors provide high-quality staining figures.<br /> 4. Fig2 showed that the Scx+ or Sox9+ cells was decreased in enthesis over time. At least it should be co-staining to show the distribution and frequency of double positive and single positive cell populations. However, a previous study has demonstrated this finding (PLOS ONE, 2020). It is suggested to verify some new findings by IF or IHC staining.<br /> 5. There are some conflicts about trajectory analysis. In Fig3c, RNA velocity showed that the arrow flowed from BTJ to MTJ and CTFb. However, in Fig3d, PAGA plot indicated that BTJ cells is independent of other cells. Furthermore, in supplementary figure S3, RNA velocity showed that the trajectory flowed from TC to BTJ. These figures were inconsistent with the described results. Please provide detailed explanation to avoid misleading readers.<br /> 6. Fig5 showed that C1 was the original cluster, and whether C1 cluster expressed canonical progenic/stem cell markers.<br /> 7. The authors performed cell-cell interaction based on cellchat analysis. But the cell-cell interaction was not actively examined.

Reviewer #2 (Public Review):

Diffusion MRI is sensitive to the brain microstructure, and it has been used to assess the integrity of white matter for nearly 3 decades. Its main limitation is the limited specificity, which makes it difficult to link changes in diffusion parameters to a given pathological substrate. Recently methods based on diffusion MRI that enable the estimation of axonal diameter, non invasively, have become available. This paper aims at validating one of such methods using an experimental model of neurodegeneration. The authors found a significant correlation between axonal diameter estimated by MRI and an histological marker of neurodegeneration. Although this is of great interest, as it demonstrates that this method is sensitive to neurodegeneration, a direct validation would require a measurement of axonal diameter using electron or confocal microscopy, rather than a correlation with a measure of axonal degeneration not directly related to axonal diameter. So, although these data are compelling, they do not prove that the increase in axonal diameter suggested by diffusion MRI corresponds to actual axonal swelling. The Authors also apply the same method to compare the white matter of patients with multiple sclerosis (MS) and healthy controls, showing widespread increases in axonal diameter in the patients. These data are compelling, but again, not conclusive. Other factors such as gloss could bias the MRI measurement and lead to an apparent increase in axonal diameter.

Reviewer #2 (Public Review):

The paper "Polymerization cycle of actin homolog MreB from a Gram-positive bacterium" by Mao et al. provides the second biochemical study of a gram-positive MreB, but importantly, the first study examines how gram-positive MreB filaments bind to membranes. They also show the first crystal structure of a MreB from a Gram-positive bacterium - in two nucleotide-bound forms, finally solving structures that have been missing for too long. They also elucidate what residues in Geobacillus MreB are required for membrane associations. Also, the QCM-D approach to monitoring MreB membrane associations is a direct and elegant assay.

While the above findings are novel and important, this paper also makes a series of conclusions that run counter to multiple in vitro studies of MreBs from different organisms and other polymers with the actin fold. Overall, they propose that Geobacillus MreB contains biochemical properties that are quite different than not only the other MreBs examined so far but also eukaryotic actin and every actin homolog that has been characterized in vitro. As the conclusions proposed here would place the biochemical properties of Geobacillus MreB as the sole exception to all other actin fold polymers, further supporting experiments are needed to bolster these contrasting conclusions and their overall model.

1. (Difference 1) - The predominant concern about the in vitro studies that makes it difficult to evaluate many of their results (much less compare them to other MreB/s and actin homologs) is the use of a highly unconventional polymerization buffer containing 500(!) mM KCL. As has been demonstrated with actin and other polymers, the high KCl concentration used here (500mM) is certain to affect the polymerization equilibria, as increasing salt increases the hydrophobic effect and inhibits salt bridges, and therefore will affect the affinity between monomers and filaments. For example, past work has shown that high salt greatly changes actin polymerization, causing: a decreased critical concentration, increased bundling, and a greatly increased filament stiffness(Kang et al., 2013, 2012). Similarly, with AlfA, increased salt concentrations have been shown to increase the critical concentration, decrease the polymerization kinetics, and inhibit the bundling of AlfA filaments (Polka et al., 2009). A more closely related example comes from the previous observation that increasing salt concentrations increasingly slow the polymerization kinetics of B. subtilis MreB (Mayer and Amann, 2009). Lastly, These high salt concentrations might also change the interactions of MreB(Gs) with the membrane by screening charges and/or increasing the hydrophobic effect.

Given that 500mM KCl was used throughout this paper, many (if not all) of the key experiments should be repeated in more standard salt concentration (~100mM), similar to those used in most previous in vitro studies of polymers. This would test if the many divergent properties of MreB(Gs) reported here arise from some difference in MreB(Gs) relative to other MreBs (and actin homologs), or if they arise from the 400mM difference in salt concentration between the studies. Critically, it would also allow direct comparisons to be made relative to previous studies of MreB (and other actin homologs) that used much lower salt, thereby allowing them to definitively demonstrate whether MreB(Gs) is indeed an outlier relative to other MreB and actin homologs. I would suggest using 100mM KCL, as historically, all polymerization assays of actin and numerous actin homologs have used 50-100mM KCL: 50mM KCl (for actin in F buffer) or 100mM KCl for multiple prokaryotic actin homologs and MreB (Deng et al., 2016; Ent et al., 2014; Esue et al., 2006, 2005; Garner et al., 2004; Polka et al., 2009; Rivera et al., 2011; Salje et al., 2011) Likewise, similar salt concentrations are standard for tubulin (80 mM K-Pipes) and FtsZ (100 mM KCl or 100mM KAc in HMK100 buffer).

2. (Difference 2) - One of the most important differences claimed in this paper is that MreB(Gs) filaments are straight, a result that runs counter to the curved T. Maritima and C. crescentus filaments detailed by the Löwe group (Ent et al., 2014; Salje et al., 2011). Importantly, this difference could also arise from the difference in salt concentrations used in each study (500mM here vs. 100mM in the Löwe studies), and thus one cannot currently draw any direct comparisons between the two studies.

One example of how high salt could be causing differences in filament geometry: high salts are known to greatly increase the bending stiffness of actin filaments, making them more rigid (Kang et al., 2013). Likewise, increasing salt is known to change the rigidity of membranes. As the ability of filaments to A) bend the membrane or B) Deform to the membrane depends on the stiffness of filaments relative to the stiffness of the membrane, the observed difference in the "straight vs. curved" conformation of MreB filaments might simply arise from different salt concentrations.

Thus, in order to draw several direct comparisons between their findings and those of other MreB orthologs (as done here), the studies of MreB(GS) confirmations on lipids should be repeated at the same buffer conditions as used in the Löwe papers, then allowing them to be directly compared.

3. (Difference 3) - The next important difference between MreB(Gs) and other MreBs is the claim that MreB polymers do not form in the absence of membranes.

A) This is surprising relative to other MreBs, as MreBs from 1) T. maritime (multiple studies), E.coli (Nurse and Marians, 2013), and C. crescentus (Ent et al., 2014) have been shown to form polymers in solution (without lipids) with electron microscopy, light scattering, and time-resolved multi-angle light scattering. Notably, the Esue work was able to observe the first phase of polymer formation and a subsequent phase of polymer bundling (Esue et al., 2006) of MreB in solution. 2) Similarly, (Mayer and Amann, 2009) demonstrated B. subtilis MreB forms polymers in the absence of membranes using light scattering.

B) The results shown in figure 5A also go against this conclusion, as there is only a 2-fold increase in the phosphate release from MreB(Gs) in the presence of membranes relative to the absence of membranes. Thus, if their model is correct, and MreB(Gs) polymers form only on membranes, this would require the unpolymerized MreB monomers to hydrolyze ATP at 1/2 the rate of MreB in filaments. This high relative rate of hydrolysis of monomers compared to filaments is unprecedented. For all polymers examined so far, the rate of monomer hydrolysis is several orders of magnitude less than that of the filament. For example, actin monomers are known to hydrolyze ATP 430,000X slower than the monomers inside filaments (Blanchoin and Pollard, 2002; Rould et al., 2006).

C) Thus, there is a strong possibility that MreB(Gs) polymers are indeed forming in solution in addition to those on the membrane, and these "solution polymers" may not be captured by their electron microscopy assay. For example, high salt could be interfering with the absorption of filaments to glow discharged lacking lipids.<br /> In order to definitively prove that MreB(Gs) does not have polymers in solution, the authors should:

i) conduct orthogonal experiments to test for polymers in solution. The simplest test of polymerization might be conducting pelleting assays of MreB(Gs) with and without lipids, sweeping through the concentration range as done in 2B and 5a.

ii) They also could examine if they see MreB filaments in the absence of lipids at 100mM salt (as was seen in both Löwe studies), as the high salt used here might block the charges on glow discharged grids, making it difficult for the polymer to adhere.

iii) Likewise, the claim that MreB lacking the amino-terminus and the α2β7 hydrophobic loop "is required for polymerization" is questionable as if deleting these resides blocks membrane binding, the lack of polymers on the membrane on the grid is not unexpected, as these filaments that cannot bind the membrane would not be observable. Given these mutants cannot bind the membrane, mutant polymers could still indeed exist in solution, and thus pelleting assays should be used to test if non-membrane associated filaments composed of these mutants do or do not exist.

A final note, the results shown in "Figure 1 - figure supplement 2, panel C" appear to directly refute the claim that MreB(Gs) requires lipids to polymerize. As currently written, it appears they can observe MreB(Gs) filaments on EM grids without lipids. If these experiments were done in the presence of lipids, the figure legend should be updated to indicate that. If these experiments were done in the absence of lipids, the claim that membrane association is required for MreB polymerizations should be revised.

4. (Difference 4) - The next difference between this study and previous studies of MreB and actin homologs is the conclusion that MreB(Gs) must hydrolyze ATP in order to polymerize. This conclusion is surprising, given the fact that both T. Maritima (Salje · 2011, Bean 2008) and B. subtilis MreB (Mayer 2009) have been shown to polymerize in the presence of ATP as well as AMP-PNP. Likewise, MreB polymerization has been shown to lag ATP hydrolysis in not only T. maritima MreB (Esue 2005), eukaryotic actin, and all other prokaryotic actin homologs whose polymerization and phosphate release have been directly compared: MamK (Deng et al., 2016), AlfA (Polka et al., 2009), and two divergent ParM homologs (Garner et al., 2004; Rivera et al., 2011).

Currently, the only piece of evidence supporting the idea that MreB(Gs) must hydrolyze ATP in order to polymerize comes from 2 observations: 1) using electron microscopy, they cannot see filaments of MreB(Gs) on membranes in the presence of AMP-PNP or ApCpp, and 2) no appreciable signal increase appears testing AMPPNP- MreB(Gs) using QCM-D. This evidence is by no means conclusive enough to support this bold claim: While their competition experiment does indicate AMPPNP binds to MreB(Gs), it is possible that MreB(Gs) cannot polymerize when bound to AMPPNP. For example, it has been shown that different actin homologs respond differently to different non-hydrolysable analogs: Some, like actin, can hydrolyze one ATP analog but not the other, while others are able to bind to many different ATP analogs but only polymerize with some of one of them. Thus, to further verify their "hydrolysis is needed for polymerization" conclusion, they should:<br /> A. Test if a hydrolysis deficient MreB(Gs) mutant (such as D158A) is also unable to polymerize by EM.<br /> B. They also should conduct an orthogonal assay of MreB polymerization aside from EM (pelleting assays might be the easiest). They should test if polymers of ATP, AMP-PNP, and MreB(Gs)(D158A) form in solution (without membranes) by conducting pelleting assays. These could also be conducted with and without lipids, thereby also addressing the points noted above in point 3.<br /> C. Polymers may indeed form with ATP-gamma-S, and this non-hydrolysable ATP analog should be tested.<br /> D. They could also test how the ADP-Phosphate bound MreB(Gs) polymerizes in bulk and on membranes, using beryllium phosphate to trap MreB in the ADP-Pi state. This might allow them to further refine their model.<br /> E. Importantly, the Mayer study of B. subtilis MreB found the same results in regard to nucleotides, "In polymerization buffer, MreB produced phosphate in the presence of ATP and GTP, but not in ADP, AMP, GDP or AMP-PNP, or without the readdition of any nucleotide". Thus this paper should be referenced and discussed

5. (Difference 5) - The introduction states (lines 128-130) "However, the need for nucleotide binding and hydrolysis in polymerization remains unclear due to conflicting results, in vivo and in vitro, including the ability of MreB to polymerize or not in the presence of ADP or the non-hydrolyzable ATP analog AMP-PNP."

A) While this is a great way to introduce the problem, the statement is a bit vague and should be clarified, detaining the conflicting results and appropriate references. For example, what conflicting in vivo results are they referring to? Regarding "MreB polymerization in AMP-PNP", multiple groups have shown the polymerization of MreB(Tm) in the presence of AMP-PNP, but it is not clear what papers found opposing results.

B) The statement "However, the need for nucleotide binding and hydrolysis in polymerization remains unclear due to conflicting results, in vivo and in vitro, including the ability of MreB to polymerize or not in the presence of ADP or the non-hydrolyzable ATP analog AMP-PNP" is technically incorrect and should be rephrased or further tested.

i. For all actin (or tubulin) family proteins, it is not that a given filament "cannot polymerize" in the presence of ADP but rather that the ADP-bound form has a higher critical concentration for polymer formation relative to the ATP-bound form. This means that the ADP polymers can indeed polymerize, but only when the total protein exceeds the ADP critical concentration. For example, many actin-family proteins do indeed polymerize in ADP: ADP actin has a 10-fold higher critical concentration than ATP actin, (Pollard, 1984) and the ADP critical concentrations of AlfA and ParM are 5X and 50X fold higher (respectively) than their ATP-bound forms(Garner et al., 2004; Polka et al., 2009)

ii. Likewise, (Mayer and Amann, 2009) have already demonstrated that B. subtilis MreB can polymerize in the presence of ADP, with a slightly higher critical concentration relative to the ATP-bound form.

Thus, to prove that MreB(Gs) polymers do not form in the presence of ADP would require one to test a large concentration range of ADP-bound MreB(Gs). They should test if ADP- MreB(Gs) polymerizes at the highest MreB(Gs) concentrations that can be assayed. Even if this fails, it may be the MreB(Gs) ADP polymerizes at higher concentrations than is possible with their protein preps (13uM). An even more simple fix would be to simply state MreB(Gs)-ADP filaments do not form beneath a given MreB(Gs) concentration.

Other Points to address:

1. There are several points in this paper where the work by Mayer and Amann is ignored, not cited, or readily dismissed as "hampered by aggregation" without any explanation or supporting evidence of that fact.

A) Lines 100-101 - While the irregular 3-D formations seen formed by MreB in the Dersch 2020 paper could be interpreted as aggregates, stating that the results from specifically the Gaballah and Meyer papers (and not others) were "hampered by aggregation" is currently an arbitrary statement, with no evidence or backing provided. Overall, these lines (and others in the paper) dismiss these two works without giving any evidence to that point. Thus, they should provide evidence for why they believe all these papers are aggregation, or remove these (and other) dismissive statements.

One important note - There are 2 points indicating that dismissing the Meyer and Amann work as aggregation is incorrect: 1) the Meyer work on B. subtilis MreB shows both an ATP and a slightly higher ADP critical concentration. As the emergence of a critical concentration is a steady-state phenomenon arising from the association/dissociation of monomers (and a kinetically limiting nucleation barrier), an emergent critical concentration cannot arise from protein aggregation, critical concentrations only arise from a dynamic equilibrium between monomer and polymer. 2) Furthermore, Meyer observed that increased salt slowed and reduced B. subtilis MreB light scattering, the opposite of what one would expect if their "polymerization signal" was only protein aggregation, as higher salts should increase the rate of aggregation by increasing the hydrophobic effect.

B) Lines 113-137 -The authors reference many different studies of MreB, including both MreB on membranes and MreB polymerized in solution (which formed bundles). However, they again neglect to mention or reference the findings of Meyer and Amann (Mayer and Amann, 2009), as it was dismissed as "aggregation". As B. subtilis is also a gram-positive organism, the Meyer results should be discussed.

2. Lines 387-391 state the rates of phosphate release relative to past MreB findings: "These rates of Pi release upon ATP hydrolysis (~ 1 Pi/MreB in 6 min at 53{degree sign}C) are comparable to those observed for MreBTm and MreB(Ec) in vitro". While the measurements of Pi release AND ATP hydrolysis have indeed been measured for actin, this statement does not apply to MreB and should be corrected: All MreB papers thus far have only measured Pi release alone, not ATP hydrolysis at the same time. Thus, it is inaccurate to state "rates of Pi release upon ATP hydrolysis" for any MreB study, as to accurately determine the rate of Pi release, one must measure: 1. The rate of polymer over time, 2) the rate of ATP hydrolysis, and 3) the rate of phosphate release. For MreB, no one has, so far, even measured the rates of ATP hydrolysis and phosphate release with the same sample.

3. The interpretation of the interactions between monomers in the MreB crystal should be more carefully stated to avoid confusion. While likely not their intention, the discussions of the crystal packing contacts of MreB can appear to assume that the monomer-monomer contacts they see in crystals represent the contacts within actual protofilaments. One cannot automatically assume the observations of monomer-monomer contacts within a crystal reflect those that arise in the actual filament (or protofilament).

A) They state, "the apo form of MreBGs forms less stable protofilaments than its G- homologs ." Given filaments of the Apo form of MreB(GS) or b. subtilis have never been observed in solution, this statement is not accurate: while the contacts in the crystal may change with and without nucleotide, if the protein does not form polymers in solution in the apo state, then there are no "real" apo protofilaments, and any statements about their stability become moot. Thus this statement should be rephrased or appropriately qualified.

B) Another example: while they may see that in the apo MreB crystal, the loop of domain IB makes a *single* salt bridge with IIA and none with IIB. This contrasts with every actin, MreB, and actin homolog studied so far, where domain IB interacts with IIB. This might reflect the real contacts of MreB(Gs) in the solution, or it may be simply a crystal-packing artifact. Thus, the authors should be careful in their claims, making it clear to the reader that the contacts in the crystal may not necessarily be present in polymerized filaments.

4. lines 201-202 - "Polymers were only observed at a concentration of MreB above 0.55 μM (0.02 mg/mL)". Given this concentration dependence of filament formation, which appears the same throughout the paper, the authors could state that 0.55 μM is the critical concentration of MreB on membranes under their buffer conditions. Given the lack of critical concentration measurement in most of the MreB literature, this could be an important point to make in the field.

5. Both mg/ml and uM are used in the text and figures to refer to protein concentration. They should stick to one convention, preferably uM, as is standard in the polymer field.

6. Lines 77-78 - (Teeffelen et al., 2011) should be referenced as well in regard to cell wall synthesis driving MreB motion.

7. Line 90 - "Do they exhibit turnover (treadmill) like actin filaments?". This phrase should be modified, as turnover and treadmilling are two very different things. Turnover is the lifetime of monomers in filaments, while treadmilling entails monomer addition at one end and loss at the other. While treadmilling filaments cause turnover, there are also numerous examples of non-treadmilling filaments undergoing turnover: microtubules, intermediate filaments, and ParM. Likewise, an antiparallel filament cannot directionally treadmill, as there is no difference between the two filament ends to confer directional polarity.

8. Throughout the paper, the term aggregation is used occasionally to describe the polymerization shown in many previous MreB studies, almost all of which very clearly showed "bundled" filaments, very distinct entities from aggregates, as a bundle of polymers cannot form without the filaments first polymerizing on their own. Evidence to this point, polymerization has been shown to precede the bundling of MreB(Tm) by (Esue et al., 2005).

9. lines 106-108 mention that "The N-terminal amphipathic helix of E. coli MreB (MreBEc) was found to be necessary for membrane binding. " This is not accurate, as Salje observed that one single helix could not cause MreB to mind to the membrane, but rather, multiple amphipathic helices were required for membrane association (Salje et al., 2011). The Salje results imply that dimers (or further assemblies) of MreB drive membrane association, a point that should be discussed in regard to the question "What prompts the assembly of MreB on the inner leaflet of the cytoplasmic membrane?" posed on lines 86-87.

10. On lines 414-415, it is stated, "The requirement of the membrane for polymerization is consistent with the observation that MreB polymeric assemblies in vivo are membrane-associated only." While I agree with this hypothesis, it must be noted that the presence or absence of MreB polymers in the cytoplasm has not been directly tested, as short filaments in the cytoplasm would diffuse very quickly, requiring very short exposures (<5ms) to resolve them relative to their rate of diffusion. Thus, cytoplasmic polymers might still exist but have not been tested.

11. lines 429-431 state, "but polymerization in the presence of ADP was in most cases concluded from light scattering experiments alone, so the possibility that aggregation rather than ordered polymerization occurred in the process cannot be excluded."

A) If an increased light scattering signal is initiated by the addition of ADP (or any nucleotide), that signal must come from polymerization or multimerization. What the authors imply is that there must be some ADP-dependent "aggregation" of MreB, which has not been seen thus far for any polymer. Furthermore, why would the addition of ADP initiate aggregation?

B) Likewise, the statement "Differences in the purity of the nucleotide stocks used in these studies could also explain some of the discrepancies" is unexplained and confusing. How could an impurity in a nucleotide stock affect the past MreB results, and what is the precedent for this claim?

12. lines 467-469 state, "Thus, for both MreB and actin, despite hydrolyzing ATP before and after polymerization, respectively, the ADP-Pi-MreB intermediate would be the long-lived intermediate state within the filaments."

A) For MreB, this statement is extremely speculative and unbiased, as no one has measured 1) polymerization, 2) ATP hydrolysis, and 3) phosphate release. For example, it could be that ATP hydrolysis is slow, while phosphate release is fast, as is seen in the actin from Saccharomyces cerevisiae.

B) For actin, the statement of hydrolysis of ATP of monomer occurring "before polymerization" is functionally irrelevant, as the rate of ATP hydrolysis of actin monomers is 430,000 times slower than that of actin monomers inside filaments(Blanchoin and Pollard, 2002; Rould et al., 2006).

13. Lines 442-444. "On the basis of our data and the existing literature, we propose that the requirement for ATP (or GTP) hydrolysis for polymerization may be conserved for most MreBs." Again, this statement both here (and in the prior text) is an extremely bold claim, one that runs contrary to a large amount of past work on not just MreB, but also eukaryotic actin and every actin homolog studied so far. They come to this model based on 1) one piece of suggestive data (the behavior of MreB(GS) bound to 2 non-hydrolysable ATP analogs in 500mM KCL), and 2) the dismissal (throughout the paper) of many peer-reviewed MreB papers that run counter to their model as "aggregation" or "contaminated ATP stocks ." If they want to make this bold claim that their finding invalidates the work of many labs, they must back it up with further validating experiments.

References cited.

Blanchoin L, Pollard TD. 2002. Hydrolysis of ATP by Polymerized Actin Depends on the Bound Divalent Cation but Not Profilin †. Biochemistry-us 41:597-602. doi:10.1021/bi011214b

Deng A, Lin W, Shi N, Wu J, Sun Z, Sun Q, Bai H, Pan Y, Wen T. 2016. In vitro assembly of the bacterial actin protein MamK from 'Candidatus Magnetobacterium casensis' in the phylum Nitrospirae. Protein Cell 7:267-280. doi:10.1007/s13238-016-0253-x

Dersch S, Reimold C, Stoll J, Breddermann H, Heimerl T, Soufo HJD, Graumann PL. 2020. Polymerization of Bacillus subtilis MreB on a lipid membrane reveals lateral co-polymerization of MreB paralogs and strong effects of cations on filament formation. Bmc Mol Cell Biology 21:76. doi:10.1186/s12860-020-00319-5

Ent F van den, Izoré T, Bharat TA, Johnson CM, Lowe J. 2014. Bacterial actin MreB forms antiparallel double filaments. eLife 3:e02634. doi:10.7554/elife.02634

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