Reviewer #3 (Public review):

Summary:

The authors demonstrate that activation of TFEB facilitates cholesterol clearance in cell models of Niemann-Pick type C (NPC). This is done through a variety of approaches including activation of TFEB by sulforaphane (SFN), a naturally occurring small-molecule TFEB agonist. SFN induces TFEB nuclear translocation and promotes lysosomal exocytosis. In an NPC mouse model, SFN dephosphorylates/activates TFEB in the brain and rescues the loss of Purkinje cells.

Strengths:

NPC is a severe disease and there is little in the way of treatment. The manuscript points towards some treatment options. However, the title, the title "Small-molecule activation of TFEB Alleviates Niemann-Pick Disease..." is far too strong and should be changed.

Weaknesses:

(1) The manuscript is extremely hard to read due to the writing; it needs careful editing for grammar and English.

(2) There are a number of important technical issues that need to be addressed.

(3) The TFEB influence on filipin staining in Figure 1A is somewhat subtle. In the mCherry alone panels there is a transfected cell with no filipin staining and the mCherry-TFEBS211A cells still show some filipin staining.

(4) Figure 1C is impressive for the upregulation of filipin with U18666A treatment. However, SFN is used at 15 microM. This must be hitting multiple pathways. Vauzour et al (PMID: 20166144) use SFN at 10 nM to 1microM. Other manuscripts use it in the low microM range. The authors should repeat at least some key experiments using SFN at a range of concentrations from perhaps 100 nM to 5 microM. The use of 15 microM throughout is an overall concern.

Reviewer #3 (Public review):

Summary:

The authors sought to understand social interactions both within and between groups of feral pigs, with the intent of applying their findings to models of disease transmission. The authors analyzed GPS tracking data from across various populations to determine patterns of contact that could support the transmission of a range of zoonotic and livestock diseases. The analysis then focused on the effects of sex, group dynamics, and seasonal changes on contact rates that could be used to base targeted disease control strategies that would prioritize the removal of adult males for reducing intergroup disease transmission.

Strengths:

It utilized GPS tracking data from 146 feral pigs over several years, effectively capturing seasonal and spatial variation in the social behaviors of interest. Using proximity-based social network analysis, this work provides a highly resolved snapshot of contact rates and interactions both within and between groups, substantially improving research in wildlife disease transmission. Results were highly useful and provided practical guidance for disease management, showing that control targeted at adult males could reduce intergroup disease transmission, hence providing an approach for the control of zoonotic and livestock diseases.

Weaknesses:

Despite their reliability, populations can be skewed by small sample sizes and limited generalizability due to specific environmental and demographic characteristics. Further validation is needed to account for additional environmental factors influencing social dynamics and contact rates

Reviewer #3 (Public review):

Summary:

The authors describe an exciting 400-drug screening using a MMV pathogen box to select compounds that effectively affect the medically important Toxoplasma parasite bradyzoite stage. This work utilises a bradyzoites culture technique that was published recently by the same group. They focused on compounds that affected directly the mitochondria electron transport chain (mETC) bc1-complex and compared them with other bc1 inhibitors described in the literature such as atovaquone and HDQs. They further provide metabolomics analysis of inhibited parasites which serves to provide support for the target and to characterise the outcome of the different inhibitors.

Strengths:

This work is important as, until now, there are no effective drugs that clear cysts during T. gondii infection. So, the discovery of new inhibitors that are effective against this parasite stage in culture and thus have the potential to battle chronic infection is needed. The further metabolic characterization provides indirect target validation and highlights different metabolic outcomes for different inhibitors. The latter forms the basis for new studies in the field to understand the mode of inhibition and mechanism of bc1-complex function in detail.

The authors focused on the function of one compound, MMV1028806, that is demonstrated to have a similar metabolic outcome to burvaquone. Furthermore, the authors evaluated the importance of ATP production in tachyzoite and bradyzoites stages and under atovaquone/HDQs drugs.

Weaknesses:

Although the authors did experiments to identify the metabolomic profile of the compounds and suggested bc-1 complex as the main target of MMV1028806, they did not provide experimental validation for that.

Reviewer #3 (Public review):

Summary:

In this study, Wang et al utilized the available GTEx data to compile a comprehensive analysis that attempt to reveal aging-related sex-dimorphic gene expression as well as alternative splicing changes in humans.

The key conclusions based on their analysis are that

(1) extensive sex-dimorphisms during aging with distinct patterns of change in gene expression and alternative splicing (AS), and

(2) the male-biased age-associated AS events have a stronger association with Alzheimer's disease, and

(3) the female-biased events are often regulated by several sex-biased splicing factors that may be controlled by estrogen receptors. They further performed break-point analysis and revealed that in males there are two main breakpoints around ages 35 and 50, while in females, there is only one breakpoint at 45.

Strengths:

This study sets an ambitious goal, leveraging the extensive GTEx dataset to investigate aging-related, sex-dimorphic gene expression and alternative splicing changes in humans. The research addresses a significant question, as our understanding of sex-dimorphic gene expression in the context of human aging is still in its early stages. Advancing our knowledge of these molecular changes is vital for identifying therapeutic targets for age-related diseases and extending the human health span. The study is highly comprehensive, and the authors are commendable for their attempted thorough analysis of both gene expression and alternative splicing - an area often overlooked in similar studies.

Weaknesses:

Due to the inherent noise within the GTEx dataset - which includes numerous variables beyond aging and sex - there are significant technical concerns surrounding this study. Additionally, the lack of cross-validation with independent, existing data raises questions about whether the observed gene expression changes genuinely reflect those associated with human aging. For instance, the break-point analysis in this study identifies two major breakpoints in males around ages 35 and 50, and one breakpoint in females at age 45; however, these findings contradict a recent multi-omics longitudinal study involving 108 participants aged 25 to 75 years, where breakpoint at 44 and 60 years was observed in both male and females (Shen et al, 2024). These issues cast doubt on the robustness of the study's conclusions. Specific concerns are outlined below:

(1) The primary method used in this study is linear regression, incorporating age, sex, and age-by-sex interactions as covariates, alongside other confounding factors (such as ethnicity) as unknown variables. However, the analysis overlooks two critical known variables in the GTEx dataset: time of death (TOD) and postmortem interval (PMI). Both TOD and PMI are recorded for each sample and account for substantial variance in gene expression profiles. A recent study by Wucher et al.(Wucher et al, 2023) demonstrated the powerful impact of TOD on gene expression by using it to reconstruct human circadian and even circannual datasets. Similarly, Ferreira et al. (Ferreira et al, 2018) highlighted PMI's influence on gene expression patterns. Without properly adjusting for these two variables, confidence in the study's conclusions remains limited at best.

(2) To demonstrate that their analysis is robust and that the covariates TOD and PMI are otherwise negligible - the authors should cross-validate their findings with independent datasets to confirm that the identified gene expression changes are reproducible for some tissues. For instance, the recent study by Shen et al. (Shen et al., 2024) in Nature Aging offers an excellent dataset for cross-validation, particularly for blood samples. Comparing the GTEx-derived results with this longitudinal transcriptome dataset would enable verification of gene expression changes at both the individual gene and pathway levels. Without such validation, confidence in the study's conclusions remains limited.

(3) As a demonstration of the lack of such validation, in the Shen et al. study (Shen et al., 2024), breakpoints at 44 and 60 years were observed in both males and females, while this study identifies two major breakpoints in males around ages 35 and 50, and one breakpoint in females at age 45. What caused this discrepancy?

(4) Although the alternative splicing analysis is intriguing, the authors did not differentiate between splicing events that alter the protein-coding sequence and those that do not. Many splicing changes occurring in the 5' UTR and 3' UTR regions do not impact protein coding, so it is essential to filter these out and focus specifically on alternative splicing events that can modify protein-coding sequences.

(5) One of the study's main conclusions - that "male-biased age-associated AS events have a stronger association with Alzheimer's disease" - is not supported by the data presented in Figure 4A, which shows an association with "regulation of amyloid precursor formation" only in female, not male, alternative splicing genes. Additionally, the gene ontology term "Alzheimer's disease" is absent from the unbiased GO analysis in Figure S6. These discrepancies suggest that the focus on Alzheimer's disease may reflect selective data interpretation rather than results driven by an unbiased analysis.

(6) The experimental data presented in Figures 5E - I merely demonstrate that estrogen receptor regulates the expression of two splicing factors, SRSF1 and SRSF7, in an estradiol-dependent manner. However, this finding does not support the notion that this regulation actually contributes to sex-dimorphic alternative splicing changes during human aging. Notably, the authors do not provide evidence that SRSF1 and SRSF7 expression changes actually occur in a sex-dependent manner with human aging (in a manner similar to TIA1). As such, this experimental dataset is disconnected from the main focus of the study and does not substantiate the conclusions on sex-dimorphic splicing during human aging. The authors performed RNA-seq in wild-type and ER mutant cells, and they should perform a comprehensive analysis of ER-dependent alternative splicing and compare the results with the GTEx data. It should be straightforward.

References:

Ferreira PG, Muñoz-Aguirre M, Reverter F, Sá Godinho CP, Sousa A, Amadoz A, Sodaei R, Hidalgo MR, Pervouchine D, Carbonell-Caballero J et al (2018) The effects of death and post-mortem cold ischemia on human tissue transcriptomes. Nature Communications 9: 490.

Shen X, Wang C, Zhou X, Zhou W, Hornburg D, Wu S, Snyder MP (2024) Nonlinear dynamics of multi-omics profiles during human aging. Nature Aging.

Wucher V, Sodaei R, Amador R, Irimia M, Guigó R (2023) Day-night and seasonal variation of human gene expression across tissues. PLOS Biology 21: e3001986.

Reviewer #3 (Public review):

Summary:

Bos et al study a computational model of cortical circuits with excitatory (E) and two subtypes of inhibition - parvalbumin (PV) and somatostatin (SOM) expressing interneurons. They perform stability and gain analysis of simplified models with nonlinear transfer functions when SOM neurons are perturbed. Their analysis suggests that in a specific setup of connectivity, instability and gain can be untangled, such that SOM modulation leads to both increases in stability and gain, in contrast to the typical direction in neuronal networks where increased gain results in decreased stability.

Strengths:

- Analysis of the canonical circuit in response to SOM perturbations. Through numerical simulations and mathematical analysis, the authors have provided a rather comprehensive picture of how SOM modulation may affect response changes.<br /> - Shedding light on two opposing circuit motifs involved in the canonical E-PV-SOM circuitry - namely, direct inhibition (SOM -> E) vs disinhibition (SOM -> PV -> E). These two pathways can lead to opposing effects, and it is often difficult to predict which one results from modulating SOM neurons. In simplified circuits, the authors show how these two motifs can emerge and depend on parameters like connection weights.<br /> - Suggesting potentially interesting consequences for cortical computation. The authors suggest that certain regimes of connectivity may lead to untangling of stability and gain, such that increases in network gain are not compromised by decreasing stability. They also link SOM modulation in different connectivity regimes to versatile computations in visual processing in simple models.

Weaknesses

Computationally, the analysis is solid, but it's very similar to previous studies (del Molino et al, 2017). Many studies in the past few years have done the perturbation analysis of a similar circuitry with or without nonlinear transfer functions (some of them listed in the references). This study applies the same framework to SOM perturbations, which is a useful computational analysis, in view of the complexity of the high-dimensional parameter space.

Link to biology: the most interesting result of the paper with regard to biology is the suggestion of a regime in which gain and stability can be modulated in an unconventional way - however, it is difficult to link the results to biological networks:<br /> - A general weakness of the paper is a lack of direct comparison to biological parameters or experiments. How different experiments can be reconciled by the results obtained here, and what new circuit mechanisms can be revealed? In its current form, the paper reads as a general suggestion that different combinations of gain modulation and stability can be achieved in a circuit model equipped with many parameters (12 parameters). This is potentially interesting but not surprising, given the high dimensional space of possible dynamical properties. A more interesting result would have been to relate this to biology, by providing reasoning why it might be relevant to certain circuits (and not others), or to provide some predictions or postdictions, which are currently missing in the manuscript.<br /> - For instance, a nice motivation for the paper at the beginning of the Results section is the different results of SOM modulation in different experiments - especially between L23 (inhibition) and L4 (disinhibition). But no further explanation is provided for why such a difference should exist, in view of their results and the insights obtained from their suggested circuit mechanisms. How the parameters identified for the two regimes correspond to different properties of different layers?<br /> - One of the key assumptions of the model is nonlinear transfer functions for all neuron types. In terms of modelling and computational analysis, a thorough analysis of how and when this is necessary is missing (an analysis similar to what has been attempted in Figure 6 for synaptic weights, but for cellular gains). A discussion of this, along with the former analysis to know which nonlinearities would be necessary for the results, is needed, but currently missing from the study. The nonlinearity is assumed for all subtypes because it seems to be needed to obtain the results, but it's not clear how the model would behave in the presence or absence of them, and whether they are relevant to biological networks with inhibitory transfer functions.<br /> - Tuning curves are simulated for an individual orientation (same for all), not considering the heterogeneity of neuronal networks with multiple orientation selectivity (and other visual features) - making the model too simplistic.

Reviewer #3 (Public review):

Summary:

The authors report a novel, direct interaction of Spt6p tSH2 domain to Tom1p. This extends the function of Spt6p from communication with factors associated with RNAPII transcription to processes of ubiquitination. Tom1p is known to ubiquitinate a large variety of substrates, but it is unknown how substrate recognition is done in a specific manner. The team identified a conserved central acidic region of Tom1p which is essential for in vivo functions and binds to histones and nucleosomes, as well as Spt6p. They further describe the Tom1p occupancy pattern on chromatin, assigning it a stabilizing effect on nucleosomes near promotors and a destabilizing effect on nucleosomes within the gene bodies. The authors were able to resolve two different conformational states of Tom1p which are likely connected to its activity, and possibly substrate selectivity.

Overall, the authors show that an intrinsically disordered region in Tom1p is important for substrate interaction and function of Tom1p. The protein is further involved in chromatin architecture and structural transitions control its activity.

Strengths:

By revealing the interaction of Spt6p and Tom1p, the authors discover a novel connection between transcriptional elongation and processes of ubiquitination.<br /> In recent years, disordered regions of MDa protein complexes have become a focus of research projects. The effects of disordered regions on protein localization and specificity of binding interactions have been discussed in great extent, including proteins that are involved in chromatin remodeling and transcription. Adding to these current efforts, the authors assign a function to a highly conserved disordered region of Tom1p in technically clean experiments. Furthermore, with their data, they pin down a specific functional region in Tom1p which is relevant for the previously observed temperature sensitivity caused by Tom1p deletion in yeast.<br /> The team performs a thorough and complete analysis of the cryo-EM structure and they nicely model the hinge motion and details of an open and closed conformation.

Weaknesses:

Despite the high number of interesting findings, there is little connection between the individual sections of the manuscript. For example, many experiments are not related to Spt6p binding although this protein is presented as a major actor in this manuscript during the introduction. Furthermore, the structural analysis is well done, but it is also not quite clear how structural rearrangements are connected to Spt6 binding or chromatin remodeling. Some experimental results lack novelty, as similar data has previously been presented for the human homolog.

To confirm the novel, direct binding interaction of Spt6p and Tom1p, no orthogonal binding assays (SPR, MST, ITC) have been performed to confirm the interaction. To me, this is insufficient, especially since the team has purified both proteins to high quality levels, or could use peptides to test the function of the relevant regions.<br /> Additionally, interaction of Tom1p with Spt6p in the context of transcription elongation is proposed. Yet it is not clear on the mechanistic level how this is regulated if Tom1p and Rpb1p bind in a competitive manner. How is Tom1p tethered to the elongation complex if not through Spt6p? In addition to WT vs. knockout, the authors should further perform the genetic analyses with the intΔ11 mutant. This way they might be able pin down which interactions on chromatin are mediated by Spt6 vs. by other factors and could strengthen the overall model involving Spt6P.

Although the authors try to describe a final model in the discussion, this section is not easy to follow and needs more explanation, ideally drawn as a Figure of the proposed mechanism.

Reviewer #3 (Public review):

This paper discusses how non-sensory and latent, sensory-like attentional templates are represented during attentional preparation. Using multivariate pattern analysis, they found that visual impulses can enhance the decoding generalization from perception to attention tasks in the preparatory stage in the visual cortex. Furthermore, the emergence of the sensory-like template coincided with enhanced information connectivity between V1 and frontoparietal areas and was associated with improved behavioral performance. It is an interesting paper with supporting evidence for the latent, sensory-like attentional template, but several problems still need to be solved.

(1) The title is "Dual-format Attentional Template," yet the supporting evidence for the non-sensory format and its guiding function is quite weak. The author could consider conducting further generalization analysis from stimulus selection to preparation stages to explore whether additional information emerges.

(2) In Figure 2, the author did not find any decodable sensory-like coding in IPS and PFC, even during the impulse-driven session, indicating that these regions do not represent sensory-like information. However, in the final section, the author claimed that the impulse-driven sensory-like template strengthens informational connectivity between sensory and frontoparietal areas. This raises a question: how can we reconcile the lack of decodable coding in these frontoparietal regions with the reported enhancement in network communication? It would be helpful if the author provided a clearer explanation or additional evidence to bridge this gap.

(3) Given that the impulse-driven sensory-like template facilitated behavior, the author proposed that it might also enhance network communication. Indeed, they observed changes in informational connectivity. However, it remains unclear whether these changes in network communication have a direct and robust relationship with behavioral improvements.

(4) I'm uncertain about the definition of the sensory-like template in this paper. Is it referring to the Ping impulse-driven condition or the decodable performance in the early visual cortex? If it is the former, even in working memory, whether pinging identifies an activity-silent mechanism is currently debated. If it's the latter, the authors should consider whether a causal relationship - such as "activating the sensory-like template strengthens the informational connectivity between sensory and frontoparietal areas" - is reasonable.

Reviewer #3 (Public review):

Summary:

Cryptococcus neoformans is a global critical threat pathogen and the manuscript by Mota et al demonstrates that the pathogen's N-glycan-dependent protein quality control system regulates the capacity of the fungus to cause disease. The system makes sure that glycoproteins are folded correctly. The system is involved in the fitness and virulence of the fungus by regulating aspects of cellular robustness and the trafficking of virulence-associated compounds outside of the cell via transport in extracellular vesicles.

Strengths:

The investigators use multiple modalities to demonstrate that the system is involved in cryptococcal pathogenesis. The investigators generated mutant C. neoformans to explore the role of genes involved in the protein folding system. Basic microbiology, genetic analyses, proteomics, fluorescence and transmission microscopy, nanotracking analyses, and murine studies were performed. The validity of the findings are thus very high. Hypotheses are robustly demonstrated.

Weaknesses:

Aspects of the results should be better explained. Some results are extrapolated in their meaning beyond the extent of the data.

Reviewer #3 (Public review):

Summary:

Here Wengert et al., establish a rodent model of KCNC1 (Kv3.1) epilepsy by introducing the A421V mutation. The authors perform video-EEG, slice electrophysiology, and in vivo 2P imaging of calcium activity to establish disease mechanisms involving impairment in the excitability of fast-spiking parvalbumin (PV) interneurons in the cortex and thalamic PV cells.

Outside-out nucleated patch recordings were used to evaluate the biophysical consequence of the A421V mutation on potassium currents and showed a clear reduction in potassium currents. Similarly, action potential generation in cortical PV interneurons was severely reduced. Given that both potassium currents and action potential generation were found to be unaffected in excitatory pyramidal cells in the cortex the authors propose that loss of inhibition leads to hyperexcitability and seizure susceptibility in a mechanism similar to that of Dravet Syndrome.

Strengths:

This manuscript establishes a new rodent model of KCNC1-developmental and epileptic encephalopathy. The manuscript provides strong evidence that parvabumin-type interneurons are impaired by the A421V Kv3.1 mutation and that cortical excitatory neurons are not impaired. Together these findings support the conclusion that seizure phenotypes are caused by reduced cortical inhibition.

Weaknesses:

The manuscript identifies a partial mechanism of disease that leaves several aspects unresolved including the possible role of the observed impairments in thalamic neurons in the seizure mechanism. Similarly, while the authors identify a reduction in potassium currents and a reduction in PV cell surface expression of Kv3.1 it is not clear why these impairments would lead to a more severe disease phenotype than other loss-of-function mutations which have been characterized previously. Lastly, additional analysis of video-EEG data would be helpful for interpreting the extent of the seizure burden and the nature of the seizure types caused by the mutation.

Reviewer #3 (Public review):

Summary:

The obligate intracellular bacterium Chlamydia trachomatis (Ct) divides by binary fission. It lacks FtsZ, but still has many other proteins that regulate the synthesis of septal peptidoglycan, including FtsW and FtsI (PBP3) as well as divisome proteins that recruit and activate them, such as FtsK and FtsQLB. Interestingly, MreB is also required for the division of Ct cells, perhaps by polymerizing to form an FtsZ-like scaffold. Here, Harpring et al. show that MreB does not act early in division and instead is recruited to a protein complex that includes FtsK and PBP2/PBP3. This indicates that Ct cell division is organized by a chimera between conserved divisome and elongasome proteins. Their work also shows convincingly that FtsK is the earliest known step of divisome activity, potentially nucleating the divisome as a single protein complex at the future division site. This is reminiscent of the activity of FtsZ, yet fundamentally different.

Strengths:

The study is very well written and presented, and the data are convincing and rigorous. The data underlying the proposed localization dependency order of the various proteins for cell division is well justified by several different approaches using small molecule inhibitors, knockdowns, and fluorescent protein fusions. The proposed dependency pathway of divisome assembly is consistent with the data and with a novel mechanism for MreB in septum synthesis in Ct.

Weaknesses:

The paper could be improved by including more information about FtsK, the "focus" of this study. For example, if FtsK really is the FtsZ-like nucleator of the Ct divisome, how is the Ct FtsK different sequence-wise or structurally from FtsK of, e.g. E. coli? Is the N-terminal part of FtsK sufficient for cell division in Ct like it is in E. coli, or is the DNA translocase also involved in focus formation or localization? Addressing those questions would put the proposed initiator role of FtsK in Ct in a better context and make the conclusions more attractive to a wider readership.

Another weakness is that the title of the paper implies that FtsK alone initiates divisome assembly. However, the data indicate only that FtsK is important at an early stage of divisome assembly, not that it is THE initiator. I suggest modifying the title to account for this--perhaps "FtsK is required to initiate....".

Reviewer #3 (Public review):

Summary:

This manuscript by Duan and Song interrogates the gating mechanisms and specifically force transmission in mechanosensitive NOMPC channels using steered molecular dynamics simulations. They propose that the ankyrin spring can transmit force to the gate through torsional forces adding molecular detail to the force transduction pathways in this channel.

3. Constant velocity or constant force<br /> For the SMD the authors write "and a constant velocity or constant force". It's unclear from this reviewer's perspective what is used to generate the simulation data.

Strengths:

Detailed, rigorous simulations coupled with a novel model for force transduction.

Weaknesses:

Experimental validation of reduced mechanosensitivity through mutagenesis of proposed ankyrin/TRP domain coupling interactions would greatly enhance the manuscript. I have some additional questions documented below:

(1) The membrane-parallel torsion force can open NOMPC<br /> How does the TRP domain interact with the S4-S5 linker? In the original structural studies, the coordination of lipids in this region seems important for gating. In this manner does the TRP domain and S4-S5 linker combined act like an amphipathic helix as suggested first for MscL (Bavi et al., 2016 Nature Communications) and later identified in many MS channels (Kefauver et al., 2020 Nature).

(2) Torsional forces on shorter ankyrin repeats of mammalian TRP channels<br /> Is it possible torsional forces applied to the shorter ankyrin repeats of mammalian TRPs may also convey force in a similar manner?

(3) Constant velocity or constant force<br /> For the SMD the authors write "and a constant velocity or constant force". It's unclear from this reviewer's perspective which is used to generate the simulation data.

Reviewer #3 (Public review):

Summary:

The paper begins by analyzing the drift in individual behavior over time. Specifically, it quantifies the circling direction of freely walking flies in an arena. The main takeaway from this dataset is that while flies exhibit an individual turning bias (when averaged over time), their preferences fluctuate over slow timescales.

To understand whether genetic or neuromodulatory mechanisms influence the drift in individual preference, the authors test different fly strains concluding that both genetic background and the neuromodulator serotonin contribute to the degree of drift.

Finally, the authors use theoretical approaches to identify the range of environmental conditions under which drift in individual bias supports population growth.

Strengths:

The model provides a clear prediction of the environmental fluctuations under which a drift in bias should be beneficial for population growth.

The approach attempts to identify genetic and neurophysiological mechanisms underlying drift in bias.

Weaknesses:

Different behavioral assays are used and are differently analysed, with little discussion on how these behaviors and analyses compare to each other.

Some of the model assumptions should be made more explicit to better understand which aspects of the behaviors are covered.

Reviewer #3 (Public review):

Summary:

The paper by Zhu et al is on an important topic in visual neuroscience, the emergence in the visual cortex of signals about figures and ground. This topic also goes by the name border ownership. The paper utilizes modern recording techniques very skillfully to extend what is known about border ownership. It offers new evidence about the prevalence of border ownership signals across different cortical layers in V1 cortex. Also, it uses pairwise cross-correlation to study signal flow under different conditions of visual stimulation that include the border ownership paradigm.

Strengths:

The paper's strengths are its use of multi-electrode probes to study border ownership in many neurons simultaneously across the cortical layers in V1, and its innovation of using cross-correlation between cortical neurons -- when they are viewing border-ownership patterns or instead are viewing grating patterns restricted to the classical receptive field (CRF).

Weaknesses:

The paper's weaknesses are its largely incremental approach to the study of border ownership and the lack of a critical analysis of the cross-correlation data. The paper as it is now does not advance our understanding of border ownership; it mainly confirms prior work, and it does not challenge or revise consensus beliefs about mechanisms. However, it is possible that, in the rich dataset the authors have obtained, they do possess data that could be added to the paper to make it much stronger.

Critique:

The border ownership data on V1 offered in the paper replicates experimental results obtained by Zhou and von der Heydt (2000) and confirms the earlier results using the same analysis methods as Zhou. The incremental addition is that the authors found border ownership in all cortical layers extending Zhou's results that were only about layer 2/3.

The cross-correlation results show that the pattern of the cross-correlogram (CCG) is influenced by the visual pattern being presented. However, the results are not analyzed mechanistically, and the interpretation is unclear. For instance, the authors show in Figure 3 (and in Figure S2) that the peak of the CCG can indicate layer 2/3 excites layer 4C when the visual stimulus is the border ownership test pattern, a large square 8 deg on a side. But how can layer 2/3 excite layer 4C? The authors do not raise or offer an answer to this question. Similar questions arise when considering the CCG of layer 4A/B with layer 2/3. What is the proposed pathway for layer 2/3 to excite 4A/B? Other similar questions arise for all the interlaminar CCG data that are presented. What known functional connections would account for the measured CCGs?

The problems in understanding the CCG data are indirectly caused by the lack of a critical analysis of what is happening in the responses that reveal the border ownership signals, as in Figure 2. Let's put it bluntly - are border ownership signals excitatory or inhibitory? The reason I raise this question is that the present authors insightfully place border ownership as examples of the action of the non-classical receptive field (nCRF) of cortical cells. Most previous work on the nCRF (many papers cited by the authors) reveal the nCRF to be inhibitory or suppressive. In order to know whether nCRF signals are excitatory or inhibitory, one needs a baseline response from the CRF, so that when you introduce nCRF signals you can tell whether the change with respect to the CRF is up or down. As far as I know, prior work on border ownership has not addressed this question, and the present paper doesn't either. This is where the rich dataset that the present authors possess might be used to establish a fundamental property of border ownership.

Then we must go back to consider what the consequences of knowing the sign of the border ownership signal would mean for interpreting the CCG data. If the border ownership signals from extrastriate feedback or, alternatively, from horizontal intrinsic connections, are excitatory, they might provide a shared excitatory input to pairs of cells that would show up in the CCG as a peak at 0 delay. However, if the border ownership manuscript signals are inhibitory, they might work by exciting only inhibitory neurons in V1. This could have complicated consequences for the CCG. The interpretation of the CCG data in the present version of the m is unclear (see above). Perhaps a clearer interpretation could be developed once the authors know better what the border ownership signals are.

My critique of the CCG analysis applies to Figure 5 also. I cannot comprehend the point of showing a very weak correlation of CCG asymmetry with Border Ownership Index, especially when what CCG asymmetry means is unclear mechanistically. Figure 5 does not make the paper stronger in my opinion.

In Figure 3, the authors show two CCGs that involve 4C--4C pairs. It would be nice to know more about such pairs. If there are any 6--6 pairs, what they look like also would be interesting. The authors also in Figure 3 show CCG's of two 4C--4A/B pairs and it would be quite interesting to know how such CCGs behave when CRF and nCRF stimuli are compared. In other words, the authors have shown us they have many data but have chosen not to analyze them further or to explain why they chose not to analyze them. It might help the paper if the authors would present all the CCG types they have. This suggestion would be helpful when the authors know more about the sign of border ownership signals, as discussed at length above.

Reviewer #3 (Public review):

Summary:

Fahrenfort et al. investigate how liberal or conservative criterion placement in a detection task affects the construct validity of neural measures of unconscious cognition and conscious processing. Participants identified instances of "seen" or "unseen" in a detection task, a method known as post hoc sorting. Simulation data convincingly demonstrate that, counterintuitively, a conservative criterion inflates effect sizes of neural measures compared to a liberal criterion. While the impact of criterion shifts on effect size is suggested by signal detection theory, this study is the first to address this explicitly within the consciousness literature. Decoding analysis of data from two EEG experiments further shows that different criteria lead to differential effects on classifier performance in post hoc sorting. The findings underscore the pervasive influence of experimental design and participants report on neural measures of consciousness, revealing that criterion placement poses a critical challenge for researchers.

Strengths and Weaknesses:

One of the strengths of this study is the inclusion of the Perceptual Awareness Scale (PAS), which allows participants to provide more nuanced responses regarding their perceptual experiences. This approach ensures that responses at the lowest awareness level (selection 0) are made only when trials are genuinely unseen. This methodological choice is important as it helps prevent the overestimation of unconscious processing, enhancing the validity of the findings.

A potential area for improvement in this study is the use of single time-points from peak decoding accuracy to generate current source density topography maps. While we recognize that the decoding analysis employed here differs from traditional ERP approaches, the robustness of the findings could be enhanced by exploring current source density over relevant time windows. Event-related peaks, both in terms of timing and amplitude, can sometimes be influenced by noise or variability in trial-averaged EEG data, and a time-window analysis might provide a more comprehensive and stable representation of the underlying neural dynamics.

It is helpful that the authors show the standard error of the mean for the classifier performance over time. A similar indication of a measure of variance in other figures could improve clarity and transparency.<br /> That said, the paper appears solid regarding technical issues overall. The authors also do a commendable job in the discussion by addressing alternative paradigms, such as wagering paradigms, as a possible remedy to the criterion problem (Peters & Lau, 2015; Dienes & Seth, 2010). Their consideration of these alternatives provides a balanced view and strengthens the overall discussion.

Impact of the Work:

This study effectively demonstrates a phenomenon that has been largely unexplored within the consciousness literature. Subjective measures may not reliably capture the construct they aim to measure due to criterion confounds. Future research on neural measures of consciousness should account for this issue, and no-report measures may be necessary until the criterion problem is resolved.

Reviewer #3 (Public review):

Summary:

The paper by Li et al explored the role of Estrogen receptor 1 (Esr1) expressing neurons in the pontine micturition center (PMC), a brainstem region also known as Barrington's nucleus (Hou et al 2016, Keller et al 2018). First, the author conducted bulk Ca2+ imaging/unit recording from PMCESR1 to investigate the correlations of PMCESR1 neural activity to voiding behavior in conscious mice and bladder pressure/external urethral muscle activity in urethane anesthetized mice. Next, the authors conducted optogenetics inactivation/activation of PMCESR1 to confirm the contribution to the voiding behavior also conducted peripheral nerve transection together with optogenetics activation to confirm the independent control of bladder pressure and urethral sphincter muscle.

Weaknesses:

(1) The study demonstrates that pelvic nerve transection reduces urinary volume triggered by PMCESR1+ cell photoactivation in freely moving mice. Could the role of pudendal nerve transection also be examined in awake mice to provide a more comprehensive understanding of neural involvement?

(2) While the paper primarily focuses on PMCESR1+ cells in bladder-sphincter coordination, the analysis of PMCESR1+-DGC/SPN neural circuits - given their distinct anatomical projections in the sacral spinal cord - feels underexplored. How do these circuits influence bladder and sphincter function when activated or inhibited? Also, do you have any tracing data to confirm whether bladder-sphincter innervation comes from distinct spinal nuclei?

(3) Although the paper successfully identifies the physiological role of PMCESR1+ cells in bladder-sphincter coordination, the study falls short in examining the electrophysiological properties of PMCESR1+-DGC/SPN cells. A deeper investigation here would strengthen the findings.

(4) The parameters for photoactivation (blue light pulses delivered at 25 Hz for 15 ms, every 30 s) and photoinhibition (pulses at 50 Hz for 20 ms) vary. What drove the selection of these specific parameters? Moreover, for photoactivation experiments, the change in pressure (ΔP = P5 sec - P0 sec) is calculated differently from photoinhibition (Δpressure = Ppeak - Pmin). Can you clarify the reasoning behind these differing approaches?

(5) The discussion could further emphasize how PMCESR1+ cells coordinate bladder contraction and sphincter relaxation to control urination, highlighting their central role in the initiation and suspension of this process.

(6) In Figure 8, The authors analyze the temporal sequence of bladder pressure and EUS bursting during natural voiding and PMC activation-induced voiding. It would be acceptable to consider the existence of a lower spinal reflex circuit, however, the interpretation of the data contains speculation. Bladder pressure measurement is hard to say reflecting efferent pelvic nerve activity in real time. (As a biological system, bladder contraction is mediated by smooth muscle, and does not reflect real-time efferent pelvic nerve activity. As an experimental set-up, bladder pressure measurement has some delays to reflect bladder pressure because of tubing, but EUS bursting has no delay.) Especially for the inactivation experiment, these factors would contribute to the interpretation of data. This reviewer recommends a rewrite of the section considering these limitations. Most of the section is suitable for the results.

Reviewer #3 (Public Review):

Summary:

The article is an interesting approach to determining the MPOX receptor using "in silico" tools. The results show the presence of two regions of the H3 protein with a high probability of being involved in the interaction with the HS cell receptor. However, the α-helical region seems to be the most probable, since modifications in this region affect the virus binding to the HS receptor.

Strengths:

In my opinion, it is an informative article with interesting results, generated by a combination of "in silico" and wet science to test the theoretical results. This is a strong point of the article.

Comments on revised version:

After a review of the changes to the manuscript and the author's responses, no further changes are needed.

Reviewer #3 (Public review):

Summary:

In this manuscript, Hirai et al investigated the release properties of glutamate/GABA co-transmission at SuM-GC synapses and reported that glutamate/GABA co-transmission exhibits distinct short-term plasticity with segregated postsynaptic targets. Using optogenetics, whole-cell patch-clamp recordings, and immunohistochemistry, the authors reveal distinct transmission modes of glutamate/GABA co-release as frequency-dependent filters of incoming SuM inputs.

Strengths:

Overall, this study is well-designed and executed; conclusions are supported by the results. This study addressed a long-standing question of whether GABA and glutamate are packaged in the same vesicles and co-released in response to the same stimuli in the SuM-GC synapses (Pedersen et al., 2017; Hashimotodani et al., 2018; Billwiller et al., 2020; Chen et al., 2020; Li et al., 2020; Ajibola et al., 2021). Knowledge gained from this study advances our understanding of neurotransmitter co-release mechanisms and their functional roles in the hippocampal circuits.

Comments on revisions:

The authors have addressed my comments, and now the manuscript is in a good form as it currently stands.

Reviewer #3 (Public review):

Summary:

In the present work, Zhang et al investigate involvement of the bacterial DNA damage repair SOS response in the evolution of beta-lactam drug resistance evolution in Escherichia coli. Using a combination of microbiological, bacterial genetics, laboratory evolution, next-generation, and live-cell imaging approaches, the authors propose short-term (transient) drug resistance evolution can take place in RecA-deficient cells in an SOS response-independent manner. They propose the evolvability of drug resistance is alternatively driven by the oxidative stress imposed by accumulation of reactive oxygen species and compromised DNA repair. Overall, this is a nice study that addresses a growing and fundamental global health challenge (antimicrobial resistance).

Strengths:

The authors introduce new concepts to antimicrobial resistance evolution mechanisms. They show short-term exposure to beta-lactams can induce durably fixed antimicrobial resistance mutations. They propose this is due to comprised DNA repair and oxidative stress. Antibiotic resistance evolution under transient stress is poorly studied, so the authors' work is a nice mechanistic contribution to this field.

Weaknesses:

The authors do not show any direct evidence of altered mutation rate or accumulated DNA damage in their model.

Reviewer #3 (Public review):

Summary:

In this manuscript, Costa and colleagues investigate how asymmetry in dorsal root ganglion (DRG) neurons is established. The authors developed an in vitro system that mimics the pseudo-unipolar morphology and asymmetry of DRG neurons during the regeneration of the peripheral and central branch axons. They suggest that central-like DRG axons exhibit a higher density of growing microtubules. By reducing the polymerization of microtubules in these central-like axons, they were able to eliminate the asymmetry in DRG neurons.

Strengths:

The authors point out a distinct microtubule-associated protein signature that differentiates between DRG neurons' central and peripheral axonal branches. Experimental results demonstrate that genetic deletion of spastin eliminated the differences in microtubule dynamics and axon regeneration between the central and peripheral branches.

Weaknesses:

While some of the data are compelling, experimental evidence only partially supports the main claims.

In its current form, the study is primarily descriptive and lacks convincing mechanistic insights. It misses important controls and further validation using 3D in vitro models.

Given the heterogeneity of dorsal root ganglion (DRG) neurons, it is unclear whether the in vitro model described in this study can be applied to all major classes of DRG neurons. Also unclear is the inconsistency with embryonic DRG cultures with embryonic (E)16 from rats and E13 from mice (spastin knockout and wild-type controls). Furthermore, the authors stated (line 393) that only a small subset of cultured DRG neurons exhibited a pseudo-unipolar morphology. The authors should include the percentage of the neurons that exhibit a pseudo-unipolar morphology.

The significance of studying microtubule polymerization to DRG asymmetry in vitro is questionable, especially considering the model's validity. The authors might consider eliminating the in vitro data and instead focus on characterizing DRG asymmetry in vivo both before and after a conditioning lesion. If the authors choose to retain the in vitro data, classifying the central and peripheral-like branches in cultured DRG neurons will require further in-depth characterization. Additional validation should be performed in adult DRG neuron cultures not aged in vitro.

The comparison of asymmetry associated with a regenerative response between in vitro and in vivo paradigms has significant limitations due to the nature of the in vitro culture system. When cultured in isolation, DRG neurons fail to form functional connections with appropriate postsynaptic target neurons (the central branch) or to differentiate the peripheral domains associated with the innervation of target organs. Rather than growing neurons on a flat, hard surface like glass, more physiologically relevant substrates and/or culturing conditions should be considered. This approach could help eliminate potential artifacts caused by plating adult DRG neurons on a flat surface. Additionally, the authors should consider replicating their findings in a 3D culture model or using dorsal root ganglia explants, where both centrally and peripherally projecting axons are present.

Panels 5H-J require additional processing with astrocyte markers to accurately define the lesion borders. Furthermore, including a lower magnification would facilitate a direct comparison of the lesion site. The use of cholera toxin subunit B (CTB) to trace dorsal column sensory axons is prone to misinterpretation, as the tracer accumulates at the axon's tip. This limitation makes it extremely challenging to distinguish between regenerating and degenerating axons.

Reviewer #3 (Public review):

Summary:

A long noncoding RNA, lnc-FANCI-2, was reported to be regulated by HPV E7 oncoprotein and a cell transcription factor, YY1 by this group. The current study focuses on the function of lnc-FANCI-2 in HPV-16 positive cervical cancer is to intrinsically regulate RAS signaling, thereby facilitating our further understanding of additional cellular alterations during HPV oncogenesis. The authors used advanced technical approaches such as KO, transcriptome and (IRPCRP) and LC- MS/MS analyses in the current study and concluded that KO Inc-FANCI-2 significantly increases RAS signaling, especially phosphorylation of Akt and Erk1/2.

Strengths:

(1) HPV E6E7 are required for full immortalization and maintenance of the malignant phenotype of cervical cancer, but they are NOT sufficient for full transformation and tumorigenesis. This study helps further understanding of other cellular alterations in HPV oncogenesis.

(2) lnc-FANCI-2 is upregulated in cervical lesion progression from CIN1, CIN2-3 to cervical cancer, cancer cell lines, and HPV transduced cell lines.

(3) Viral E7 of high-risk HPVs and host transcription factor YY1 are two major factors promoting lnc-FANCI-2 expression.

(4) Proteomic profiling of cytosolic and secreted proteins showed inhibition of MCAM, PODXL2, and ECM1 and increased levels of ADAM8 and TIMP2 in KO cells.

(5) RNA-seq analyses revealed that KO cells exhibited significantly increased RAS signaling but decreased IFN pathways.

(6) Increased phosphorylated Akt and Erk1/2, IGFBP3, MCAM, VIM, and CCND2 (cyclin D2) and decreased RAC3 were observed in KO cells.

Weaknesses:

(1) The authors observed the increased Inc-FANCI-2 in HPV 16 and 18 transduced cells, and other cervical cancer tissues as well, HPV-18 positive HeLa cells exhibited different expressions of Inc-FANCI-2.

(2) Previous studies and data in the current showed a steadily increased Inc-FANCI-2 during cancer progression, however, the authors did not observe significant changes in cell behaviors (both morphology and proliferation) in KO Inc-FANCI-2.

(3) The authors observed the significant changes of RAS signaling (downstream) in KO cells, but they provided limited interpretations of how these results contributed to full transformation or tumorigenesis in HPV-positive cancer.

Reviewer #3 (Public review):

The paper from Liang and Guan details the calculation of the potential mean force for the transition between two key states of the melibiose (Mel) transporter MelB. The authors used the string method along with replica-exchange umbrella sampling to model the transition between the outward and inward-facing Mel-free states, including the binding and subsequent release of Mel. They find a barrier of ~6.8 kcal/mol and an overall free-energy difference of ~6.4 kcal/mol. They also investigate the same process without the co-transported Na+, finding a higher barrier, while in the D59C mutant, the barrier is nearly eliminated.

I found this to be an interesting and technically competent paper. I was disappointed actually to see that the authors didn't try to complete the cycle. I realize this is beyond the scope of the study as presented.

The results are in qualitative agreement with expectations from experiments. Could the authors try to make this comparison more quantitative? For example, by determining the diffusivity along the path, the authors could estimate transition rates.

Relatedly, could the authors comment on how typical concentration gradients of Mel and Na+ would affect these numbers?

Reviewer #3 (Public review):

This study aimed to investigate the impact of seasonality on the malaria parasite population genetic. To achieve this, the researchers conducted a longitudinal study in a region characterized by seasonal malaria transmission. Over a 2.5-year period, blood samples were collected from 1,516 participants residing in four villages in the Upper River Region of The Gambia and tested the samples for malaria parasite positivity. The parasites from the positive samples were genotyped using a genetic barcode and/or whole genome sequencing, followed by a genetic relatedness analysis.

The study identified three key findings:

(1) The parasite population continuously recombines, with no single genotype dominating, in contrast to viral populations;

(2) The relatedness of parasites is influenced by both spatial and temporal distances; and

(3) The lowest genetic relatedness among parasites occurs during the transition from low to high transmission seasons. The authors suggest that this latter finding reflects the increased recombination associated with sexual reproduction in mosquitoes.

The results section is well-structured, and the figures are clear and self-explanatory. The methods are adequately described, providing a solid foundation for the findings. While there are no unexpected results, it is reassuring to see the anticipated outcomes supported by actual data. The conclusions are generally well-supported; however, the discussion on the burden of asymptomatic infections falls outside the scope of the data, as no specific analysis was conducted on this aspect and was not stated as part of the aims of the study. Nonetheless, the recommendation to target asymptomatic infections is logical and relevant.

Reviewer #3 (Public review):

Summary:

The purpose of the current manuscript was to investigate a magnetic cell steering technique for efficiency and tissue-specific targeting, using two types of stem cells, in a mouse model of glaucoma. As the authors point out, trabecular meshwork (TM) cell therapy is an active area of research for treating elevated intraocular pressure as observed in glaucoma. Thus, further studies determining the ideal cell choice for TM cell therapy is warranted. The experimental protocol of the manuscript involved the injection of either human adipose derived mesenchymal stem cells (hAMSCs) or induced pluripotent cell derivatives (iPSC-TM cells) into a previously reported mouse glaucoma model, the transgenic MYOCY437H mice and wild-type littermates followed by the magnetic cell steering. Numerous outcome measures were assessed and quantified including IOP, outflow facility, TM cellularity, retention of stem cells, and the inner wall BM of Schlemm's canal.

Strengths:

All of these analyses were carefully carried out and appropriate statistical methods were employed. The study has clearly shown that the hAMSCs are the cells of choice over the iPSC-TM cells, the latter of which caused tumors in the anterior chamber. The hAMSCs were shown to be retained in the anterior segment over time and this resulted in increased cellular density in the TM region and a reduction in IOP and outflow facility. These are all interesting findings and there is substantial data to support it.

Weaknesses:

However, where the study falls short is in the MYOCY437H mouse model of glaucoma that was employed. The authors clearly state that a major limitation of the study is that this model, in their hands, did not exhibit glaucomatous features as previously reported, such as a significant increase in IOP, which was part of the overall purpose of the study. The authors state that it is possible that "the transgene was silenced in the original breeders". The authors did not show PCR, western blot, or immuno of angle tissue of the tg to determine transgenic expression (increased expression of MYOC was shown in the angle tissue of the transgenics in the original paper by Zode et al, 2011). This should be investigated given that these mice were rederived. Thus, it is clearly possible that these are not transgenic mice. If indeed they are transgenics, the authors may want to consider the fact that in the Zode paper, the most significant IOP elevation in the mutant mice was observed at night and thus this could be examined by the authors. Other glaucomatous features of these mice could also have been investigated such as loss of RGCs, to further determine their transgenic phenotype. Finally, while increased cellular density in the TM region was observed, proliferative markers could be employed to determine if the transplanted cells are proliferating.

Reviewer #3 (Public review):

Summary:

In this study, several novel class IIb microcin biosynthetic gene clusters have been discovered by specific homology searches and manual curation. Using a specific E. coli expression system, the microcins were expressed and conjugated to monoglycosylated enterobactin as siderophore moiety. While this synthetic biology approach cannot account for other siderophores being coupled to the microcin core peptide in the original producing strains, it nonetheless allows for a general screening for the activity of the heterologously produced compounds. Through this approach, the activity of several predicted microcins has been confirmed and three novel class IIb microcin clades were identified.

Strengths:

The experimental design is sound, the results are corroborated by suitable controls, and the findings have a high level of novelty and significance. Furthermore, the comments of the initial round of peer review have been answered satisfactorily by the authors.

Reviewer #3 (Public review):

This study attempted to investigate the relations between processing in the human brain during movie watching and corresponding thought processes. This is a highly interesting question, as movie watching presents a semi-constrained task, combining naturally occurring thoughts and common processing of sensory inputs across participants. This task is inherently difficult because in order to know what participants are thinking at any given moment, one has to interrupt the same thought process which is the object of study.

This study attempts to deal with this issue by aggregating staggered experience sampling data across participants in one behavioral study and using the population level thought patterns to model brain activity in different participants in an open access fMRI dataset.

The behavioral data consist of 120 participants who watched 3 11-minute movie clips. Participants responded to the mDES questionnaire: 16 visual scales characterizing ongoing thought 5 times, two minutes apart, in each clip. The 16 items are first reduced to 4 factors using PCA, and their levels are compared across the different movies. The factors are "episodic knowledge", "intrusive distraction", "verbal detail", and "sensory engagement". The factors differ between the clips, and distraction is negatively correlated with movie comprehension and sensory engagement is positively correlated with comprehension.

The components are aggregated across participants (transforming single subject mDES answers into PCA space and concatenating responses of different participants) and are used as regressors in a GLM analysis. This analysis identifies brain regions corresponding to the components. The resulting brain maps reveal activations that are consistent with the proposed mental processes (e.g. negative loading for intrusion in frontoparietal network, positive loadings for visual and auditory cortices for sensory engagement).

Then, the coordinates for brain regions which were significant for more than one component are entered into a paper search in neurosynth. It is not clear what this analysis demonstrates beyond the fact that sensory engagement contained both visual and auditory components.

The next analysis projected group-averaged brain activation onto gradients (based on previous work) and used gradient timecourses to predict the behavioral report timecourses. This revealed that high activations in gradient 1 (sensory→association) predicted high sensory engagement, and that "episodic knowledge" thought patterns were predicted by increased visual cortex activations. Then, permutation tests were performed to see whether these thought pattern related activations corresponded to well defined regions on a given cluster.

In conclusion, this study tackles a highly interesting subject and does it creatively and expertly.

Reviewer #3 (Public review):

Summary:

Individuals with Down syndrome (DS) have high rates of autoimmunity and can have exaggerated immune responses to infection that can unfortunately cause significant medical complications. Prior studies from these authors and others have convincingly demonstrated that individuals with DS have immune dysregulation including increased Type I IFN activity, elevated production of inflammatory cytokines (hypercytokinemia), increased autoantibodies, and populations of dysregulated adaptive immune cells that pre-dispose to autoimmunity. Prior studies have demonstrated that using JAK inhibitors to treat patient samples in vitro, in small case series of patients, and in mouse models of DS leads to improvement of immune phenotype and/or clinical disease. This manuscript provides two major advances in our understanding of the immune dysregulation and therapy for patients. First, they perform deep immune phenotyping on several hundred individuals with DS and demonstrate that immune dysregulation is present from infancy. Second, they report promising interim analysis of a Phase II clinical trial of a JAK inhibitor in 10 people with DS and moderate to severe skin autoimmunity.

Strengths and weaknesses:

The relatively large cohort and careful clinical annotation here provides new insights into the immune phenotype of patients with DS. For example, it is interesting that regardless of autoimmune disease or autoantibody status, individuals with DS have elevated cytokines and CRP. Analysis of the cohorts by age demonstrated that some cytokines are significant elevated in people with DS starting in infancy (e.g., IL-9 and IL-17C). Nearly all adults with DS in this study had autoantibodies (98%) and most had six or more autoantibodies (63%), which differed significantly from euploid study participants. This implies that all patients with DS might benefit from early intervention with therapy to reduce inflammation. However, it is also worth considering that an alternative interpretation that since hypercytokinemia does not vary based on disease state in individuals with DS, that this may not be a key factor driving autoimmunity (although it may be relevant for other clinical symptoms such as neuroinflammation).

Small case series have suggested the benefit of JAK inhibitors to treat autoimmunity in DS. This is the first report of a prospective clinical trial to test a JAK inhibitor in this setting. The clinical trial entry criteria included moderate to severe autoimmune skin disease in patients aged 12-50 years with DS, and treatment was with the JAK1/3 inhibitor tofacitinib. This clinical trial is a critically important step for the field. The early results support that treatment is well tolerated with improvement of interferon scores in patients and reduction of autoantibodies. Most patients experienced clinical improvement, with alopecia areata having the greatest response. Treatment may not affect all skin disease equally, for example of the 5 patients with hidradenitis suppurativa, only 1 showed clinical improvement based on skin score. While very promising, the clinical trial results reported here are preliminary and based on interim analysis of 10 patients at 16 weeks. Individuals with DS have a lifelong risk of immune dysregulation and thus it is unclear how long therapy, if of benefit, would need to be continued. Results of longer-term therapy will be informative when considering the risks/benefits of this therapy.

Comments on revised version:

The authors have made appropriate revisions to this important contribution to the literature.

Reviewer #3 (Public review):

Summary:

The manuscript by Pooja Popli and co-authors tested the importance of Atg14 in the female reproductive tract by conditionally deleting Atg14 use PrCre and also Foxj1cre. The authors showed that loss of Atg14 leads to infertility due to the retention of embryos within the oviduct. The authors further concluded that the retention of embryos within the oviduct is due to pyroptosis in oviduct cells leading to defective cellular integrity. The revised manuscript has included new experimental data (Figs. S2B, 5B, 5C, and S3) that satisfied the concerns of this reviewer. The manuscript should provide important advancement to the field.

Reviewer #3 (Public review):

Summary:

In this paper, Arimura et al report a new method, termed MagIC-Cryo-EM, which refers to the method of using magnetic beads to capture specific proteins out of a lysate via, followed immunoprecipitation and deposition on EM grids. The so-enriched proteins can be analzyed structurally. Importantly, the nanoparticles are further functionalized with protein-based spacers, to avoid a distorted halo around the particles. This is a very elegant approach and allows the resolution of the stucture of small amounts of native proteins at atomistic resolution.<br /> Here, the authors apply this method to study the chromatosome formation from nucleosomes and the oocyte-specific linker histone H1.8. This allows them to resolve H1.8-containing chromatomosomes from oocyte extract in both interphase and metaphase conditions at 4.3 A resolution, which reveal a common structure with H1 placed right at the dyad and contacting both entry-and exit linker DNA.<br /> They then investigate the origin of H1.8 loss during interphase. They identify a non-nucleosomal H1.8-containing complex from interphase preparations. To resolve its structure, the authors develop a protocol (DuSTER) to exclude particles with ambiguous center, revealing particles with five-fold symmetry, that matches the chaperone NPM2. MS and WB confirms that the protein is present in interphase samples but not metaphase. The authors further separate two isoforms, an open and closed form that coexist. Additional densities in the open form suggest that this might be bound H1.8.

Strengths:

Together this is an important addition to the suite of cryoEM methods, with broad applications. The authors demonstrate the method using interesting applications, showing that the methods work and they can get high resolution structures from nucleosomes in complex with H1 from native environments.

Weaknesses:

The structures of the NPM2 chaperone is less well resolved, and some of the interpretation in this part seems only weakly justified.

Reviewer #3 (Public review):

Summary:

The authors were interested in how Ankle2 regulates nuclear envelope reformation after cell division. Other published manuscripts, including those from the authors, show without a doubt that Ankle2 plays a role in this critical process. However, the mechanism by which Ankle2 functions was unclear. Previous work using worms and humans (Asencio et al., 2012) established that human ANKLE2 could bind endogenous PP2A subunits. The binding was direct and was mediated through a region before and including the first ankyrin repeat in human ANKLE2. In addition to its interaction with PP2A, Asencio et al., 2012 also show that ANKLE2 regulates VRK1 kinase activity. Together PP2A and VRK1 regulate BAF phosphorylation for proper nuclear envelope reformation. Here, the authors provide more evidence for interaction with PP2A by also mapping the domain of interaction to the ankyrin repeat in Drosophila. In addition, the ankyrin repeat is essential for nuclear envelope reformation after division. They show that Ankle2 can bind in a PP2A complex without other known regulatory subunits of PP2A. The authors also identify a novel interaction with ER protein Vap33, but functional relevance for this interaction in nuclear envelope reformation is not provided in the manuscript, which the authors explicitly state. This manuscript does not comment on the activity of Ballchen/VRK1 in relation to Ankle2 loss and BAF phosphorylation or nuclear envelope reformation, even though links were previously shown by multiple studies (Asencio et al., Link et al., Apridita Sebastian et al.,). Nuclear envelope defects were rescued by the reduction of VRK1 in two of these manuscripts. It is possible that BAF phosphorylation phenotypes can be contributed by both PP2A inactivity and VRK1 overactivity due to the loss of Ankle2.

Strengths:

This manuscript is a useful finding linking Ankle2 function during nuclear envelope reformation to the PP2A complex. The authors present solid data showing that Ankle2 can form a complex with PP2A-29B and Mts and generate a phosphoproteomic resource that is fundamentally important to understanding Ankle2 biology.

Weaknesses:

However, the main findings/conclusions about subcellular localization might be incomplete since they are drawn from overexpression experiments. In addition, throughout the text, some conclusions are overstated or are not supported by data.

Reviewer #3 (Public review):

In this very thorough study, the authors characterize the function of a novel Drosophila gene, which they name Sakura. They start with the observation that sakura expression is predicted to be highly enriched in the ovary and they generate an anti-sakura antibody, a line with a GFP-tagged sakura transgene, and a sakura null allele to investigate sakura localization and function directly. They confirm the prediction that it is primarily expressed in the ovary and, specifically, that it is expressed in germ cells, and find that about 2/3 of the mutants lack germ cells completely and the remaining have tumorous ovaries. Further investigation reveals that Sakura is required for piRNA-mediated repression of transposons in germ cells. They also find evidence that sakura is important for germ cell specification during development and germline stem cell maintenance during adulthood. However, despite the role of sakura in maintaining germline stem cells, they find that sakura mutant germ cells also fail to differentiate properly such that mutant germline stem cell clones have an increased number of "GSC-like" cells. They attribute this phenotype to a failure in the repression of Bam by dpp signaling. Lastly, they demonstrate that sakura physically interacts with otu and that sakura and otu mutants have similar germ cell phenotypes. Overall, this study helps to advance the field by providing a characterization of a novel gene that is required for oogenesis. The data are generally high-quality and the new lines and reagents they generated will be useful for the field. However, there are some weaknesses and I would recommend that they address the comments in the Recommendations for the authors section below.

Reviewer #3 (Public review):

Summary:

Recently, the off-target activity of antibiotics on human mitoribosome has been paid more attention in the mitochondrial field. Hafner et al applied mitoribosome profilling to study the effect of antibiotics on protein translation in mitochondria as there are similarities between bacterial ribosome and mitoribosome. The authors conclude that some antibiotics act on mitochondrial translation initiation by the same mechanism as in bacteria. On the other hand, the authors showed that chloramphenicol, linezolid and telithromycin trap mitochondrial translation in a context-dependent manner. More interesting, during deep analysis of 5' end of ORF, the authors reported the alternative start codon for ND1 and ND5 proteins instead of previously known one. This is a novel finding in the field and it also provides another application of the technique to further study on mitochondrial translation.

Strengths:

This is the first study which applied mitoribosome profiling method to analyze mutiple antibiotics treatment cells.<br /> The mitoribosome profiling method had been optimized carefully and has been suggested to be a novel method to study translation events in mitochondria. The manuscript is constructive and written well.

Weaknesses:

This is a novel and interesting study, however, most of the conclusion comes from mitoribosome profiling analysis, as a result, the manuscript lacks the cellular biochemical data to provide more evidence and support the findings.

Reviewer #3 (Public review):

Summary:

Rayshubskiy et al. performed whole-cell recordings from descending neurons (DNs) of fruit flies to characterize their role in steering. Two DNs implicated in "walking control" and "steering control" by previous studies (Namiki et al., 2018, Cande et al., 2018, Chen et al., 2018) were chosen by the authors for further characterization. In-vivo whole-cell recordings from DNa01 and DNa02 showed that their activity predicts spontaneous ipsilateral turning events. The recordings also showed that while DNa02 predicts transient turns DNa01 predicts slow sustained turns. However, optogenetic activation or inactivation showed relatively subtle phenotypes for both neurons (consistent with data in other recent preprints, Yang et al 2023 and Feng et al 2024). The authors also further characterized DNa02 with respect to its inputs and showed a functional connection with olfactory and thermosensory inputs as well as with the head-direction system. DNa01 is not characterized to this extent.

Strengths:

(1) In-vivo recordings and especially dual recordings are extremely challenging in Drosophila and provide a much higher resolution DN characterization than other recent studies that have relied on behavior or calcium imaging. Especially impressive are the simultaneous recordings from bilateral DNs (Figure 3). These bilateral recordings show clearly that DNa02 cells not only fire more during ipsilateral turning events but that they get inhibited during contralateral turns. In line with this observation, the difference between left and right DNa02 neuronal activity is a much better predictor of turning events compared to individual DNa02 activity.

(2) Another technical feat in this work is driving local excitation in the head-direction neuronal ensemble (PEN-1 neurons), while simultaneously imaging its activity and performing whole-cell recordings from DNa02 (Figure 4). This impressive approach provided a way to causally relate changes in the head-direction system to DNa02 activity. Indeed, DNa02 activity could predict the rate at which an artificially triggered bump in the PEN-1 ring attractor returns to its previous stable point.

(3) The authors also support the above observations with connectomics analysis and provide circuit motifs that can explain how the head direction system (as well as external olfactory/thermal stimuli) communicated with DNa02. All these results unequivocally put DNa02 as an essential DN in steering control, both during exploratory navigation as well as stimulus-directed turns.

Weaknesses:

(1) I understand that the first version of this preprint was already on biorxiv in 2020, and some of the "weaknesses" I list are likely a reflection of the fact that I'm tasked to review this manuscript in late 2024 (more than 4 years later). But given this is a 2024 updated version it suffers from laying out the results in contemporary terms. For instance, the manuscript lacks any reference to the DNp09 circuit implicated in object-directed turning and upstream to DNa02 even though the authors cite one of the papers where this was analyzed (Braun et al, 2024). More importantly, these studies (both Braun et al 2024 and Sapkal et al 2024) along with recent work from the authors' lab (Yang et al 2023) and other labs (Feng et al 2024) provide a view that the entire suite of leg kinematics changes required for turning are orchestrated by populations of heterogeneous interconnected DNs. Moreover, these studies also show that this DN-DN network has some degree of hierarchy with some DNs being upstream to other DNs. In this contemporary view of steering control, DNa02 (like DNg13 from Yang et al 2023) is a downstream DN that is recruited by hierarchically upstream DNs like DNa03, DNp09, etc. In this view, DNa02 is likely to be involved in most turning events, but by itself unable to drive all the motor outputs required for the said events. This reasoning could be used while discussing the lack of major phenotypes with DNa02 activation or inactivation observed in the current study, which is in stark contrast to strong phenotypes observed in the case of hierarchically upstream DNs like DNp09 or DNa03. In the section, "Contributions of single descending neuron types to steering behavior": the authors start off by asking if individual DNs can make measurable contributions to steering behavior. Once more, any citations to DNp09 or DNa03 - two DNs that are clearly shown to drive strong turning-on activation (Bidaye et al, 2020, Feng et al 2024) - are lacking. Besides misleading the reader, such statements also digress the results away from contemporary knowledge in the field. I appreciate that the brief discussion in the section titled "Ensemble codes for steering" tries to cover these recent updates. However, I think this would serve a better purpose in the introduction and help guide the results.

(2) The second major weakness is the lack of any immunohistochemistry (IHC) images quantifying the expression of the genetic tools used in these studies. Even though the main split-Gal4 tools for DNa01 and DNa02 were previously reported by Namiki et al, 2018, it is important to document the expression with the effectors used in this work and explicitly mention the expression in any ectopic neurons. Similarly, for any experiments where drivers were combined together (double recordings, functional connectivity) or modified for stochastic expression (Figure 8), IHC images are absolutely necessary. Without this evidence, it is difficult to trust many of the results (especially in the case of behavioral experiments in Figure 8). For example, the DNa01 genetic driver used by the authors is also expressed in some neurons in the nerve cord (as shown on the Flylight webpage of Janelia Research Campus). One wonders if all or part of the results described in Figure 8 are due to DNa01 manipulation or manipulation of the nerve cord neurons. The same applies for optic lobe neurons in the DNa02 driver.

(3) The paper starts off with a comparative analysis of the roles of DNa01 and DNa02 during steering. Unfortunately, after this initial analysis, DNa01 is largely ignored for further characterization (e.g. with respect to inputs, connectomics, etc.), only to return in the final figure for behavioral characterization where DNa01 seems to have a stronger silencing phenotype compared to DNa02. I couldn't find an explanation for this imbalance in the characterization of DNa01 versus DNa02. Is this due to technical reasons? Or was it an informed decision due to some results? In addition to being a biased characterization, this also results in the manuscript lacking a coherent thread, which in turn makes it a bit inaccessible to the non-specialist.

(4) There seems to be a discrepancy with regard to what is emphasized in the main text and what is shown in Figures S3/S4 in relation to the role of these DNs in backward walking. There are only two sentences in the main text where these figures are cited.<br /> a) "DNa01 and DNa02 firing rate increases were not consistently followed by large changes in forward velocity (Figs. 1G and S3)."<br /> b) "We found that rotational velocity was consistently related to the difference in right-left firing rates (Fig. 3B). This relationship was essentially linear through its entire dynamic range, and was consistent across paired recordings (Fig. 3C). It was also consistent during backward walking, as well as forward walking (Fig. S4)."<br /> These main text sentences imply the role of the difference between left and right DNa02 in turning. However, the actual plots in the Figures S3 and S4 and their respective legends seem to imply a role in "backward walking". For instance, see this sentence from the legend of Figure S3 "When (ΔvoltageDNa02>>ΔvoltageDNa01), the fly is typically moving backward. When (firing rateDNa02>>firing rateDNa01), the fly is also often moving backward, but forward movement is still more common overall, and so the net effect is that forward velocity is small but still positive when (firing rateDNa02>>firing rateDNa01). Note that when we condition our analysis on behavior rather than neural activity, we do see that backward walking is associated with a large firing rate differential (Fig. S4)." This sort of discrepancy in what is emphasized in the text, versus what is emphasized in the figures, ends up confusing the reader. More importantly, I do not agree with any of these conclusions regarding the implication of backward walking. Both Figures S3 and S4 are riddled with caveats, misinterpretations, and small sample sizes. As a result, I actually support the authors' decision to not infer too much from these figures in the "main text". In fact, I would recommend going one step further and removing/modifying these figures to focus on the role of "rotational velocity". Please find my concerns about these two figures below:<br /> a) In Figures S3 and S4, every heat map has a different scale for the same parameter: forward velocity. S3A is -10 to +10mm/s. S3B is -6 to +6 S4B (left) is -12 to +12 and S4B (right) is -4 to +4. Since the authors are trying to depict results based on the color-coding this is highly problematic.<br /> b) Figure S3A legend "When (ΔvoltageDNa02>>ΔvoltageDNa01), the fly is typically moving backward." There are also several instances when ΔvoltageDNa02= ΔvoltageDNa01 and both are low (lower left quadrant) when the fly is typically moving backwards. So in my opinion, this figure in fact suggests DNa02 has no role in backward velocity control.<br /> c) Based on the example traces in S4A, every time the fly walks backwards it is also turning. Based on this it is important to show absolute rotational velocity in Figure S4C. It could be that the fly is turning around the backward peak which would change the interpretation from Figure S4C. Also, it is important to note that the backward velocities in S4A are unprecedentedly high. No previous reports show flies walking backwards at such high velocities (for example see Chen et al 2018, Nat Comm. for backward walking velocities on a similar setup).<br /> d) In my opinion, Figure S4D showing that right-left DNa02 correlates with rotational velocity, regardless of whether the fly is in a forward or backward walking state, is the only important and conclusive result in Figures S3/S4. These figures should be rearranged to only emphasize this panel.

(5) Figure 3 shows a really nice analysis of the bilateral DNa02 recordings data. While Figure S5 shows that authors have a similar dataset for DNa01, a similar level analysis (Figures 3D, E) is not done for DNa01 data. Is there a reason why this is not done?

(6) In Figure 4 since the authors have trials where bump-jump led to turning in the opposite direction to the DNa02 being recorded, I wonder if the authors could quantify hyperpolarization in DNa02 as is predicted from connectomics data in Figure 7.

(7) Figure 6 suggests that DNa02 contains information about latent steering drives. This is really interesting. However, in order to unequivocally claim this, a higher-resolution postural analysis might be needed. Especially given that DNa02 activation does not reliably evoke ipsilateral turning, these "latent" steering events could actually contain significant postural changes driven by DNa02 (making them "not latent"). Without this information, at least the authors need to explicitly mention this caveat.

(8) Figure 7 would really benefit from connectome data with synapse numbers (or weighted arrows) and a corresponding analysis of DNa01.

(9) In Figure 8E, the most obvious neuronal silencing phenotype is decreased sideways velocity in the case of DNa01 optogenetic silencing. In Figure S2, the inverse filter for sideways velocity for DNa01 had a higher amplitude than the rotational velocity filter. Taken together, does this point at some role for DNa01 in sideways velocity specifically?

(10) In Figure 8G, the effect on inner hind leg stance prolongation is very weak, and given the huge sample size, hard to interpret. Also, it is not clear how this fits with the role of DNa01 in slow sustained turning based on recordings.

Reviewer #3 (Public review):

Summary:

In this work, the authors investigated the molecular and cellular basis of sour taste perception in Drosophila melanogaster, focusing on identifying receptors that mediate attractive responses to certain carboxylic acids. It builds on previous work from the same group that had identified the IR co-receptors IR25a and IR76b for this sensory process, screening a set of mutants in IRs to identify three, IR51b, IR94a, and IR94h, required for feeding preference responses to some or all of the tested acids.

Strengths:

The work is of interest because it assigns sensory roles to IRs of previously unknown function, in particular IR94a and IR94h, and points to pharyngeal neurons in which these receptors are expressed as the relevant sensory neurons (potentially with different roles for IR94a- and IR94h-expressing neurons). The work combines elegant genetics, simple but effective feeding and taste assays, chemo-/opto-genetic activation, and some calcium imaging. Overall the presented data look solid and well-controlled.

Weaknesses:

The in situ expression analysis relies entirely on transgenic driver lines for IR94a and IR94h (which had been previously described, though not fully cited in this work). Importantly, given that many of the behavioral experiments (genetic rescue, physiology, artificial activation) use the IR94a and IR94h GAL4 driver lines, it would be helpful to validate that these faithfully reflect IR94a and IR94h expression (as far as I can tell, such validation wasn't done in the original papers describing these lines as part of a large collection of IR drivers). For IR51b, pharyngeal expression is concluded indirectly from non-quantitative RT-PCR analysis (genetic reporters did not work). The lack of direct detection of gene/protein expression (for example, through RNA FISH, immunofluorescence, or protein tagging) would have made for a more complete characterization of these receptors (for example, there is no direct evidence that they also express IR25a and IR76b, as one might expect). Finally, the relationship of IR94a and IR94h neurons to other types of pharyngeal neurons remains unclear, as are their projection patterns in the SEZ.

Conceptually, the work is of interest mostly to those in the immediate field; there have been a very large number of studies in the past decade (several from this lab) characterizing the contributions of different IRs to various chemosensory processes. The current work doesn't lend much insight into the nature of the minimal functional unit of gustatory IRs (reconstitution of a functional IR in a heterologous neuron/cell has not been achieved here, but this is a limitation of many other previous studies), nor to how different pharyngeal sensory pathways might collaborate to control behavior. Nevertheless, the findings provide a useful contribution to the literature.

Reviewer #3 (Public review):

Summary:

Sukhina et al are trying to understand the impacts of malnutrition on immunity. They model malnutrition with a diet switch from ad libitum to 40% caloric restriction (CR) in post-weaned mice. They test impacts on immune function with listeriosis. They then test whether re-feeding corrects these defects and find aspects of emergency myelopoiesis that remain defective after a precedent period of 40% CR. Overall, this is a very interesting observational study on the impacts of sudden prolonged exposure to less caloric intake.

Strengths:

The study is rigorously done. The observation of lasting defects after a bout of 40% CR is quite interesting. Overall, I think the topic and findings are of interest.

Weaknesses:

While the observations are interesting, in this reviewer's opinion, there is both a lack of mechanistic understanding of the phenomena and also some lack of resolution/detail about the phenomena itself. Addressing the following major issues would be helpful towards aspects of both:

(1) Is it calories, per se, or macro/micronutrients that drive these phenotypes observed with 40% CR. At the least, I would want to see isocaloric diets (primarily protein, fat, or carbs) and then some of the same readouts after 40% CR. Ie does low energy with relatively more eg protein prevent immunosuppression (as is commonly suggested)? Micronutrients would be harder to test experimentally and may be out of the scope of this study. However, it is worth noting that many of the malnutrition-associated diseases are micronutrient deficiencies.

(2) Is immunosuppression a function of a certain weight loss threshold? Or something else? Some idea of either the tempo of immunosuppression (happens at 1, in which weight loss is detected; vs 2-3, when body length and condition appear to diverge; or 5 weeks), or grade of CR (40% vs 60% vs 80%) would be helpful since the mechanism of immunosuppression overall is unclear (but nailing it may be beyond the scope of this communication).

(3) Does an obese mouse that gets 40% CR also become immunodeficient? As it stands, this ad libitum --> 40% CR model perhaps best models problems in the industrial world (as opposed to always being 40% CR from weaning, as might be more common in the developing world), and so modeling an obese person losing a lot of weight from CR (like would be achieved with GLP-1 drugs now) would be valuable to understanding generalizability.

(4) Generalizing this phenomenon as "bacterial" with listeriosis, which is more like a virus in many ways (intracellular phase, requires type I IFN, etc.) and cannot be given by the natural route of infection in mice, may not be most accurate. I would want to see an experiment with E.Coli, or some other bacteria, to test the statement of generalizability (ie is it bacteria, or type I IFN-pathway dominant infections, like viruses). If this is unique listeriosis, it doesn't undermine the story as it is at all, but it would just require some word-smithing.

(5) Previous reports (which the authors cite) implicate Leptin, the levels of which scale with fat mass, as "permissive" of a larger immune compartment (immune compartment as "luxury function" idea). Is their phenotype also leptin-mediated (ie leptin AAV)?

(6) The inability of re-feeding to "rescue" the myeloid compartment is really interesting. Can the authors do a bone marrow transplantation (CR-->ad libitum) to test if this effect is intrinsic to the CR-experienced bone marrow?

(7) Is the defect in emergency myelopoiesis a defect in G-CSF? Ie if the authors injected G-CSF in CR animals, do they equivalently mobilize neutrophils? Does G-CSF supplementation (as one does in humans) rescue host defense against Listeria in the CR or re-feeding paradigms?

Reviewer #3 (Public review):

Summary:

This study established a single-cell RNA sequencing atlas of pipefish embryos. The results obtained identified unique gene expression patterns for pipefish-specific characteristics, such as fgf22 in the tip of the palatoquadrate and Meckel's cartilage, broadly informing the genetic mechanisms underlying morphological novelty in teleost fishes. The data obtained are unique and novel, potentially important in understanding fish diversity. Thus, I would enthusiastically support this manuscript if the authors improve it to generate stronger and more convincing conclusions than the current forms.

Weakness:

Regarding the expression of sfrp1a and bmp4 dorsal to the elongating ethmoid plate and surrounding the ceratohyal: Are their expression patterns spatially extended or broader compared to the pipefish ancestor? Is there a much closer species available to compare gene expression patterns with pipefish? Did the authors consider using other species closely related to pipefish for ISH? Sfrp1a and bmp4 may be expressed in the same regions of much more closely related species without face elongation. I understand that embryos of such species are not always accessible, but it is also hard to argue responsible genes for a specific phenotype by only comparing gene expression patterns between distantly related species (e.g., pipefish vs. zebrafish). Due to the same reason, I would not directly compare/argue gene expression patterns between pipefish and mice, although I should admit that mice gene expression patterns are sometimes helpful to make a hypothesis of fish evolution. Alternatively, can the authors conduct ISH in other species of pipefish? If the expression patterns of sfrp1a and bmp4 are common among fishes with face elongation, the conclusion would become more solid. If these embryos are not available, is it possible to reduce the amount of Wnt and BMP signal using Crispr/Cas, MO, or chemical inhibitor? I do think that there are several ways to test the Wnt and/or BMP hypothesis in face elongation.

Reviewer #3 (Public review):

Summary:

Ruan and colleagues consider a branching process model (in their terminology the "Haldane model") and the most basic Wright-Fisher model. They convincingly show that offspring distributions are usually non-Poissonian (as opposed to what's assumed in the Wright-Fisher model), and can depend on short-term ecological dynamics (e.g., variance in offspring number may be smaller during exponential growth). The authors discuss branching processes and the Wright-Fisher model in the context of 3 "paradoxes" --- 1) how Ne depends on N might depend on population dynamics; 2) how Ne is different on the X chromosome, the Y chromosome, and the autosomes, and these differences do match the expectations base on simple counts of the number of chromosomes in the populations; 3) how genetic drift interacts with selection. The authors provide some theoretical explanations for the role of variance in the offspring distribution in each of these three paradoxes. They also perform some experiments to directly measure the variance in offspring number, as well as perform some analyses of published data.

Strengths:

- The theoretical results are well-described and easy to follow.<br /> - The analyses of different variances in offspring number (both experimentally and analyzing public data) are convincing that non-Poissonian offspring distributions are the norm.<br /> - The point that this variance can change as the population size (or population dynamics) change is also very interesting and important to keep in mind.<br /> - I enjoyed the Density-Dependent Haldane model. It was a nice example of the decoupling of census size and effective size.<br /> - Equation (10) is a nice result (but see below)

Weaknesses:

- I am not convinced that these types of effects cannot just be absorbed into some time-varying Ne and still be well-modeled by the Wright-Fisher process. As a concrete example, Mohle and Sagitov 2001 show that a "coalescent Ne" for the WF model should be (N-1)/Var(K). This resolves the exponentially growing bacteria "paradox" raised in the present paper --- when the bacteria are growing Var(K) ~ 0, and hence there should be very little drift. This exactly resolves the "paradox" raised by the authors. Instead, it merely underscores that Ne does not need to be equal to (or even positively correlated!) with N. I absolutely do not see this as a failure of the WF model. Whether one finds branching processes or the WF model more biologically intuitive is a matter of taste, but to say that WF models cannot explain this "paradox" is false, when a well-known paper from more than 20 years ago does just that.<br /> - Along these lines, the result that Ne in the Wright-Fisher process might not be related to N in any straightforward (or even positively correlated) way are well-known (e.g., Neher and Hallatschek 2012; Spence, Kamm, and Song 2016; Matuszewski, Hildebrandt, Achaz, and Jensen 2018; Rice, Novembre, and Desai 2018; the work of Lounès Chikhi on how Ne can be affected by population structure; etc...)<br /> - I was also missing some discussion of the relationship between the branching process and the Wright-Fisher model (or more generally Cannings' Exchangeable Models) when conditioning on the total population size. In particular, if the offspring distribution is Poisson, then conditioned on the total population size, the branching process is identical to the Wright-Fisher model.<br /> - Given that Cannings' exchangeable models decouple N and Ne, it would not surprise me if something like equation (10) could be derived under such a model. I have not seen such a derivation, and the authors' result is nice, but I do not see it as proof that WF-type models (i.e., Cannings' models) are irreparably broken.

Editors' comments (Public review):

Weaknesses:

(1) Figure 7C, 7E, 7I, and "Figure7-figure supplement 1 ": All data in these data panels are based on only n=3, which is insufficient. Sample sizes of n=3 are too low to correctly assess normality of distribution and, as a consequence, do not allow to select the appropriate parametric/non-parametric tests. Accordingly, no statistical comparison can be performed and all p values and symbols currently indicating statistically significant differences between groups must be removed.

(2) Figure 3A, 3B, or 3C: No information about n numbers per group. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

(3) Figure 4 E and 4F: No information about n numbers per group. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

(4) Figure 5: No information about n numbers per group is provided. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

Reviewer #3 (Public review):

Summary:

The study aims to determine whether the endosomal protein SNX4 performs a role in neurotransmitter release and synaptic vesicle recycling. The authors exploited a newly generated conditional knockout mouse to allow them to interrogate SNX4 function. A series of basic parameters were assessed, with an observed impact on neurotransmitter release and active zone morphology. The work is interesting, however as things currently stand, the work is descriptive with little mechanistic insight. There are a number of places where some of the conclusions require further validation.

Strengths:

The strengths of the work are the state-of-the-art methods to monitor presynaptic function.

Weaknesses:

The weaknesses are the fact that the work is largely descriptive, with no mechanistic insight into the role of SNX4.

Comments on revisions:

The authors have addressed a couple of the more major concerns with the manuscript, however many of the original weaknesses remain. The primary weakness being the lack of mechanism. It is disappointing that real-time VAMP2 trafficking was not investigated, and the authors justification as to why the experiment was not performed was not convincing (especially since this is the approach that all other groups employ to examine SV cargo trafficking). In a number of instances "contractual constraints" are referred to as an explanation for not performing additional experiments. It was unclear whether this refers to licencing issues with the mouse line or the lack of personnel to perform the work. Regardless it still leaves this work as somewhat incomplete.

Reviewer #3 (Public review):

Summary of the review process from the Reviewing Editor:

The authors and the reviewers did not agree on several important points made in this paper. The reviewers were critical of the operationalisation of the concept of visual homogeneity (VH), and questioned its validity. For instance, they found it unsatisfying that VH was not calculated on the basis of images themselves, but on the basis of reaction times instead. The authors responded by providing further explanation and argumentation for the importance of this novel concept, but the reviewers were not persuaded. The reviewers also pointed out some data features that did not fit the theory (e.g., overlapping VH between present and absent stimuli), which the authors acknowledge as a point that needs further refining. Finally, the reviewers pointed out that the new so-called visual homogeneity brain region does not overlap very much in the two studies, to which the authors have responded that it is remarkable that there is even partial overlap, given the many confounding differences between the two studies. Altogether, the authors have greatly elaborated their case for VH as an important concept, but the reviewers were not persuaded, and we conclude that the current evidence does not yet meet the high bar for declaring that a novel image property, visual homogeneity, is computed in a localised brain region.

Reviewer #3 (Public review):

Summary:

In this study, the authors set out to investigate whether GPRC6A mediates kokumi taste initiated by the amino acid L-ornithine. They used Wistar rats, a standard laboratory strain, as the primary model and also performed an informative taste test in humans, in which miso soup was supplemented with various concentrations of L-ornithine. The findings are valuable and overall the evidence is solid. L-Ornithine should be considered to be a useful test substance in future studies of kokumi taste and the class C G protein-coupled receptor known as GPRC6A (C6A) along with its homolog, the calcium-sensing receptor (CaSR) should be considered candidate mediators of kokumi taste.

Strengths:

The overall experimental design is solid based on two bottle preference tests in rats. After determining the optimal concentration for L-Ornithine (1 mM) in the presence of MSG, it was added to various tastants, including inosine 5'-monophosphate; monosodium glutamate (MSG); mono-potassium glutamate (MPG); intralipos (a soybean oil emulsion); sucrose; sodium chloride (NaCl); citric acid and quinine hydrochloride. Robust effects of ornithine were observed in the cases of IMP, MSG, MPG, and sucrose, and little or no effects were observed in the cases of sodium chloride, citric acid, and quinine HCl. The researchers then focused on the preference for Ornithine-containing MSG solutions. The inclusion of the C6A inhibitors Calindol (0.3 mM but not 0.06 mM) or the gallate derivative EGCG (0.1 mM but not 0.03 mM) eliminated the preference for solutions that contained Ornithine in addition to MSG. The researchers next performed transections of the chord tympani nerves (with sham operation controls) in anesthetized rats to identify the role of the chorda tympani branches of the facial nerves (cranial nerve VII) in the preference for Ornithine-containing MSG solutions. This finding implicates the anterior half-two thirds of the tongue in ornithine-induced kokumi taste. They then used electrical recordings from intact chorda tympani nerves in anesthetized rats to demonstrate that ornithine enhanced MSG-induced responses following the application of tastants to the anterior surface of the tongue. They went on to show that this enhanced response was insensitive to amiloride, selected to inhibit 'salt tastant' responses mediated by the epithelial Na+ channel, but eliminated by Calindol. Finally, they performed immunohistochemistry on sections of rat tongue demonstrating C6A positive spindle-shaped cells in fungiform papillae that partially overlapped in its distribution with the IP3 type-3 receptor, used as a marker of Type-II cells, but not with (i) gustducin, the G protein partner of Tas1 receptors (T1Rs), used as a marker of a subset of type-II cells; or (ii) 5-HT (serotonin) and Synaptosome-associated protein 25 kDa (SNAP-25) used as markers of Type-III cells.

Weaknesses:

The researchers undertook what turned out to be largely confirmatory studies in rats with respect to their previously published work on Ornithine and C6A in mice (Mizuta et al Nutrients 2021).

The authors point out that animal models pose some difficulties of interpretation in studies of taste and raise the possibility in the Discussion that umami substances may enhance the taste response to ornithine (Line 271, Page 9). They miss an opportunity to outline the experimental results from the study that favor their preferred interpretation that ornithine is a taste enhancer rather than a tastant.

At least two other receptors in addition to C6A might mediate taste responses to ornithine: (i) the CaSR, which binds and responds to multiple L-amino acids (Conigrave et al, PNAS 2000), and which has been previously reported to mediate kokumi taste (Ohsu et al., JBC 2010) as well as responses to Ornithine (Shin et al., Cell Signaling 2020); and (ii) T1R1/T1R3 heterodimers which also respond to L-amino acids and exhibit enhanced responses to IMP (Nelson et al., Nature 2001). While the experimental results as a whole favor the authors' interpretation that C6A mediates the Ornithine responses, they do not make clear either the nature of the 'receptor identification problem' in the Introduction or the way in which they approached that problem in the Results and Discussion sections. It would be helpful to show that a specific inhibitor of the CaSR failed to block the ornithine response. In addition, while they showed that C6A-positive cells were clearly distinct from gustducin-positive, and thus T1R-positive cells, they missed an opportunity to clearly differentiate C6A-expressing taste cells and CaSR-expressing taste cells in the rat tongue sections.

It would have been helpful to include a positive control kokumi substance in the two-bottle preference experiment (e.g., one of the known gamma-glutamyl peptides such as gamma-glu-Val-Gly or glutathione), to compare the relative potencies of the control kokumi compound and Ornithine, and to compare the sensitivities of the two responses to C6A and CaSR inhibitors.

The results demonstrate that enhancement of the chorda tympani nerve response to MSG occurs at substantially greater Ornithine concentrations (10 and 30 mM) than were required to observe differences in the two bottle preference experiments (1.0 mM; Figure 2). The discrepancy requires careful discussion and if necessary further experiments using the two-bottle preference format.

Reviewer #3 (Public review):

Summary:

This study helps to clarify the mixed literature on dopamine responses to aversive stimuli. While it is well accepted that dopamine in the ventral striatum increases in response to various rewarding and appetitive stimuli, aversive stimuli have been shown to evoke phasic increases or decreasing depending on the exact aversive stimuli, behavioral paradigm, and/or dopamine recording method and location examined. Here the authors use a well-designed set of experiments to show differential responses to an appetitive primary reward (sucrose) that later becomes a conditioned aversive stimulus (sucrose previously paired with lithium chloride in a conditioned taste aversion paradigm). The results are interesting and add valuable data to the question of how the mesolimbic dopamine system encodes aversive stimuli, however, the conclusions are strongly stated given that the current data do not necessarily align with prior conflicting data in terms of recording location, and it is not clear exactly how to interpret the generally biphasic dopamine response to the CTA-sucrose which also evolves over exposures within a single session.

Strengths:

• The authors nicely demonstrate that their two aversive stimuli examined, quinine and sucrose following CTA, evoked aversive facial expressions and paw movements that differed from those following rewarding sucrose to support that the stimuli experienced by the rats differ in valence.

• Examined dopamine responses to the exact same sensory stimuli conditioned to have opposing valences, avoiding standard confounds of appetitive and aversive stimuli being sensed by different sensory modalities (i.e., sweet taste vs. electric shock).

• The authors examined multiple measurements of dopamine activity - cell body calcium (GCaMP6f) in midbrain and release in NAc (Grab-DA2h), which is useful as the prior mixed literature on aversive dopamine responses comes from a variety of recording methods.

• Correlations between sucrose preference and dopamine signals demonstrate behavioral relevance of the differential dopamine signals.

• The delayed testing experiment in Figure 7 nicely controls for the effect of time to demonstrate that the "rewarding" dopamine response to sucrose only recovers after multiple extinction sucrose exposures to extinguish the CTA.

Weaknesses for consideration:

• Regional differences in dopamine signaling to aversive stimuli are mentioned in the introduction and discussion. For instance, the idea that dopamine encodes salience is strongly argued against in the discussion, but the paper cited as arguing for that (Kutlu et al. 2021) is recording from the medial core in mice. Given other papers cited in the text about the regional differences in dopamine signaling in the NAc and from different populations of dopamine neurons in midbrain, it's important to mention this distinction wrt to salience signaling. Relatedly, the text says that the lateral NAc shell was targeted for accumbens recordings, but the histology figure looks like the majority of fibers were in the anterior lateral core of NAc. For the current paper to be a convincing last word on the issue, it would be extremely helpful to have similar recordings done in other parts of the NAc to do a more thorough comparison against other studies.

• Dopamine release in the NAc never dips below baseline for the conditioned sucrose. Is it possible to really consider this as a signal for valence per se, as opposed to it being a weaker response relative to the original sucrose response?

• Related to this, the main measure of the dopamine signal here, "mean z-score," obscures the temporal dynamics of the aversive dopamine response across a trial. This measure is used to claim that sucrose after CTA is "suppressing" dopamine neuron activity and release, which is true relative to the positive valence sucrose response. However, both GRAB-DA and cell-body GCaMP measurements show clear increases after onset of sucrose infusion before dipping back to baseline or slightly below in the average of all example experiments displayed. One could point to these data to argue either that aversive stimuli cause phasic increases in dopamine (due to the initial increase) or decreases (due to the delayed dip below baseline) depending on the measurement window. Some discussion of the dynamics of the response and how it relates to the prior literature would be useful.<br /> - Would this delayed below-baseline dip be visible with a shorter infusion time?<br /> - Does the max of the increase or the dip of the decrease better correlate with the behavioral measures of aversion (orofacial, paw movements) or sucrose preference than "mean z-score" measure used here?<br /> - The authors argue strongly in the discussion against the idea that dopamine is encoding "salience." Could this initial peak (also seen in the first few trials of quinine delivery, fig 1c color plot) be a "salience" response?

• Related to this, the color plots showing individual trials show a reduction in the increases to positive valence sucrose across conditioning day trials and a flip from infusion-onset increase to delayed increases across test day trials. This evolution across days makes it appear that the last few conditioning day trials would be impossible to discriminate from the first few test day trials in the CTA-paired. Presumably, from strength of CTA as a paradigm, the sucrose is already aversive to the animals at the first trial of test day. Why do the authors think the response evolves across this session?

• Given that most of the work is using a conditioned aversive stimulus, the comparison to a primary aversive tastant quinine is useful. However, the authors saw basically no dopamine response to a primary aversive tastant quinine (measured only with GRAB-DA) and saw less noticeable decreases following CTA for NAc recordings with GRAB-DA2h than with cell body GCaMP. Given that they are using the high-affinity version of the GRAB sensor, this calls into question whether this is a true difference in release vs. soma activity or issue of high affinity release sensor making decreases in dopamine levels more difficult to observe.

Reviewer #3 (Public review):

Summary:

The authors use high-depth, full-length scRNA-Seq analysis of fetal human retina to identify novel regulators of photoreceptor specification and retinoblastoma progression.

Strengths:

The use of high-depth, full-length scRNA-Seq to identify functionally important alternatively spliced variants of transcription factors controlling photoreceptor subtype specification, and identification of SYK as a potential mediator of RB1-dependent cell cycle reentry in immature cone photoreceptors.

Human developing fetal retinal tissue samples were collected between 13-19 gestational weeks and this provides a substantially higher depth of sequencing coverage, thereby identifying both rare transcripts and alternative splice forms, and thereby representing an important advance over previous droplet-based scRNA-Seq studies of human retinal development.

Weaknesses:

The weaknesses identified are relatively minor. This is a technically strong and thorough study, that is broadly useful to investigators studying retinal development and retinoblastoma.

Reviewer #3 (Public review):

Summary:

The decrease in estrogen levels is strongly associated with postmenopausal hypertension. Dr. Yao Li and colleagues aimed to investigate the metabolomic mechanisms of underlying postmenopausal hypertension using OVX and OVX+E2 rat models. They successfully established a correlation between reduced estrogen levels and the development of hypertension in rats. They identified L-alpha-aminobutyric acid (AABA) as a potential marker for postmenopausal hypertension. The research explored the metabolic alterations in aortic tissues and proposed several potential mechanisms contributing to postmenopausal hypertension.

Strengths:

The group performed a comprehensive enrichment analysis and various statistical analyses of the metabolomics data.

Weaknesses:

(1) The manuscript is descriptive in nature, although they mentioned their primary objective is to explore the potential mechanisms linking low estrogen levels with postmenopausal hypertension. No mechanism insights have been interrogated in this study, which has been mentioned by the authors in the discussion. The connection between E2, AABA, and macrophage needs to be validated in endothelial cells, vascular smooth muscle cells, and other aortic tissue cells. Without such verification, the manuscript predominantly raises hypotheses only based on metabolomic data.

(2) The serum contains three forms of estrogen: Estradiol, Estrone, and Estriol. The authors used the Rat E2 ELISA kit. Ideally, all three forms of estrogen should be measured.

Reviewer #3 (Public review):

Summary:

At the abandoned replication fork, loading of DnaB helicase requires assistance from PriABC, repA, and other protein partners, but it does not require replication initiator protein, DnaA. In contrast, nucleotide-dependent DnaA binding at the specific functional elements is fundamental for helicase loading, leading to the DUE region's opening. However, the authors questioned in this study that in case of impeding replication at the bacterial chromosomal origins, oriC, a strategy similar to an abandoned replication fork for loading DnaB via bypassing the DnaA interaction step could be functional. The study by Yoshida et al. suggests that PriC could promote DnaB helicase loading on the chromosomal oriC ssDNA without interacting with the DnaA protein. However, the conclusions drawn from the primarily qualitative data presented in the study could be slightly overwhelming and need supportive evidence.

Strengths:

Understanding the mechanism of how DNA replication restarts via reloading the replisomes onto abandoned DNA replication forks is crucial. Notably, this knowledge becomes crucial to understanding how bacterial cells maintain DNA replication from a stalled replication fork when challenging or non-permissive conditions prevail. This critical study combines experiments to address a fundamental question of how DnaB helicase loading could occur when replication initiation impedes at the chromosomal origin, leading to replication restart.

Weaknesses:

The term colony formation used for a spotting assay could be misleading for apparent reasons. Both assess cell viability and growth; while colony formation is quantitative, spotting is qualitative. Particularly in this study, where differences appear minor but draw significant conclusions, the colony formation assays representing growth versus moderate or severe inhibition are a more precise measure of viability.

Figure 2<br /> The reduced number of two oriC copies per cell in the dnaA46priC-deficient strain was considered moderate inhibition. When combined with the data suggested by the dnaAC2priC-deficient strain containing two origins in cells with or without PriC (indicating no inhibition)-the conclusion was drawn that PriC rescue blocked replication via assisting DnaC-dependent DnaB loading step at oriC ssDNA.

The results provided by Saifi B, Ferat JL. PLoS One. 2012;7(3):e33613 suggests the idea that in an asynchronous DnaA46 ts culture, the rate by which dividing cells start accumulating arrested replication forks might differ (indicated by the two subpopulations, one with single oriC and the other with two oriC). DnaA46 protein has significantly reduced ATP binding at 42C, and growing the strain at 42C for 40-80 minutes before releasing them at 30 C for 5 minutes has the probability that the two subpopulations may have differences in the active ATP-DnaA. The above could be why only 50% of cells contain two oriC. Releasing cells for more time before adding rifampicin and cephalexin could increase the number of cells with two oriCs. In contrast, DnaC2 cells have inactive helicase loader at 42 C but intact DnaA-ATP population (WT-DnaA at 42 or 30 C should not differ in ATP-binding). Once released at 30 C, the reduced but active DnaC population could assist in loading DnaB to DnaA, engaged in normal replication initiation, and thus should appear with two oriC in a PriC-independent manner.

Broadly, the evidence provided by the authors may support the primary hypothesis. Still, it could call for an alternative hypothesis: PriC involvement in stabilizing the DnaA-DnaB complex (this possibility could exist here). To prove that the conclusions made from the set of experiments in Figures 2 and 3, which laid the foundations for supporting the primary hypothesis, require insights using on/off rates of DnaB loading onto DnaA and the stability of the complexes in the presence or absence of PriC, I have a few other reasons to consider the latter arguments.

Figure 3<br /> One should consider the fact that dnA46 is present in these cells. Overexpressing pdnaAFH could produce mixed multimers containing subunits of DnaA46 (reduced ATP binding) and DnaAFH (reduced DnaB binding). Both have intact DnaA-DnaA oligomerization ability. The cooperativity between the two functions by a subpopulation of two DnaA variants may compensate for the individual deficiencies, making a population of an active protein, which in the presence of PriC could lead to the promotion of the stable DnaA: DnaBC complexes, able to initiate replication. In the light of results presented in Hayashi et al. and J Biol Chem. 2020 Aug 7;295(32):11131-11143, where mutant DnaBL160A identified was shown to be impaired in DnaA binding but contained an active helicase function and still inhibited for growth; how one could explain the hypothesis presented in this manuscript. If PriC-assisted helicase loading could bypass DnaA interaction, then how growth inhibition in a strain carrying DnaBL160A should be described. However, seeing the results in light of the alternative possibility that PriC assists in stabilizing the DnaA: DnaBC complex is more compatible with the previously published data.

Figure 4<br /> Overexpression of DiaA could contribute to removing a higher number of DnaA populations. This could be more aggravated in the absence of PriC (DiaA could titrate out more DnaA)- the complex formed between DnaA: DnaBC is not stable, therefore reduced DUE opening and replication initiation leading to growth inhibition (Fig. 4A ∆priC-pNA135). Figure 7C: Again, in the absence of PriC, the reduced stability of DnaA: DnaBC complex leaves more DnaA to titrate out by DiaA, and thus less Form I*. However, adding PriC stabilizes the DnaA: DnaBC hetero-complexes, with reduced DnaA titration by DiaA, producing additional Form I*. Adding a panel with DnaBL160A that does not interact with DnaA but contains helicase activity could be helpful. Would the inclusion of PriC increase the ability of mutant helicase to produce additional Form I*?

Figure 5<br /> The interpretation is that colony formation of the Left-oriC ∆priC double mutant was markedly compromised at 37˚C (Figure 5B), and 256 the growth defects of the Left-oriC mutant at 25{degree sign}C and 30{degree sign}C were aggravated. However, prima facia, the relative differences in the growth of cells containing and lacking PriC are similar. Quantitative colony-forming data is required to claim these results. Otherwise, it is slightly confusing.

A minor suggestion is to include cells expressing PriC using plasmid DNA to show that adding PriC should reverse the growth defect of dnaA46 and dnaC2 strains at non-permissive temperatures. The same should be added at other appropriate places.

Reviewer #3 (Public review):

Summary:

Obray et al. investigate the long-lasting effects of adolescent intermittent ethanol (AIE) in rats, a model of alcohol dependence, on a neural circuit within the prefrontal cortex. The studies are focused on inputs from the basolateral amygdala (BLA) onto parvalbumin (PV) interneurons and pyramidal cells that project to the periaqueductal gray (PAG). The authors found that AIE increased BLA excitatory drive onto parvalbumin interneurons and increased BLA feedforward inhibition onto PAG-projecting neurons.

Strengths:

Fully powered cohorts of male and female rodents are used, and the design incorporates both AIE and an acute pain model. The authors used several electrophysiological techniques to assess synaptic strength and excitability from a few complimentary angles. The design and statistical analysis are sound, and the strength of evidence supporting synaptic changes following AIE results is solid.

Weaknesses:

(1) There is incomplete evidence supporting some of the conclusions drawn in this manuscript. The authors claim that the changes in feedforward inhibition onto pyramidal cells are due to the changes in parvalbumin interneurons, but evidence is not provided to support that idea. PV cells do not spontaneously fire action potentials spontaneously in slices (nor do they receive high levels of BLA activity while at rest in slices). It is possible that spontaneous GABA release from PV cells is increased after AIE but the authors did not report sIPSC frequency. Second, the authors did not determine that PV cells mediate the feedforward BLA op-IPSCs and changes following AIE (this would require manipulation to reduce/block PV-IN activity). This limitation in results and interpretation is important because prior work shows BLA-PFC feedforward IPSCs can be driven by somatostatin cells. Cholecystokinin cells are also abundant basket cells in PFC and have been recently shown to mediate feedforward inhibition from the thalamus and ventral hippocampus, so it's also possible that CCK cells are involved in the effects observed here.

(2) The authors conclude that the changes in this circuit likely mediate long-lasting hyperalgesia, but this is not addressed experimentally. In some ways, the focused nature of the study is a benefit in this regard, as there is extensive prior literature linking this circuit with pain behaviors in alternative models (e.g., SNI), but it should be noted that these studies have not assessed hyperalgesia stemming from prior alcohol exposure. While the current studies do not include a causative behavioral manipulation, the strength of the association between BLA-PL-PAG function and hyperalgesia could be bolstered by current data if there were relationships detected between electrophysiological properties and hyperalgesia. Have the authors assessed this? In addition, this study is limited by not addressing the specificity of synaptic adaptations to the BLA-PL-PAG circuit. For instance, PL neurons send reciprocal projections to BLA and send direct projections to the locus coeruleus (which the authors note is an important downstream node of the PAG for regulating pain).

(3) I have some concerns about methodology. First, 5-ms is a long light pulse for optogenetics and might induce action-potential independent release. Does TTX alone block op-EPSCs under these conditions? Second, PV cells express a high degree of calcium-permeable AMPA receptors, which display inward rectification at positive holding potentials due to blockade from intracellular polyamines. Typically, this is controlled/promoted by including spermine in the internal solution, but I do not believe the authors did that. Nonetheless, the relatively low A/N ratios for this cell type suggest that CP-AMPA receptors were not sampled with the +40/+40 design of this experiment, raising concerns that the majority of AMPA receptors in these cells were not sampled during this experiment. Finally, it should be noted that asEPSC frequency can also reflect changes in a number of functional/detectable synapses. This measurement is also fairly susceptible to differences in inter-animal differences in ChR2 expression. There are other techniques for assessing presynaptic release probability (e.g., PPR, MK-801 sensitivity) that would improve the interpretation of these studies if that is intended to be a point of emphasis.

(4) In a few places in the manuscript, results following voluntary drinking experiments (especially Salling et al. and Sicher et al.) are discussed without clear distinction from prior work in vapor models of dependence

(5) Discussion (lines 416-420). The authors describe some differing results with the literature and mention that the maximum current injection might be a factor. To me, this does not seem like the most important factor and potentially undercuts the relevance of the findings. Are the cells undergoing a depolarization block? Did the authors observe any changes in the rheobase or AP threshold? On the other hand, a more likely difference between this and previous work is that the proportion of PAG-projecting cells is relatively low, so previous work in L5 likely sampled many types of pyramidal cells that project to other areas. This is a key example where additional studies by the current group assessing a distinct or parallel set of pyramidal cells would aid in the interpretation of these results and help to place them within the existing literature. Along these lines, PAG-projecting neurons are Type A cells with significant hyperpolarization sag. Previous studies showed that adolescent binge drinking stunts the development of HCN channel function and ensuing hyperpolarization sag. Have the authors observed this in PAG-projecting cells? Another interesting membrane property worth exploring with the existing data set is the afterhyperpolarization / SK channel function.

Reviewer #3 (Public Review):

This neuroimaging study investigated how brain activity related to visual pattern-based reasoning changes over the adult lifespan, addressing the topic of functional compensation in older age. To this end, the authors employed a version of the Cattell task, which probes visual pattern recognition for identifying commonalities and differences within sets of abstract objects in order to infer the odd object among a given set. Using a state-of-the-art univariate analysis approach on fMRI data from a large lifespan sample, the authors identified brain regions in which the activation contrast between hard and easy Cattell task conditions was modulated by both age and performance. Regions identified comprised prefrontal areas and bilateral cuneus. Applying a multivariate decoding approach to activity in these regions, the authors went on to show that only in older adults, the cuneus, but not the prefrontal regions, carried information about the task condition (hard vs. easy) beyond that already provided by activity patterns of voxels that showed a univariate main effect of task difficulty. This was taken as compelling evidence for task-specific compensatory activity in the cuneus in advanced age.

The study is well-motivated and well-written. The authors used appropriate, rigorous methods that allowed them to control for a range of possible confounds or alternative explanations. Laudable aspects include the large sample with a wide and even age distribution, the validation of the in-scanner task performance against previous results obtained with a more standard version outside the scanner, and the control for vascular age-related differences in hemodynamic activity via a BOLD signal amplitude measure obtained from a separate resting-state fMRI scan. Overall, the conclusions are well-supported by the data.

Comment from Reviewing Editor: The revised manuscript has addressed the points raised during the review of the original submission.

Reviewer #3 (Public review):

Summary:

The authors are showing evidence that they claim establishes the controversial epigenetic mark, DNA 6mA, as promoting genome stability.

Strengths:

The identification of a poorly understood protein, METTL3, and its subsequent characterization in DDR is of high quality and interesting.

Weaknesses:

(1) The very presence of 6mA (DNA) in mammalian DNA is still highly controversial and numerous studies have been conclusively shown to have reported the presence of 6mA due to technical artifacts and bacterial contamination. Thus, to my knowledge there is no clear evidence for 6mA as an epigenetic mark in mammals, and consequently, no evidence of writers and readers of 6mA. None of this is mentioned in the introduction. Much of the introduction can be reduced, but a paragraph clearly stating the controversy and lack of evidence for 6mA in mammals needs to be added, otherwise, the reader is given an entirely distorted view of the field.

These concerns must also be clearly in the limitations section and even in the results section which fails to nuance the authors' findings.

(2) What is the motivation for using HT-29 cells? Moreover, the materials and methods do not state how the authors controlled for bacterial contamination, which has been the most common cause of erroneous 6mA signals to date. Did the authors routinely check for mycoplasma?

(3) The single cell imaging of 6mA in various cells is nice. The results are confirmed by mass spec as an orthogonal approach. Another orthogonal and quantitative approach to assessing 6mA levels would be PacBio. Similarly, it is unclear why the authors have not performed dot-blots of 6mA for genomic DNA from the given cell lines.

(4) The results of Figure 3 need further investigation and validation. If the results are correct the authors are suggesting that the majority of 6mA in their cell lines is present in the DNA, and not the RNA, which is completely contrary to every other study of 6mA in mammalian cells that I am aware of. This could suggest that the antibody is not, in fact, binding to 6mA, but to unmodified adenine, which would explain why the signal disappears after DNAse treatment. Indeed, binding of 6mA to unmethylated DNA is a commonly known problem with most 6mA antibodies and is well described elsewhere.

(5) Given the lack of orthologous validation of the observed DNA 6mA and the lack of evidence supporting the presence of 6mA in mammalian DNA and consequently any functional role for 6mA in mammalian biology, the manuscript's conclusions need to be toned down significantly, and the inherent difficulty in assessing 6mA accurately in mammals acknowledged throughout.

Reviewer #3 (Public review):

This manuscript reports a detailed model of the rat non-barrel somatosensory cortex, consisting of 4.2 million morphologically and biophysically detailed neuron models, arranged in space and connected according to highly sophisticated rules informed by diverse experimental data. Due to its breadth and sophistication the model will undoubtedly be of interest to the community, and the reporting of anatomical details of modeling in this paper is important for understanding all the assumptions and procedures involved in constructing the model. While a useful contribution to this field, the model and the manuscript could be improved by employing data more directly and comparing simple features of the model's connectivity - in particular, connection probabilities - with relevant experimental data.

The manuscript is overall well-written, but contains a substantial number of confusing or unclear statements, and some important information is not provided.

Comments on revisions:

The authors mostly addressed all my points and improved the paper substantially. I do not have further extensive comments except one general point below.

Regarding section 2.3 and metrics of connectivity like pairwise connection probabilities, it is great that the authors rewrote that section and added comparisons with experimental data in Figs. 4 and S9. Unfortunately, what one finds when direct comparisons are made is that the modeled pairwise connectivity is quite different from the data. Fig. S9 shows that the model's results do not agree with data in about half of the cases (purple and red arrows). Similarly large discrepancies can be seen for some other metrics, like in Fig. S10B and S10C1,C2. (And similar concerns apply to thalamocortical connections in section 2.5, where it looks like little to no data are available to verify the pairwise connectivity between the thalamic and cortical neurons via a direct comparison.)

This is concerning since this model forms the basis for multiple other studies of cortical dynamics and function by the same group and potentially others in the community, with multiple papers relying on it, whereas basic properties of connectivity are apparently not captured well.

On the other hand, this is also a "glass half full" situation, showing that the sophisticated algorithms for establishing connections, developed by the authors, are working well in at least half of the connection types explored. It is therefore imperative that the authors continue refining these algorithms to capture the remaining half in future iterations and producing improved models that the community can better rely on.

Please also note that Fig. S11 does not have a caption.

Reviewer #3 (Public review):

Summary:

The submission from Cronshagen and colleagues describes the application of a previously described method (selection linked integration) to the systematic study of PfEMP1 trafficking in the human malaria parasite Plasmodium falciparum. PfEMP1 is the primary virulence factor and surface antigen of infected red blood cells and is therefore a major focus of research into malaria pathogenesis. Since the discovery of the var gene family that encodes PfEMP1 in the late 1990s, there have been multiple hypotheses for how the protein is trafficked to the infected cell surface, crossing multiple membranes along the way. One difficulty in studying this process is the large size of the var gene family and the propensity of the parasites to switch which var gene is expressed, thus preventing straightforward gene modification-based strategies for tagging the expressed PfEMP1. Here the authors solve this problem by forcing the expression of a targeted var gene by fusing the PfEMP1 coding region with a drug-selectable marker separated by a skip peptide. This enabled them to generate relatively homogenous populations of parasites all expressing tagged (or otherwise modified) forms of PfEMP1 suitable for study. They then applied this method to study various aspects of PfEMP1 trafficking.

Strengths:

The study is very thorough, and the data are well presented. The authors used SLI to target multiple var genes, thus demonstrating the robustness of their strategy. They then perform experiments to investigate possible trafficking through PTEX, they knock out proteins thought to be involved in PfEMP1 trafficking and observe defects in cytoadherence, and they perform proximity labeling to further identify proteins potentially involved in PfEMP1 export. These are independent and complimentary approaches that together tell a very compelling story.

Weaknesses:

(1) When the authors targeted IT4var19, they were successful in transcriptionally activating the gene, however, they did not initially obtain cytoadherent parasites. To observe binding to ICAM-1 and EPCR, they had to perform selection using panning. This is an interesting observation and potentially provides insights into PfEMP1 surface display, folding, etc. However, it also raises questions about other instances in which cytoadherence was not observed. Would panning of these other lines have been successfully selected for cytoadherent infected cells? Did the authors attempt panning of their 3D7 lines? Given that these parasites do export PfEMP1 to the infected cell surface (Figure 1D), it is possible that panning would similarly rescue binding. Likewise, the authors knocked out PTP1, TryThrA, and EMPIC3 and detected a loss of cytoadhesion, but they did not attempt panning to see if this could rescue binding. To ensure that the lack of cytoadhesion in these cases is not serendipitous (as it was when they activated IT4var19), they should demonstrate that panning cannot rescue binding.

(2) The authors perform a series of trafficking experiments to help discern whether PfEMP1 is trafficked through PTEX. While the results were not entirely definitive, they make a strong case for PTEX in PfEMP1 export. The authors then used BioID to obtain a proxiome for PfEMP1 and identified proteins they suggest are involved in PfEMP1 trafficking. However, it seemed that components of PTEX were missing from the list of interacting proteins. Is this surprising and does this observation shed any additional light on the possibility of PfEMP1 trafficking through PTEX? This warrants a comment or discussion.

Reviewer #3 (Public review):

The authors aim to understand how gene pleiotropy affects parallel evolutionary changes among independent replicates of adaptation to a new hot environment of a set of experimental lines of Drosophila simulans using experimental evolution. The flies were RNAsequenced after more than 100 generations of lab adaptation and the changes in average gene expression were obtained relative to ancestral expression levels from reconstructed ancestral lines. Parallelism of gene expression change among lines is evaluated as variance in differential gene expression among lines relative to error variance. Similarly, the authors ask how the standing variation in gene expression estimated from a handful of flies from a reconstructed outbred line affects parallelism. The main findings are that parallelism in gene expression responses is positively associated with pleiotropy and negatively associated with expression variation. Those results are in contradiction with theoretical predictions and empirical findings. To explain those seemingly contradictory results the authors invoke the role of synergistic pleiotropy and correlated selection, although they do not attempt to measure either.

Strengths:

(1) The study uses highly replicated outbred laboratory lines of Drosophila simulans evolved in the lab under a constant hot regime for over 100 generations. This allows for robust comparisons of evolutionary responses among lines.

(2) The manuscript is well written and the hypotheses are clearly delineated at the onset.

(3) The authors have run a causal analysis to understand the causal dependencies between pleiotropy and expression variation on parallelism.

(4) The use of whole-body RNA extraction to study gene expression variation is well justified.

Weaknesses:

(1) It is unclear how well phenotypic variation in gene expression of the evolved lines has been estimated by the sample of 20 males from a reconstructed outbred line not directly linked to the evolved lines under study. I see this as a general weakness of the experimental design.

(2) There are no estimates of standing genetic variation of expression levels of the genes under study, only phenotypic variation. I wished the authors had been clear about that limitation and had discussed the consequences of the analysis. This also constitutes a weakness of the study.

(3) Moreover, since the phenotype studied is gene expression, its genetic basis extends beyond expressed sequences. The phenotypic variation of a gene's expression may thus likely misrepresent the genetic variation available for its evolution. The genetic variation of gene expression phenotypes could be estimated from a cross or pedigree information but since individuals were pool-sequenced (by batches of 50 males), this type of analysis is not possible in this study.

(4) The authors have not attempted to estimate synergistic pleiotropy among genes, nor how selection acts on gene expression modules. It makes any conclusion regarding the role of synergistic pleiotropy highly speculative.

I don't understand the reason why the analysis would be restricted to significantly differentially expressed genes only. It is then unclear whether pleiotropy, parallelism, and expression variation do play a role in adaptation because the two groups of adaptive and non-adaptive genes have not been compared. I recommend performing those comparisons to help us better understand how "adaptive" genes differentially contribute to adaptation relative to "non-adaptive" genes relative to their difference in population and genetic properties.

There is a lack of theoretical groundings on the role of so-called synergistic pleiotropy for parallel genetic evolution. The Discussion does not address this particular prediction. It could be removed from the Introduction.

Reviewer #3 (Public review):

Summary:

Heterochromatin is characterized by low transcription activity and late replication timing, both dependent on the NAD-dependent protein deacetylase Sir2, the founding member of the sirtuins. This manuscript addresses the mechanism by which Sir2 delays replication timing at the rDNA in budding yeast. Previous work from the same laboratory (Foss et al. PLoS Genetics 15, e1008138) showed that Sir2 represses transcription-dependent displacement of the Mcm helicase in the rDNA. In this manuscript, the authors show convincingly that the repositioned Mcms fire earlier and that this early firing partly depends on the ATPase activity of the nucleosome remodeler Fun30. Using read-depth analysis of sorted G1/S cells, fun30 was the only chromatin remodeler mutant that somewhat delayed replication timing in sir2 mutants, while nhp10, chd1, isw1, htl1, swr1, isw2, and irc5 had no effect. The conclusion was corroborated with orthogonal assays including two-dimensional gel electrophoresis and analysis of EdU incorporation at early origins. Using an insightful analysis with an Mcm-MNase fusion (Mcm-ChEC), the authors show that the repositioned Mcms in sir2 mutants fire earlier than the Mcm at the normal position in wild type. This early firing at the repositioned Mcms is partially suppressed by Fun30. In addition, the authors show Fun30 affects nucleosome occupancy at the sites of the repositioned Mcm, providing a plausible mechanism for the effect of Fun30 on Mcm firing at that position. However, the results from the MNAse-seq and ChEC-seq assays are not fully congruent for the fun30 single mutant. Overall, the results support the conclusions providing a much better mechanistic understanding how Sir2 affects replication timing at rDNA,

Strengths:

(1) The data clearly show that the repositioned Mcm helicase fires earlier than the Mcm in the wild type position.

(2) The study identifies a specific role for Fun30 in replication timing and an effect on nucleosome occupancy around the newly positioned Mcm helicase in sir2 cells.

Comments on revisions:

In the previous revision the authors addressed my concerns and improved the manuscript and the presentation of the data. All my recommendations were implemented.

Reviewer #3 (Public review):

Significance:

About 5% of metastatic castration-resistant prostate cancers (mCRPC) display genomic alterations in the transcriptional kinase CDK12. The mechanisms by which CDK12 alterations drive tumorigenesis in this molecularly-defined subset of mCRPC have remained elusive. In particular, some studies have suggested that CDK12 loss confers a homologous recombination deficiency (HRd) phenotype, However, clinical studies have not borne out the benefit to PARP inhibitors in patients with CDK12 alterations, despite the fact that these agents are typically active against tumors with HRd.

In this study, Frank et al. reconcile these findings by showing that: (1) tumors with biallelic CDK12 alterations do not have genomic features of HRd; (2) in vitro, HR gene downregulation occurs with acute depletion of CDK12 but is far less pronounced with chronic CDK12 loss; (3) CDK12-altered cells are uniquely sensitive to genetic or pharmacologic inhibition of CDK13.

Strengths:

Overall, this is an important study that reconciles disparate experimental and clinical observations. The genomic analyses are comprehensive and conducted with a high degree of rigor and represent an important resource to the community regarding the features of this molecular subtype of mCRPC.

Weaknesses:

(1) It is generally assumed that CDK12 alterations are inactivating, but it is noteworthy that homozygous deletions are comparatively uncommon (Figure 1a). Instead many tumors show missense mutations on either one or both alleles, and many of these mutations are outside of the kinase domain (Figure 1b). It remains possible that the CDK12 alterations that occur in some tumors may retain residual CDK12 function, or may confer some other neomorphic function, and therefore may not be accurately modeled by CDK12 knockout or knockdown in vitro. This would also reconcile the observation that knockout of CDK12 is cell-essential while the human genetic data suggest that CDK12 functions as a tumor suppressor gene.

(2) It is not entirely clear whether CDK12 altered tumors may require a co-occurring mutation to prevent loss of fitness, either in vitro or in vivo (e.g. perhaps one or more of the alterations that occur as a result of the TDP may mitigate against the essentiality of CDK12 loss).

Reviewer #4 (Public review):

Summary:

Li and Chouhan et al. follow up on a previous publication describing the role of anterior-posterior (ap) and medial (m) ɑ′/β′ Kenyon cells in mediating sleep-dependent and sleep-independent memory consolidation, respectively, based on feeding state in Drosophila melanogaster. The authors sequenced bulk RNA of ap ɑ′/β′ Kenyon cells 1h after flies were either trained-fed, trained-starved or untrained-fed and find a small number of genes (59) differentially expressed (3 upregulated, 56 downregulated) between trained-fed and trained-starved conditions. Many of these genes encode proteins involved in the regulation of gene expression. The authors then screened these differentially expressed genes for sleep phenotypes by expressing RNAi hairpins constitutively in ap ɑ′/β′ Kenyon cells and measuring sleep patterns. Two hits were selected for further analysis: Polr1F, which promoted sleep, and Regnase-1, which reduced sleep. The pan-neuronal expression of Polr1F and Regnase-1 RNAi constructs was then temporally restricted to adult flies using the GeneSwitch system. Polr1F sleep phenotypes were still observed, while Regnase-1 sleep phenotypes were not, indicating developmental defects. Appetitive memory was then assessed in flies with constitutive knockdown of Polr1F and Regnase-1 in ap ɑ′/β′ Kenyon cells. Polr1F knockdown did not affect sleep-dependent or sleep-independent memory, while Regnase-1 knockdown disrupted sleep-dependent memory, sleep-independent memory, as well as learning. Polr1F knockdown increased pre-ribosomal RNA transcripts in the brain, as measured by qPCR, in line with its predicted role as part of the RNA polymerase I complex. A puromycin incorporation assay to fluorescently label newly synthesized proteins also indicated higher levels of bulk translation upon Polr1F knockdown. Regnase-1 knockdown did not lead to observable changes in measurements of bulk translation.

Strengths:

The proposed involvement of RNA processing genes in regulating sleep and memory processes is interesting, and relatively unexplored. The methods are satisfactory.

Weaknesses:

The main weakness of previous versions of the paper was the over-interpretation of results, particularly relating to the proposed link between sleep and memory consolidation. This has now been appropriately addressed, as reflected in the change of title and incorporation of alternative interpretations of the data in the text.

Reviewer #3 (Public review):

Yan et al. ("When do visual category representations emerge in infant brains?") present an EEG study of category-specific visual responses in infancy from 3 to 15 months of age. In their experiment, infants viewed visually controlled images of faces and several non-face categories in a steady state evoked potential paradigm. The authors find visual responses at all ages, but face responses only at 4-6 months and older, and other category-selective responses at later ages. They find that spatiotemporal patterns of response can discriminate faces from other categories at later ages.

Overall, I found the study well-executed and a useful contribution to the literature. The study advances prior work by using well-controlled stimuli, subgroups at different ages, and new analytic approaches. The data and analyses support their conclusions regarding developmental change in neural responses to high-level visual stimuli.

Reviewer #3 (Public review):

Summary:

Non-opioid analgesics derived from human amniotic membrane (AM) product represents a novel and unique approach to analgesia that may avoid the traditional harms associated with opioids. Here, the study investigators demonstrate that HC-HAPTX3 is the primary bioactive component of the AM product FLO responsible for anti-nociception in mouse-model and in-vitro dorsal root ganglion (DRG) cell culture experiments. The mechanism is demonstrated to be via CD44 with an acute cytoskeleton rearrangement that is induced that inhibits Na+ and Ca++ current through ion channels. Taken together, the studies reported in the manuscript provide supportive evidence clarifying the mechanisms and efficacy of HC-HAPTX3 antinociception and analgesia.

Strengths:

Extensive experiments including murine behavioral paw withdrawal latency and Catwalk test data demonstrating analgesic properties. Breadth and depth of experimental data are clearly supporting mechanisms and antinociceptive properties.

Weaknesses:

None. Only a few minor directed changes to the text of the manuscript.<br /> P4 last sentence - "Our findings highlight the potential of a naturally derived biologic from human birth tissues as an effective non-opioid treatment for post-surgical pain and unravel the underlying mechanisms." - another sentence clause is required before "unravel"<br /> P7 second paragraph - please edit the following sentence for clarity: "Since HC-HA/PTX3 mimics FLO in producing pain inhibition, and it has high-purity and is more water-soluble than FLO, making it suitable for probing cellular mechanisms."

Reviewer #3 (Public review):

Summary:

In the study presented by Burnicka-Turek et al., the authors generated for the first time a mouse model to cause the combined conditional deletion of Tbx3 and Tbx5 genes. This has been impossible to achieve to date due to the proximity of these genes in chromosome 5, preventing the generation of loss of function strategies to delete simultaneously both genes. It is known that both Tbx3 and Tbx5 are required for the development of the cardiac conduction system by transcription factor-specific but also overlapping roles as seen in the common and diverse cardiac defects found in patients with mutations for these genes. After validating the deletion efficiency and specificity of the line, the authors characterised the cardiac phenotype associated with the cardiac conduction system (CCS)-specific combined deletion of Tbx5 and Tbx3 in the adult by inducing the activation of the CCS-specific tamoxifen-inducible Cre recombination (MinK-creERT) at 6 weeks after birth. Their analysis of 8-9-week-old animals did not identify any major morphological cardiac defects. However, the authors found conduction defects including prolonged PR and QTR intervals and ventricular tachycardia causing the death of the double mutants, which do not survive more than 3 months after tamoxifen induction. Molecular and optical mapping analysis of the ventricular conduction system (VCS) of these mutants concluded that, in the absence of Tbx5 and Tbx3 function, the cells forming the ventricular conduction system (VCS) become working myocardium and lose the specific contractile features characterising VCS cells. Altogether, the study identified the critical combined role of Tbx3 and Tbx5 in the maintenance of the VCS in adulthood.

Strengths:

The study generated a new animal model to study the combined deletion of Tbx5 and Tbx3 in the cardiac conduction system. This unique model has provided the authors with the perfect tool to answer their biological questions. The study includes top-class methodologies to assess the functional defects present in the different mutants analysed, and gathered very robust functional data on the conduction defects present in these mutants. They also applied optical action potential (OAP) methods to demonstrate the loss of conduction action potential and the acquisition of working myocardium action potentials in the affected cells because of Tbx5/Tbx3 loss of function. The study used simpler molecular and morphological analysis to demonstrate that there are no major morphological defects in these mutants and that indeed, the conduction defects found are due to the acquisition of working myocardium features by the VCS cells. Altogether, this study identified the critical role of these transcription factors in the maintenance of the VCS in the adult heart.

Weaknesses:

In the opinion of this reviewer, the weakness in the study lies in the morphological and molecular characterization. The morphological analysis simply described the absence of general cardiac defects in the adult heart, however, whether the CCS tissues are present or not was not investigated. Lineage tracing analysis using the reporter lines included in the crosses described in the study will determine if there are changes in CCS tissue composition in the different mutants studied. Similarly, combining this reporter analysis with the molecular markers found to be dysregulated by qPCR and western blot, will demonstrate that indeed the cells that were specified as VCS in the adult heart, become working myocardium in the absence of Tbx3 and Tbx5 function.

Reviewer #3 (Public review):

Summary:

The manuscript by Patel et al investigates the hypothesis that CDHR1a on photoreceptor outer segments is the binding partner for PCDH15 on the calyceal processes, and the absence of either adhesion molecule results in separation between the two structures, eventually leading to degeneration. PCDH15 mutations cause Usher syndrome, a disease of combined hearing and vision loss. In the ear, PCDH15 binds CDH23 to form tip links between stereocilia. The vision loss is less understood. Previous work suggested PCDH15 is localized to the calyceal processes, but the expression of CDH23 is inconsistent between species. Patel et al suggest that CDHR1a (formerly PCDH21) fulfills the role of CDH23 in the retina.

The experiments are mainly performed using the zebrafish model system. Expression of Pcdh15b and Cdhr1a protein is shown in the photoreceptor layer through standard confocal and structured illumination microscopy. The two proteins co-IP and can induce aggregation in vitro. Loss of either Cdhr1a or Pcdh15, or both, results in degeneration of photoreceptor outer segments over time, with cones affected primarily.

The idea of the study is logical given the photoreceptor diseases caused by mutations in either gene, the comparisons to stereocilia tip links, and the protein localization near the outer segments. The work here demonstrates that the two proteins interact in vitro and are both required for ongoing outer segment maintenance. The major novelty of this paper would be the demonstration that Pcdh15 localized to calyceal processes interacts with Cdhr1a on the outer segment, thereby connecting the two structures. Unfortunately, the data presented are inadequate proof of this model.

Strengths:

The in vitro data to support the ability of Pcdh15b and Cdhr1a to bind is well done. The use of pcdh15b and cdhr1a single and double mutants is also a strength of the study, especially being that this would be the first characterization of a zebrafish cdhr1a mutant.

Weaknesses:

(1) The imaging data in Figure 1 is insufficient to show the specific localization of Pcdh15 to calyceal processes or Cdhr1a to the outer segment membrane. The addition of actin co-labelling with Pcdh15/Cdhr1a would be a good start, as would axial sections. The division into rod and cone-specific imaging panels is confusing because the two cell types are in close physical proximity at 5 dpf, but the cone Cdhr1a expression is somehow missing in the rod images. The SIM data appear to be disrupted by chromatic aberration but also have no context. In the zebrafish image, the lines of Pcdh15/Cdhr1a expression would be 40-50 um in length if the scale bar is correct, which is much longer than the outer segments at this stage and therefore hard to explain.

(2) Figure 3E staining of Cdhr1a looks very different from the staining in Figure 1. It is unclear what the authors are proposing as to the localization of Cdhr1a. In the lab's previous paper, they describe Cdhr1a as being associated with the connecting cilium and nascent OS discs, and fail to address how that reconciles with the new model of mediating CP-OS interaction. And whether Cdhr1a localizes to discrete domains on the disc edges, where it interacts with Pcdh15 on individual calyceal processes.

(3) The authors state "In PRCs, Pcdh15 has been unequivocally shown to be localized in the CPs". However, the immunostaining here does not match the pattern seen in the Miles et al 2021 paper, which used a different antibody. Both showed loss of staining in pcdh15b mutants so unclear how to reconcile the two patterns.

(4) The explanation for the CRISPR targets for cdhr1a and the diagram in Figure 3 does not fit with crRNA sequences or the mutation as shown. The mutation spans from the latter part of exon 5 to the initial portion of exon 6, removing intron 5-6. It should nevertheless be a frameshift mutation but requires proper documentation.

(5) There are complications with the quantification of data. First, the number of fish analyzed for each experiment is not provided, nor is the justification for performing statistics on individual cell measurements rather than using averages for individual fish. Second, all cone subtypes are lumped together for analysis despite their variable sizes. Third, t-tests are inappropriately used for post-hoc analysis of ANOVA calculations.

(6) Unclear how calyceal process length is being measured. The cone measurements are shown as starting at the external limiting membrane, which is not equivalent to the origin of calyceal processes, and it is uncertain what defines the apical limit given the multiple subtypes of cones. In Figure 5, the lines demonstrating the measurements seem inconsistently placed.

(7) The number of fish analyzed by TEM and the prevalence of the phenotype across cells are not provided. A lower magnification view would provide context. Also, the authors should explain whether or not overgrowth of basal discs was observed, as seen previously in cdhr1-null frogs (Carr et al., 2021).

(8) The statement describing the separation between calyceal processes and the outer segment in the mutants is not backed up by the data. TEM or co-labelling of the structures in SIM could be done to provide evidence.

(9) "Based on work in the murine model and our own observations of rod CPs, we hypothesize that zebrafish rod CPs only extend along the newly forming OS discs and do not provide structural support to the ROS." Unclear how murine work would support that conclusion given the lack of CPs in mice, or what data in the manuscript supports this conclusion.

(10) The authors state "from the fact that rod CPs are inherently much smaller than cone CPs" without providing a reference. In the manuscript, the measurements do show rod CPs to be shorter, but there are errors in the cone measurements, and it is possible that the RPE pigment is interfering with the rod measurements.

(11) The discussion should include a better comparison of the results with ocular phenotypes in previously generated pcdh15 and cdhr1 mutant animals.

(12) The images in panels B-F of the Supplemental Figure are uncannily similar, possibly even of the same fish at different focal planes.

Reviewer #3 (Public review):

Summary:

Transcriptionally silent HIV-1 genomes integrated into the host`s genome represent the main obstacle to an HIV-1 cure. Therefore, agents aimed at promoting HIV transcription, the so-called latency reactivating agents (LRAs) might represent useful tools to render these hidden proviruses visible to the immune system. The authors successfully identified, through multiple techniques, INTS12, a component of the Integrator complex involved in 3' processing of small nuclear RNAs U1 and U2, as a factor promoting HIV-1 latency and hindering elongation of the HIV RNA transcripts. This factor synergizes with a previously identified combination of LRAs, one of which, AZD5582, has been validated in the macaque model for HIV persistence during therapy (https://pubmed.ncbi.nlm.nih.gov/37783968/). The other compound, I-BET151, is known to synergize with AZD5582, and is a inhibitor of BET, factors counteracting the elongation of RNA transcripts.

Strengths:

The findings were confirmed through multiple screens and multiple techniques. The authors successfully mapped the identified HIV silencing factor at the HIV promoter.

Weaknesses:

(1) Initial bias:<br /> In the choice of the genes comprised in the library, the authors readdress their previous paper (Hsieh et al.) where it is stated: "To specifically investigate host epigenetic regulators involved in the maintenance of HIV-1 latency, we generated a custom human epigenome specific sgRNA CRISPR library (HuEpi). This library contains sgRNAs targeting epigenome factors such as histones, histone binders (e.g., histone readers and chaperones), histone modifiers (e.g., histone writers and erasers), and general chromatin associated factors (e.g., RNA and DNA modifiers) (Fig 1B and 1C)".

From these figure panels, it clearly appears that the genes chosen are all belonging to the indicated pathways. While I have nothing to object to on the pertinence to HIV latency of the pathways selected, the authors should spend some words on the criteria followed to select these pathways. Other pathways involving epigenetic modifications and containing genes not represented in the indicated pathways may have been left apart.

(2) Dereplication:<br /> From Figure 1 it appears that INTS12 alone reactivates HIV -1 from latency alone without any drug intervention as shown by the MACGeCk score of DMSO-alone controls. If INTS12 knockdown alone shows antilatency effects, why, then were they unable to identify it in their previous article (Hsieh et al., 2023)? The authors should include some words on the comparison of the results using DMSO alone with those of the previous screen that they conducted.

(3) Translational potential:<br /> In order to propose a protein as a drug target, it is necessary to adhere to the "primum non nocere" principle in medicine. It is therefore fundamental to show the effects of INTS12 knockdown on cell viability/proliferation (and, advisably, T-cell activation). These data are not reported in the manuscript in its current form, and the authors are strongly encouraged to provide them.

Finally, as many readers may not be very familiar with the general principles behind CRISPR Cas9 screening techniques, I suggest addressing them in this excellent review: https://pmc.ncbi.nlm.nih.gov/articles/PMC7479249/.

Reviewer #3 (Public review):

In this manuscript, Fang et al. describe a new oncogenic function of the STAMBPL1 protein in triple-negative breast cancer (TNBC). STAMBPL1 is a deubiquitinase that has been poorly studied in cancer. Previous reports identify it as a promoter of epithelial to mesenchymal transition or an inhibitor of cisplatin-induced cell death, but its participation to other cancer phenotypes has not been investigated. Fang et al. find that in cell line models of TNBC, STAMBPL1 promotes expression of the transcription factor HIF-1a and its downstream target VEGF, with the consequence of stimulating neo-angiogenesis in vitro and in vivo. Mechanistically, the authors find that this occurs via a non-enzymatic and indirect mechanism, that is by promoting the expression of GRHL3, a transcription factor that in turn binds to the HIF-1a promoter to stimulate its transcription. Interestingly, the way by which STAMPB1 promotes GRHL3 expression is by facilitating the transcriptional activity of FOXO1, a known regulator of GRHL3. Because the authors find that STAMBPL1 and FOXO1 interact, they suggest that STAMBPL1 may promote the formation of an active transcriptional complex containing FOXO1, perhaps by facilitating the recruitment of transcriptional coactivators.<br /> In conclusion, these data position for the first time the STAMBPL1 deubiquitinase in a FOXO-GRHL3 regulatory axis for the control of VEGF expression and tumor angiogenesis.<br /> The main weaknesses of this work are that the relevance of this molecular axis to the pathogenesis of TNBC is not clear, and it is not clearly established whether this is a regulatory pathway that occurs in hypoxic conditions or independently of oxygen levels.<br /> With respect to the first point, both FOXO1 and GRHL3 have been previously described as tumor suppressors, with reports of FOXO1 inhibiting tumor angiogenesis. Therefore, this works describes an apparently contradictory function of these proteins in TNBC. While it is not surprising that the same genes perform divergent functions in different tumor contexts, a stronger evidence in support of the oncogenic function of these two genes should be provided to make the data more convincing. As an example, the data in support of high STAMBPL1, FOXO and GRHL3 gene expression in TNBC TCGA specimens provided in Figure 8 is not very strong and it is not clear what the non-TNBC specimens are (whether other breast cancers or other tumors, perhaps those tumors whether these genes perform tumor suppressive functions). To strengthen the notion that STAMBPL1, FOXO and GRHL3 are overexpressed in TNCB, the authors could provide a comparison with normal tissue, as well as the analysis of other publicly available datasets (like the NCI Clinical Proteomic Tumor Analysis Consortium as an example). Finally, is it not clear what are the basal protein expression levels of STAMBPL1 in the cell lines used in this study, as based on the data presented in Figures 2D and F it appears that the protein is not expressed if not exogenously overexpressed. It would be helpful if the authors addressed this issue and provided further evidence of STAMBPL1 expression in TNBC cell lines.<br /> Linked to these considerations is the second major criticism, namely that it is not made clear if this new regulatory axis is proposed to act in normoxic or hypoxic conditions. The experiments presented in this paper are performed in both conditions but a clear explanation as to why cells are exposed to hypoxia is not given and would be necessary being that HIF-1a transcription and not protein stability is being analyzed. Also, different hypoxic conditions are sometimes used, resulting in different mRNA levels of HIF-1a and its downstream targets and quite significant fluctuations within the same cell line from one experimental setting to the next. The authors should provide an explanation as to why experimental conditions are changed and, more importantly, the experiments presented in Figure 2 should be performed also in normoxia.<br /> Another critical point is that necessary experimental controls are sometimes missing, and this is reducing the strength of some of the conclusions enunciated by the authors. As examples, experiments where overexpression of STAMBPL1 is coupled to silencing of FOXO1 to demonstrate dependency lack FOXO1silencing the absence of STAMBPL1 overexpression. Because diminishing FOXO1 expression affects HIF-1a/VEGF transcription even in the absence of STAMBPL1 (shown in Figure 7C, D), it is not clear if the data presented in Figure 7G are significant. The difference between HIF-1a expression upon FOXO1 silencing should be compared in the presence or absence of STAMBPL1 overexpression to understand if FOXO1 impacts HIF-1a transcription dependently or independently of STAMBPL1.

In addition, some minor comments to improve the quality of this manuscript are provided.<br /> (1) As a general statement, the manuscript is extremely synthetic. While this is not necessarily a negative feature, sometimes results are discussed in the figure legends and not in the main text (as an example, western blots showing HIF-1a expression) and this makes it hard to read thought the data in an easy and enjoyable manner.<br /> (2) The effect of STAMBPL1 overexpression on HIF-1a transcription is minor (Figure 2) The authors should explain why they think this is the case and whether hypoxia may provide a molecular environment that is more permissive to this type of regulation.<br /> (3) HIF-1a does not appear upregulated at the protein level protein by STAMBPL1 or GRLH3 overexpression, even though this is stated in the legends of Figures 2 and 6. The authors should show unsaturated western blots images and provide quantitative data of independent experiments to make this point.<br /> In summary, adding necessary controls and performing additional experiments to substantiate the oncogenic function of these genes in TNCB would strengthen the authors' conclusions.

Reviewer #3 (Public review):

In this manuscript, the authors examine circulating and bone parameters in patients with T2DM or obesity vs control subjects. Based on their findings they conclude that increased inflammation in bone of subjects with T2DM and obesity is negatively correlated with Wnt pathway signaling and bone strength.

Overall, this is a well done clinical study that provides further insights into the pathogenesis of bone loss associated with T2DM. However, there are a number of issues that the authors should address:

(1) The major conceptual problem is that the alterations in circulating and bone factors they observed would predominantly affect bone turnover and thus, bone mass. But bone mass is preserved in T2DM (as their own data show). They postulate that their findings lead to impaired bone quality, but it is not clear how this would occur. For example, the impairment in bone quality could be due to the accumulation of AGEs in bone in T2DM, and the correlations observed be true but unrelated. Along these lines, were serum or bone AGEs measured - and if not, is it possible for the authors to do so? At the least, this issue should be fully addressed in the Discussion if the authors are unable to provide additional data to address this.

(2) The T2DM patients were extremely well controlled. This may have limited some of the differences between groups. Was it not possible to select a group of less well-controlled patients - that is more the norm? This may also explain why the biomechanical indices in Table 3 were only marginally different in the T2DM vs the other groups. This point should also be addressed.

(3) The authors found some interesting differences in bone sclerostin levels. Were circulating sclerostin levels measured? This data would be of interest and should be provided.

(4) Fig 4A - the correlation between TNFa and SOST seems to be driven by one highly influential point. What happens if this point is removed? Is this point a formal statistical outlier? Please check this.

Reviewer #3 (Public review):

This paper addresses through experiment and simulation the combined effects of bacterial circular swimming near no-slip surfaces and chemotaxis in simple linear gradients. The authors have constructed a microfluidic device in which a gradient of L-aspartate is established to which bacteria respond while swimming while confined in channels of different widths. There is a clear effect that the chemotactic drift velocity reaches a maximum in channel widths of about 8 microns, similar in size to the circular orbits that would prevail in the absence of side walls. Numerical studies of simplified models confirm this connection.

The experimental aspects of this study are well executed. The design of the microfluidic system is clever in that it allows a kind of "multiplexing" in which all the different channel widths are available to a given sample of bacteria.

While the data analysis is reasonably convincing, I think that the authors could make much better use of what must be voluminous data on the trajectories of cells by formulating the mathematical problem in terms of a suitable Fokker-Planck equation for the probability distribution of swimming directions. In particular, I would like to see much more analysis of how incipient circular trajectories are interrupted by collisions with the walls and how this relates to enhanced chemotaxis. In essence, there needs to be a much clearer control analysis of trajectories without sidewalls to understand the mechanism in their presence.

The authors argue that these findings may have relevance to a number of physiological and ecological contexts. Yet, each of these would be characterized by significant heterogeneity in pore sizes and geometries, and thus it is very unclear whether or how the findings in this work would carry over to those situations.

Reviewer #3 (Public review):

Hong et al. used a model they previously developed to study the impact of horizontal gene transfer (HGT) on microbial multispecies communities. They investigated the effect of HGT on the existence of alternative stable states in a community. The model most closely resembles HGT through the conjugation of incompatible plasmids, where the transferred genes confer independent growth-related fitness effects. For this type of HGT, the authors find that increasing the rate of HGT leads to an increasing number of stable states. This effect of HGT persists when the model is extended to include multiple competitive niches (under a shared carrying capacity) or spatially distinct patches (that interact in a grid-like fashion). Instead, if the mobile gene is assumed to reduce between-species competition, increasing HGT leads to a smaller region of multistability and fewer stable states. Similarly, if the mobile gene is deleterious an increase in HGT reduces the parameter region that supports multistability.

This is an interesting and important topic, and I welcome the authors' efforts to explore these topics with mathematical modeling. The manuscript is well written and the analyses seem appropriate and well-carried out. However, I believe the model is not as general as the authors imply and more discussion of the assumptions would be helpful (both to readers + to promote future theoretical work on this topic). Also, given the model, it is not clear that the conclusions hold quite so generally as the authors claim and for biologically relevant parameters. To address this, I would recommend adding sensitivity analyses to the manuscript.

Specific points

(1) The model makes strong assumptions about the biology of HGT, that are not adequately spelled out in the main text or methods, and will not generally prove true in all biological systems. These include:<br /> a) The process of HGT can be described by mass action kinetics. This is a common assumption for plasmid conjugation, but for phage transduction and natural transformation, people use other models (e.g. with free phage that adsorp to all populations and transfer in bursts).<br /> b) A subpopulation will not acquire more than one mobile gene, subpopulations can not transfer multiple genes at a time, and populations do not lose their own mobilizable genes. [this may introduce bias, see below].<br /> c) The species internal inhibition is independent of the acquired MGE (i.e. for p1 the self-inhibition is by s1).<br /> These points are in addition to the assumptions explored in the supplementary materials, regarding epistasis, the independence of interspecies competition from the mobile genes, etc. I would appreciate it if the authors could be more explicit in the main text about the range of applicability of their model, and in the methods about the assumptions that are made.

(2) I am not surprised that a mechanism that creates diversity will lead to more alternative stable states. Specifically, the null model for the absence of HGT is to set gamma to zero, resulting in pij=0 for all subpopulations (line 454). This means that a model with N^2 classes is effectively reduced to N classes. It seems intuitive that an LV-model with many more species would also allow for more alternative stable states. For a fair comparison, one would really want to initialize these subpopulations in the model (with the same growth rates - e.g. mu1(1+lambda2)) but without gene mobility.

(3) I am worried that the absence of double gene acquisitions from the model may unintentionally promote bistability. This assumption is equivalent to an implicit assumption of incompatibility between the genes transferred from different species. A highly abundant species with high HGT rates could fill up the "MGE niche" in a species before any other species have reached appreciable size. This would lead to greater importance of initial conditions and could thus lead to increased multistability.

This concern also feels reminiscent of the "coexistence for free" literature (first described here http://dx.doi.org/10.1016/j.epidem.2008.07.001 ) which was recently discussed in the context of plasmid conjugation models in the supplementary material (section 3) of https://doi.org/10.1098/rstb.2020.0478 .

(4) The parameter values tested seem to focus on very large effects, which are unlikely to occur commonly in nature. If I understand the parameters in Figure 1b correctly for instance, lambda2 leads to a 60% increase in growth rate. Such huge effects of mobile genes (here also assumed independent from genetic background) seem unlikely except for rare cases. To make this figure easier to interpret and relate to real-world systems, it could be worthwhile to plot the axes in terms of the assumed cost/benefit of the mobile genes of each species.

Something similar holds for the HGT rate (eta): given that the population of E. coli or Klebsiella in the gut is probably closer to 10^9 than 10^12 (they make up only a fraction of all cells in the gut), the assumed rates for eta are definitely at the high end of measured plasmid transfer rates (e.g. F plasmid transfers at a rate of 10^-9 mL/CFU h-1, but it is derepressed and considered among the fastest - https://doi.org/10.1016/j.plasmid.2020.102489 ). To adequately assess the impact of the HGT rate on microbial community stability it would need to be scanned on a log (rather than a linear) scale. Considering the meta-analysis by Sheppard et al. it would make sense to scan it from 10^-7 to 1 for a community with a carrying capacity around 10^9.

(5) It is not clear how sensitive the results (e.g. Figure 2a on the effect of HGT) are to the assumption of the fitness effect distribution of the mobile genes. This is related to the previous point that these fitness effects seem quite large. I think some sensitivity analysis of the results to the other parameters of the simulation (also the assumed interspecies competition varies from figure to figure) would be helpful to put the results into perspective and relate them to real biological systems.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors have used a combination of enzymatic, crystallographic, and in silico approaches to provide compelling evidence for substrate selectivity of SARS-CoV-2 Mpro for human TRMT1.

Strengths:

In my opinion, the authors came close to achieving their intended aim of demonstrating the structural and biochemical basis of Mpro catalysis and cleavage of human TRMT1 protein. The revised version of the manuscript has addressed most of the questions I had posed in my earlier review.

Weaknesses:

Although several new hypotheses are generated from the Mpro structural data, the manuscript falls a bit short of testing them in functional assays, which would have solidified the conclusions the authors have drawn.

Reviewer #3 (Public review):

In the report entitled "CXXC-finger protein 1 associates with FOXP3 to stabilize homeostasis and suppressive functions of regulatory T cells", the authors demonstrated that Cxxc1-deletion in Treg cells leads to the development of severe inflammatory disease with impaired suppressive function. Mechanistically, CXXC1 interacts with Foxp3 and regulates the expression of key Treg signature genes by modulating H3K4me3 deposition. Their findings are interesting and significant. However, there are several concerns regarding their analysis and conclusions.

Major concerns:

(1) Despite cKO mice showing an increase in Treg cells in the lymph nodes and Cxxc1-deficient Treg cells having normal suppressive function, the majority of cKO mice died within a month. What causes cKO mice to die from severe inflammation?

Considering the results of Figures 4 and 5, a decrease in Treg cell population due to their reduced proliferative capacity may be one of the causes. It would be informative to analyze the population of tissue Treg cells.

(2) In Figure 5B, scRNA-seq analysis indicated that Mki67+ Treg subset are comparable between WT and Cxxc1-deficient Treg cells. On the other hand, FACS analysis demonstrated that Cxxc1-deficient Treg shows less Ki-67 expression compared to WT in Figure 5I. The authors should explain this discrepancy.

In addition, the authors concluded on line 441 that CXXC1 plays a crucial role in maintaining Treg cell stability. However, there appears to be no data on Treg stability. Which data represent the Treg stability?

(3) The authors found that Cxxc1-deficient Treg cells exhibit weaker H3K4me3 signals compared to WT in Figure 7. This result suggests that Cxxc1 regulates H3K4me3 modification via H3K4 methyltransferases in Treg cells. The authors should clarify which H3K4 methyltransferases contribute to the modulation of H3K4me3 deposition by Cxxc1 in Treg cells.

Furthermore, it would be important to investigate whether Cxxc1-deletion alters Foxp3 binding to target genes.

(4) In Figure 7, the authors concluded that CXXC1 promotes Treg cell homeostasis and function by preserving the H3K4me3 modification since Cxxc1-deficient Treg cells show lower H3K4me3 densities at the key Treg signature genes. Are these Cxxc1-deficient Treg cells derived from mosaic mice? If Cxxc1-deficient Treg cells are derived from cKO mice, the gene expression and H3K4me3 modification status are inconsistent because scRNA-seq analysis indicated that expression of these Treg signature genes was increased in Cxxc1-deficient Treg cells compared to WT (Figure 5F and G).

Reviewer #4 (Public Review):

In this work, Tee et al. study the implications of Heparan Sulfate (HS) binding mutations observed on the Enterovirus A71 (EV-A71) capsid. HS-binding mutations are observed for several virus infections and are often presumed to be a cell culture adaptation. However, in the case of EV-A71, the presence of HS-binding mutations in clinical samples and the contradictory findings in animal studies have made the clinical relevance of HS-binding a subject of debate. Therefore, to better understand the role of HS-binding in EV-A71, the authors use a mouse-adapted EV-A71 variant (MP4) and compare it to a cell-adapted strong HS-binder (MP4-97R/167G). Using these two variants, the authors show that the strong HS-binder does not require acidification for uncoating and genome release. Furthermore, it is demonstrated that the capsid stability of the HS-binding variant is compromised, resulting in pH-independent uncoating. Overall, this study provides new insights demonstrating that seemingly beneficial mutations increasing viral replication may be counterbalanced by other unintended consequences.

Strengths:

The thoroughness of the experiments performed to demonstrate that the HS-binding phenotype results in pH-independent entry and capsid destabilisation is worth highlighting. In this regard, the authors have explored viral entry using a range of approaches involving lysosomotropic drugs, viral binding assays, and neutral red-labelled viruses coupled with diverse techniques such as FISH, RNAscope, and transient expression of constitutively active molecules to inhibit parts of the viral cycle. In my opinion, this is necessary to rule out the other downstream effects of the lysomotropic drugs and to confirm the role of the HS-binding mutation in the entry phase. The use of in silico analysis coupled with negative staining electron microscopy and environmental challenge assays is notable. Finally, the demonstration of some of the work using a human-relevant strain is commendable.

Weaknesses:

A major weakness in this study is the focus on using a mouse-adapted EV-A71 strain (MP4). In the introduction, it is argued that HS-binding mutations are controversial due to their occurrence in cell culture. However, due to host limitations, mice are not the natural hosts for EV-A71 and thus, the same argument can be made for a mouse-adapted strain. It is not clear how different this strain is from circulating EV-A71 strains and the relevance of these findings to the human situation is questionable. This is particularly made evident in the discussion where it is highlighted that HS-binding variants (VP1-145G/Q mutants) have been associated with severe neurological cases while the same variants show attenuated phenotypes in mice and monkeys. This contrast between clinical data and animal studies should be highlighted in the introduction, rather than later in the discussion, as currently the in vivo animal studies are presented as the optimal situation and may lead to misconstrued conclusions from the results.

An important consideration is that the results are based primarily on image analysis. The inclusion of RT-qPCR and/or plaque assays as supplementary data will help strengthen the findings. Moreover, there are suggestions of an intermediate binder having a different phenotype. As this intermediate binder is the clinical phenotype, data on the entry of this intermediate binder will be valuable.

Another weakness in the study is the lack of contextualization of the results to current EV-A71 literature. For instance, SCARB2 is referred to as the internalization receptor but a recent study has shown that SCARB2 is not required for internalization (https://doi.org/10.1128%2Fjvi.02042-21). The findings from this study are consistent with the localization of SCARB2 in the lysosomal membranes. Furthermore, the same study has highlighted host sulfation as a key factor in EV-A71 entry. Post-translational sulfation introduces negatively charged residues on host proteins including HS and SCARB2. This increases the binding of HS-binding strains to these proteins. In this regard, the reduced infectivity upon soluble SCARB2 treatment may simply be due to enhanced binding rather than capsid opening as suggested in the results. Therefore, additional experiments (e.g. nSEM following soluble SCARB2 treatment) must be performed to support the conclusion of capsid opening, due to inherent instability, upon SCARB2 binding.

In addition to the above, other existing literature on EV-A71 pathogenesis using organoids contradicts some of the explanations of differential phenotype in clinical observations versus mice models. In the introduction, it is suggested that reduced neurovirulence of HS-binding strains is due to binding to the vascular endothelia. However, the correlation of clinical severity to viremia (https://doi.org/10.1186/1471-2334-14-417) and the association of HS-binding mutants to clinical disease counteract this suggestion. Similarly, viral infection in human organoids with EV-A71 results in as low as 0.4% of the cells being infected (https://doi.org/10.1038/s41564-023-01339-5). In this case, if viral binding to (ubiquitously expressed) HS results in viral trapping then the HS-binding mutants should show lowered infectivity in organoid models rather than the observed higher infectivity (https://doi.org/10.3389/fmicb.2023.1045587, https://doi.org/10.1038/s41426-018-0077-2). Finally, EV-A71 release has also been shown to occur in exosomes (https://doi.org/10.1093%2Finfdis%2Fjiaa174) which effectively provides a protective lipid membrane. These recent findings must be incorporated into the article and will help better contextualize their findings.

Overall, the authors present new findings with convincing methodology. The manuscript can be improved in the contextualization of the findings and highlighting the weakness in translating these findings to resolve the debate surrounding the relevance of HS-binding phenotype. The inclusion of additional experiments and data recommended to the authors will also help strengthen the manuscript.

Reviewer #3 (Public Review):

I would like to congratulate the authors to an impressive piece of work highlighting important real and potential biases, which may lead to power-law distributed node degrees in protein-protein interaction networks.<br /> This manuscript is easy to follow and very well written manuscript.<br /> I truly enjoyed the concise and convincing scientific presentation.<br /> Even if some of the concerns have already been discussed or raised in the past, the manuscript assesses potential biases in PPIs in a rigorous manner.

I deem the following observations highly relevant to be communicated to the community again:<br /> (1) PL-like distributions emerge by aggregation of data sets alone.<br /> (2) Research interest in itself is PL-distributed and drives PL-like properties in PPI networks<br /> (3) Bait usage is a major driver of PL-like behaviour.<br /> (4) Accounting for biases changes the biological interpretation of the networks<br /> (5) Simulation studies further corroborate these findings.

Reviewer #3 (Public review):

The major strength of this manuscript is the "anvi-estimate-metabolism' tool, which is already accessible online, extensively documented, and potentially broadly useful to microbial ecologists. Inclusion of extensive benchmarking and validation on simulated metagenomes has further increased confidence in this approach. Further, the conceptual insights raise interesting hypotheses that could be pursued in follow-on experimental work.

Comments on revisions:

Thank you for the very thorough response and congratulations!

Reviewer #3 (Public Review):

In this manuscript the authors expand their initial identification of Fyv6 as a protein involved in the second step of pre-mRNA splicing to investigate the transcriptome-wide impact of Fyv6 on splicing and gain a deeper understanding of the mechanism of Fyv6 action.

They first use deep sequencing of transcripts in cells depleted of Fyv6 together with Upf1 (to limit loss of mis-spliced transcripts) to identify broad changes in the transcriptome due to loss of Fyv6. This includes both changes in overall gene expression, that are not deeply discussed, as well as alterations in choice of 3' splice sites - which is the focus of the rest of the manuscript

They next provide the highest resolution structure of the post-catalytic spliceosome to date; providing unparalleled insight into details of the active site and peripheral components that haven't been well characterized previously.

Using this structure they identify functionally critical interactions of Fyv6 with Syf1 but not Prp22, Prp8 and Slu7. Finally, a suppressor screen additionally provides extensive new information regarding functional interactions between these second step factors.

Overall this manuscript reports new and essential information regarding molecular interactions within the spliceosome that determine the use of the 3' splice site. It would be helpful, especially to the non-expert, to summarize these in a table, figure or schematic in the discussion.

Comments on revisions:

I'm satisfied with the changes made in the revision.

Reviewer #3 (Public review):

Protein Phosphatase 1 (PP1), a vital member of the PPP superfamily, drives most cellular serine/threonine dephosphorylation. Despite PP1's low intrinsic sequence preference, its substrate specificity is finely tuned by over 200 PP1-interacting proteins (PIPs), which employ short linear motifs (SLIMs) to bind specific PP1 surface regions. By targeting PP1 to cellular sites, modifying substrate grooves, or altering surface electrostatics, PIPs influence substrate specificity. Although many PIP-PP1-substrate interactions remain uncharacterized, the Phactr family of PIPs uniquely imposes sequence specificity at dephosphorylation sites through a conserved "RVxF-ΦΦ-R-W" motif. In Phactr1-PP1, this motif forms a hydrophobic pocket that favors substrates with hydrophobic residues at +4/+5 in acidic contexts (the "LLD motif"), a specificity that endures even in PP1-Phactr1 fusions. Neurabin/Spinophilin remodel PP1's hydrophobic groove in distinct ways, creating unique holoenzyme surfaces, though the impact on substrate specificity remains underexplored. This study investigates Neurabin/Spinophilin specificity via PDZ domain-driven interactions, showing that Neurabin/PP1 specificity is governed more by PDZ domain interactions than by substrate sequence, unlike Phactr1/PP1.

A significant strength of this work is the use of PP1-PIP fusion proteins to effectively model intact PP1•PIP holoenzymes by replicating the interactions that remodel the PP1 interface and confer site-specific substrate specificity. When combined with proteomic analyses to assess phospho-site depletion in mammalian cells, these fusions offer critical insights into holoenzyme specificity, revealing new candidate substrates for Neurabin and Spinophilin. The studies present compelling evidence that the PDZ domain of PP1-Neurabin directs its specificity, with the remodelled PP1 hydrophobic groove interactions having minimal impact. This mechanism is supported by structural analysis of the PP1-4E-BP1 substrate fusion bound to a Neurabin construct, highlighting the 4E-BP1/PDZ interaction. This work delivers crucial insights into PP1-PIP holoenzyme function, combining biochemical, proteomic, and structural approaches. It validates the PP1-PIP fusion protein model as a powerful tool, suggesting it may extend to studying additional holoenzymes. While an extremely useful model, it must be considered unlikely the PP1-PIP fusions fully recapitulate the specificity and regulation of the holoenzyme.

Reviewer #3 (Public review):

Summary:

This manuscript, "Estimating bone marrow adiposity from head MRI and identifying its genetic 2 architecture", brings together the groups of Drs. Kaufmann and Hughes in a tour de force work to develop an artificial neural network that localizes calvaria bone marrow in T1-weighted MRI head scans, with the goal of studying its composition in several large MRI datasets, and to model sex-dimorphic age trajectories, including the effect of menopause.

Strengths:

Bone marrow adiposity is a very active tissue with far-reaching implications for tissue crosstalk and human health than we had initially recognized. Although MRI has been used to measure BM, studies such as the one by these two groups are still lacking whereas very large datasets are analyzed using advanced AI machine learning tools coupled with genetic studies and a specific pathology. The groups had to develop new methods and new AI machine-learning tools for the imaging analyses.

Weaknesses:

Some aspects of the work that authors could add additional clarification.

(1) Imaging Limitations: The authors provide an excellent overview and references supporting the use of MRI as a method for assessing marrow fat, particularly with some specific modifications. However, MRI images can be affected by various factors, including the presence of other tissues as well as specific MRI settings, which are much harder to precisely control when using different datasets.

(2) The specific density of cranial bones as it relates to the types of bone marrow: Cranial bones are extremely dense structures, which naturally interfere with MRI imaging. While it is thought that cranial bones have mostly "red bone marrow", this is only true for a short time in humans. How sensitive is their system in differentiating between red and yellow BM?

(3) Both items above are further complicated by aging, but aging is not a linear event as we have learned. There are specific bursts of aging in humans around the age of 45 and early 60s. How do the system and model predict or incorporate these peaks of aging? It seems from the data shown that aging is reflected more as a linear phenomenon. Is this because additional aging datasets are needed?

(4) The authors describe in richness of detail their AI learning programming and how it extracted the data from datasets. The authors also show some important correlations with specific genes, SNPs. What is not clear is how conditions such as anemia for example. An expected finding would be that patients with chronic anemia have lower bone marrow (BM) signal intensity on MRI scans than healthy people. This is because the signal intensity of BM depends on the fat-to-cell ratio in the tissue. Furthermore, patients with a host of musculoskeletal disorders ranging from osteopenia to osteoporosis, sarcopenia, and osteosarcopenia will also have altered MRI scans. When using such large datasets how did the authors control or exclude these pathological conditions, or were all these conditions likely present?

(5) Some of the genes and SNPs although significant showed very small correlations. What is their likely physiological significance?

(6) The authors could use this excellent manuscript to expand their discussion to include the need for studies like theirs to be also complemented by multi-OMICS studies that will include proteomics and lipidomics of BM, bones, and muscles.

Reviewer #3 (Public review):

Summary:

Salmonella is interesting due to its life within a compact compartment, which we call SCV or Salmonella containing vacuole in the field of Salmonella. SCV is a tight-fitting vacuole where the acquisition of nutrients is a key factor by Salmonella. The authors among many nutrients, focussed on beta-alanine. It is also known from many other studies that Salmonella requires beta-alanine. The authors have done in vitro RAW macrophage infection assays and In vivo mouse infection assays to see the life of Salmonella in the presence of beta-alanine. They concluded by comprehending that beta-alanine modulates the expression of many genes including zinc transporters which are required for pathogenesis.

Strengths:

This study made a couple of knockouts in Salmonella and did a transcriptomic investigation to understand the global gene expression pattern.

Weaknesses:

The following questions are unanswered:

(1) It is not clear how the exogenous beta-alanine is taken up by macrophages.

(2) It is not clear how the Beta-alanine from the cytosol of the macrophage enters the SCV.

(3) It is not clear how the beta-alanine from SCV enters the bacterial cytosol.

(4) There is no clarity on the utilization of exogenous beta-alanine of the host and the de novo synthesis of beta-alanine by panD of Salmonella.

Reviewer #3 (Public review):

Summary:

The manuscript presents an ambitious and comprehensive synaptic connectome of neurosecretory cells (NSC) in the Drosophila brain, which highlights the neural circuits underlying hormonal regulation of physiology and behaviour. The authors use EM-based connectomics, retrograde tracing, and previously characterised single-cell transcriptomic data. The goal was to map the inputs to and outputs from NSCs, revealing novel interactions between sensory, motor, and neurosecretory systems. The results are of great value for the field of neuroendocrinology, with implications for understanding how hormonal signals integrate with brain function to coordinate physiology.

The manuscript is well-written and provides novel insights into the neurosecretory connectome in the adult Drosophila brain. Some, additional behavioural experiments will significantly strengthen the conclusions.

Strengths:

(1) Rigorous anatomical analysis<br /> (2) Novel insights on the wiring logic of the neurosecretory cells.

Weaknesses:

(1) Functional validation of findings would greatly improve the manuscript.

Reviewer #3 (Public review):

Summary:

This study identifies confirmational fingerprints of amylodogenic light chains, that set them apart from the non-amylodogenic ones.

Strengths:

The research employs a comprehensive combination of structural and dynamic analysis techniques, providing evidence that conformational dynamics at VL-CL interface and structural expansion are distinguished features of amylodogenic LCs.

Weaknesses:

The sample size is limited, which may affect the generalizability of the findings. Additionally, the study could benefit from deeper analysis of specific mutations driving this unique conformation to further strengthen therapeutic relevance.

Reviewer #3 (Public review):

Summary:

The study is well written, and the results are solid and well demonstrated. It shows a field that can be explored for the treatment of CDI

Strengths:

The results are really good, and the CAPE shows a good and promising alternative for treating CDI. The methodology and results are well presented, with tables and figures that corroborate them. It is solid work and very promising.

Weaknesses:

Some references are too old or missing.

Reviewer #3 (Public review):

Summary:

In this manuscript, Wirz et al use neuroimaging (fMRI) to show that counterconditioning produces a longer lasting reduction in fear conditioning relative to extinction and appears to rely on the nucleus accumbens rather than the ventromedial prefrontal cortex. These important findings are supported by convincing evidence and will be of interest to researchers across multiple subfields, including neuroscientists, cognitive theory researchers, and clinicians.

In large part, the authors achieved their aims of giving a qualitative assessment of the behavioural mechanisms of counterconditioning versus extinction, as well as investigating the brain mechanisms. The results support their conclusions and give interesting insights into the psychological and neurobiological mechanisms of the processes that underlie the unlearning, or counteracting, of threat conditioning.

Strengths:

* Mostly clearly written with interesting psychological insights<br /> * Excellent behavioural design, well-controlled and tests for a number of different psychological phenomena (e.g. extinction, recovery, reinstatement, etc).<br /> * Very interesting results regarding the neural mechanisms of each process.<br /> * Good acknowledgement of the limitations of the study.

Weaknesses:

* I think the acquisition data belongs in the main figure, so the reader can discern whether or not there are directional differences prior to CC and extinction training that could account for the differences observed. This is particularly important for the valence data which appears to differ at baseline (supplemental figure 2C).<br /> * I was confused in several sections about the chronology of what was done and when. For instance, it appears that individuals went through re-extinction, but this is just called extinction in places.<br /> * I was also confused about the data in Figure 3. It appears that the CC group maintained differential pupil dilation during CC, whereas extinction participants didn't, and the authors suggest that this is indicative of the anticipation of reward. Do reward-associated cues typically cause pupil dilation? Is this a general arousal response? If so, does this mean that the CSs become equally arousing over time for the CC group whereas the opposite occurs for the extinction group (i.e. Figure 3, bottom graphs)? It is then further confusing as to why the CC group lose differential responding on the spontaneous recovery test. I'm not sure this was adequately addressed.<br /> * I am not sure that the memories tested were truly episodic<br /> * Twice as many female participants than males<br /> * No explanation as to why shocks were varied in intensity and how (psuedo-randomly?)

Reviewer #3 (Public review):

Summary:

The cognitive striatum, also known as the dorsomedial striatum, receives input from brain regions involved in high-level cognition and plays a crucial role in processing cognitive information. However, despite its importance, the extent to which different projection pathways of the striatum contribute to this information processing remains unclear. In this paper, Bruce et al. conducted a study using various causal and correlational techniques to investigate how these pathways collectively contribute to interval timing in mice. Their results were consistent with previous research, showing that the direct and indirect striatal pathways perform opposing roles in processing elapsed time. Based on their findings, the authors proposed a revised computational model in which two separate accumulators track evidence for elapsed time in opposing directions. These results have significant implications for understanding the neural mechanisms underlying cognitive impairment in neurological and psychiatric disorders, as disruptions in the balance between direct and indirect pathway activity are commonly observed in such conditions.

Strengths:

The authors employed a well-established approach to study interval timing and employed optogenetic tagging to observe the behavior of specific cell types in the striatum. Additionally, the authors utilized two complementary techniques to assess the impact of manipulating the activity of these pathways on behavior. Finally, the authors utilized their experimental findings to enhance the theoretical comprehension of interval timing using a computational model.

Weaknesses:

The behavioral task used in this study is best suited for investigating elapsed time perception rather than interval timing. Timing bisection tasks are often employed to study interval timing in humans and animals. Given the systemic delivery of pharmacological interventions, it is difficult to conclude that the effects are specific to the dorsomedial striatum. Future studies should use the local infusion of drugs into the dorsomedial striatum.

Reviewer #3 (Public review):

Summary:

In this study, Zhang et al. reported that CHMP5 restricts bone formation by controlling endolysosome-mitochondrion-mediated cell senescence. The effects of CHMP5 on osteoclastic bone resorption and bone turnover have been reported previously (PMID: 26195726), in which study the aberrant bone phenotype was observed in the CHMP5-ctsk-CKO mouse model, using the same mouse model, Zhang et al., report a novel role of CHMP5 on osteogenesis through affecting cell senescence. Overall, it is an interesting study and provides new insights in the field of cell senescence and bone.

Strengths:

Analyzed the bone phenotype OF CHMP5-periskeletal progenitor-CKO mouse model and found the novel role of senescent cells on osteogenesis and migration.

Weaknesses:

(1) There are a lot of papers that have reported that senescence impairs osteogenesis of skeletal stem cells. In this study, the author claimed that Chmp5 deficiency induces skeletal progennitor cell senescence and enhanced osteogenesis. Can the authors explain the controversial results?

(2) Co-culture of Chmp5-KO periskeletal progenitors with WT ones should be conducted to detect the migration and osteogenesis of WT cells in response to Chmp5-KO-induced senescent cells. In addition, the co-culture of WT periskeletal progenitors with senescent cells induced by H2O2, radiation, or from aged mice would provide more information.

(3) Many EVs were secreted from Chmp5-deleted periskeletal progenitors, compared to the rarely detected EVs around WT cells. Since EVs of BMSCs or osteoprogenitors show strong effects of promoting osteogenesis, did the EVs contribute to the enhanced osteogenesis induced by Chmp5-defeciency?

(4) EVs secreted from senescent cells propagate senescence and impair osteogenesis, why do EVs secreted from senescent cells induced by Chmp5-defeciency have opposite effects on osteogenesis?

(5) The Chmp5-ctsk mice show accelerated aging-related phenotypes, such as hair loss and joint stiffness. Did Ctsk also label cells in hair follicles or joint tissue?

(6) Fifteen proteins were found to increase and five proteins to decrease in the cell supernatant of Chmp5Ctsk periskeletal progenitors. How about SASP factors in the secretory profile?

(7) D+Q treatment mitigates musculoskeletal pathologies in Chmp5 conditional knockout mice. In the previously published paper (CHMP5 controls bone turnover rates by dampening NF-κB activity in osteoclasts), inhibition of osteoclastic bone resorption rescues the aberrant bone phenotype of the Chmp5 conditional knockout mice. Whether the effects of D+Q on bone overgrowth is because of the inhibition of bone resorption?

(8) The role of VPS4A in cell senescence should be measured to support the conclusion that CHMP5 regulates osteogenesis by affecting cell senescence.

(9) Cell senescence with markers, such as p21 and H2AX, co-stained with GFP should be performed in the mouse models to indicate the effects of Chmp5 on cell senescence in vivo.

(10) ADTC5 cell as osteochondromas cells line, is not a good cell model of periskeletal progenitors. Maybe primary periskeletal progenitor cell is a better choice.

Reviewer #3 (Public review):

Summary:

The authors performed snRNA-seq in the pre-optic area (POA), a heterogeneous brain region implicated in multiple innate behaviors, comparing two species of Peromyscus mice that possess strikingly different parenting behaviors. P. polionotus shows high levels of parental care from both sexes of parent, and P. maniculatus shows lower levels of care, predominantly displayed by dams rather than sires. The overall goal of understanding the genomic basis of behavioral variation is significant and of broad interest and comparative studies in POA in these two species is an excellent approach to tackle this question. The authors correctly point out that existing studies largely compare species that are highly divergent, such as mice and humans, which confounds the association of specific neuronal populations or gene expression patterns with distinct behaviors. They identify neuronal populations with differential abundance between species and sexes and additionally report sex and species differences in gene expression within each transcriptomic cell type. Their cell type classification is aided by mapping their Peromyscus cells onto a previously existing POA single-cell dataset generated in lab mice. However, a significant fraction of the cells cannot be assigned to Mus types, which confounds their analysis. The detection and validation of previously observed sex differences in the Gal/Moxd1 cell type and species differences in Avp expression provide additional support that their data are solid. This study provides an important resource for comparative single-cell studies in the brain.

Strengths:

This is a pioneering comparative snRNA-seq study that provides a roadmap for similar approaches in non-traditional model organisms.

The authors have identified populations that may underlie sex- and species- differences in parenting behavior in rodents.

A significant strength of the manuscript is the histological validation of their most robust marker genes.

Weaknesses:

My primary concern is that the dataset is limited: 52,121 neuronal nuclei across 24 samples, which does not provide many cells per cluster to analyze comparatively across sex and species, particularly given the heterogeneity of the region dissected. The Supplementary table reports lower UMIs/genes per cell than is typically seen as well. Perhaps additional information could be obtained from the data by not restricting the analyses to cells that can be assigned to Mus types. A direct comparison of the two Peromyscus species could be valuable as would a more complete Peromyscus POA atlas.

In Supplement 7, it appears that most neurons can be assigned as excitatory or inhibitory, but then so many of these cells remain in the unassigned "gray blob" seen in panel 1E. Clustering of excitatory and inhibitory neurons separately, as in in prior cited work in Mus POA (refs 31 and 57) may boost statistical power to detect sex and species differences in cell types. Perhaps the cells that cannot be assigned to Mus contain too few reads to be useful, in which case they should be filtered out in the QC. The technical challenges of a comparative single-cell approach are considerable, so it benefits the scientific community to provide transparency about them.

The Calb1 dimorphism as observed by immunostaining, appears much more extensive in P. maniculatus compared to P. polionotus (Figures 3 E and F). This finding is not reflected in the counts of the i20:Gal/Moxd1 cluster. The use of Calb1 staining as a proxy for the Gal/Moxd1 cluster would be strengthened if the number of POA Calb1+ neurons that are found in each cluster was apparent. There may be additional Calb+ neurons in the cells that are not annotated to a Mus cluster. This clarification would add support to the overall conclusion that there is reduced sexual dimorphism in P. polionotus.

The relationship between the sex steroid receptor expression and the sex bias in gene expression would be improved if the sex bias in sex steroid receptor expression was included in Supplementary Figure 10.

There is no explanation for the finding that there is a female bias in gene expression across all cell types in P. polionotus.

Reviewer #3 (Public review):

Summary:

This is an interesting manuscript which uses state of the art experimental and simulation approaches to quantify motor unit discharge patterns in the human TA and VL. The non-linear profiles of motor unit discharge were calculated and found to have an initial acceleration phase followed by an attenuation phase. Lower threshold motor units had a larger gain of the initial acceleration whereas the higher threshold motor unit had a higher gain in the attenuation phase. These data represent a technical feat and are important for understanding how humans generate and control voluntary force.

Strengths:

The authors used rigorous, state-of-the art analyses to decompose and validate their motor unit data during a wide range of voluntary efforts.

Analyses are clearly presented, applied, and visualized.

The supplemental data provides important transparency.

Weaknesses:

Number of participants and muscles tested are relatively small - particularly given the constraints on yield. It is unclear if this will translate to other motor pools. The justification for TA and VL should be provided.

While in impressive effort was made to identify and track motor units across a range of contractions, it appears that a substantial portion of muscle force was not identified. Though high intensity contractions are challenging to decompose - the authors are commended in their technical ability in recording population motor unit discharge times with recruitment thresholds up to 75% a participant's maximal voluntary contractions. However previous groups have seen substantial recruitment motor units above 80% and even 90% maximum activation in the soleus. Given the innervation ratios of higher threshold motor units, if recruitment continued to 100%, the top quartile would likely represent a substantial portion of the traditional fast-fatigable motor units. It would be highly interesting to understand the recruitment and rate coding of the highest threshold motor units, at a minimum I would suggest using terms other than "entire range" or "full spectrum of recruitment thresholds"

The quantification of hysteresis using torque appears to make self-evident the observation that lower threshold motor units demonstrate less hysteresis with respect to torque - If there was motor unit discharge there will be force. I believe this limitation goes beyond the floor effects discussed in the manuscript. Traditionally individuals have used the discharge of a lower threshold unit as the measure on which to apply hysteresis analyses to infer ion channel function in human spinal motoneurons.

The main findings are not entirely novel. See Monster and Chan 1977 and Kanosue et al 1979

Comments on revisions:

I thank the authors for their thoughtful revision.

Just to confirm, the ranges for motor unit yield are for a single contraction. So, for example, in a participant there were 71 unique and concurrently active VL motor units able to be decomposed.

Reviewer #3 (Public review):

This study explores sensory prediction errors in sensory cortex. It focuses on the question of how these signals are shaped by non-hierarchical interactions, specifically multimodal signals arising from same level cortical areas. The authors used 2-photon imaging of mouse auditory cortex in head-fixed mice that were presented with sounds and/or visual stimuli while moving on a ball. First, responses to pure tones, visual stimuli and movement onset were characterized. Then, the authors made the running speed of the mouse predictive of sound intensity and/or visual flow (closed loop). Mismatches were created through the interruption of sound and/or visual flow for 1 second, disrupting the expected sensory signal. As a control, sensory stimuli recorded during the close loop phase were presented again decoupled from the movement (open loop). The authors suggest that auditory responses to the unpredicted interruption of the sound, which affected neither running speed nor pupil size, reflect mismatch responses. That these mismatch responses were enhanced when the visual flow was congruently interrupted, indicates cross-modal influence of prediction error signals.

This study's strengths are the relevance of the question and the design of the experiment. The authors are experts in the techniques used. The analysis explores neither the full power of the experimental design nor the population activity recorded with 2-photon, leaving open the question of to what extend what the authors call mismatch responses are not sensory responses to sound interruption (offset responses). The auditory system is sensitive to transitions and indeed responses to the interruption of the sound are similar in quality, if not quantity, in the predictive and the control situation.

Comments on revisions:

The incorporation of the analysis of the animal's running speed and the pupil size upon sound interruption improves the interpretation of the data. The authors can now conclude that responses to the mismatch are not due to behavioral effects.<br /> The issue of the relationship between mismatch responses and offset responses remains uncommented. The auditory system is sensitive to transitions, also to silence. See the work of the Linden or the Barkat labs (including the work of the first author of this manuscript) on offset responses, and also that of the Mesgarani lab (Khalighinejad et al., 2019) on responses to transitions 'to clean' (Figure 1c) in human auditory cortex. Offset responses, as the first author knows well, are modulated by intensity and stimulus length (after adaptation?). That responses to the interruption of the sound are similar in quality, if not quantity, in the closed and open loop conditions suggest that offset response might modulate the mismatch response. A mismatch response that reflects a break in predictability would presumably be less modulated by the exact details of the sensory input than an offset response. Therefore, what is the relationship between the mismatch response and the mean sound amplitude prior to the sound interruption (for example during the preceding 1 second)? And between the mismatch response and the mean firing rate over the same period?<br /> Finally, how do visual stimuli modulate sound responses in the absence of a mismatch? Is the multimodal response potentiation specific to a mismatch?

Reviewer #3 (Public review):

Summary:

In this manuscript, Bosch and colleagues describe an unexpected function of Flamingo, a core component of the planar cell polarity pathway, in cell competition in Drosophila wing and eye disc. While Flamingo depletion has no impact on tumour growth (upon induction of Ras and depletion of Scribble throughout the eye disc), and no impact when depleted in WT cells, it specifically tunes down winner clone expansion in various genetic contexts, including the overexpression of Myc, the combination of Scribble depletion with activation of Ras in clones or the early clonal depletion of Scribble in eye disc. Flamingo depletion reduces proliferation rate and increases the rate of apoptosis in the winner clones, hence reducing their competitiveness up to forcing their full elimination (hence becoming now "loser"). This function of Flamingo in cell competition is specific of Flamingo as it cannot be recapitulated with other components of the PCP pathway, does not rely on interaction of Flamingo in trans, nor on the presence of its cadherin domain. Thus, this function is likely to rely on a non-canonical function of Flamingo which may rely on downstream GPCR signaling.

This unexpected function of Flamingo is by itself very interesting. In the framework of cell competition, these results are also important as they describe, to my knowledge, one of the only genetic conditions that specifically affect the winner cells without any impact when depleted in the loser cells. Moreover, Flamingo do not just suppress the competitive advantage of winner clones, but even turn them in putative losers. This specificity, while not clearly understood at this stage, opens a lot of exciting mechanistic questions, but also a very interesting long term avenue for therapeutic purpose as targeting Flamingo should then affect very specifically the putative winner/oncogenic clones without any impact in WT cells.

The data and the demonstration are very clean and compelling, with all the appropriate controls, proper quantifications and backed-up by observations in various tissues and genetic backgrounds. I don't see any weakness in the demonstration and all the points raised and claimed by the authors are all very well substantiated by the data. As such, I don't have any suggestions to reinforce the demonstration.

While not necessary for the demonstration, documenting the subcellular localisation and levels of Flamingo in these different competition scenarios may have been relevant and provide some hints on a putative mechanism (specifically by comparing its localisation in winner and loser cells).

Also, on a more interpretative note, the absence of impact of Flamingo depletion on JNK activation does not exclude some interesting genetic interactions. JNK output can be very contextual (for instance depending on Hippo pathway status), and it would be interesting in the future to check if Flamingo depletion could somehow alter the effect of JNK in the winner cells and promote downstream activation of apoptosis (which might normally be suppressed). It would be interesting to check if Flamingo depletion could have an impact in other contexts involving JNK activation or upon mild activation of JNK in clones.

Strengths:

- A clean and compelling demonstration of the function of Flamingo in winner cells during cell competition

- One of the rare genetic conditions that affects very specifically winner cells without any impact in losers, and then can completely switch the outcome of competition (which opens an interesting therapeutic perspective on the long term)

Reviewer #3 (Public review):

Summary:

The manuscript by Jaime Tobon and Moser uses patch-clamp electrophysiology in cochlear preparations to probe the pre- and post-synaptic specializations that give rise to diverse activity of spiral ganglion afferent neurons (SGN). The experiments are quite an achievement! They use paired recordings from pre-synaptic cochlear inner hair cells (IHC) that allow precise control of voltage and therefore calcium influx, with post-synaptic recordings from type I SGN boutons directly opposed to the IHC for both presynaptic control of membrane voltage and post-synaptic measurement of synaptic function with great temporal resolution.

Any of these techniques by themselves are challenging, but the authors do them in pairs, at physiological temperatures, and in hearing animals, all of which combined make these experiments a real tour de force. The data is carefully analyzed and presented, and the results are convincing. In particular, the authors demonstrate that post-synaptic features that contribute to the spontaneous rate (SR) of predominantly monophasic post-synaptic currents (PSCs), shorter EPSC latency, and higher PSC rates are directly paired with pre-synaptic features such as a lower IHC voltage activation and tighter calcium channel coupling for release to give a higher probability of release and subsequent increase in synaptic depression. Importantly, IHCs paired with Low and High SR afferent fibers had the same total calcium currents, indicating that the same IHC can connect to both low and high SR fibers. These fibers also followed expected organizational patterns, with high SR fibers primarily contacting the pillar IHC face and low SR fibers primarily contacting the modiolar face. The authors also use in vivo-like stimulation paradigms to show different RRP and release dynamics that are similar to results from SGN in vivo recordings. Overall, this work systematically examines many features giving rise to specializations and diversity of SGN neurons.

Reviewer #3 (Public review):

Summary:

This work aims to address a fundamental biological question: how do mammalian cells achieve/lose tolerance to cold exposure? The authors first tried to establish an experimental system for cell cold exposure and evaluation of cell death and then performed genome-scale CRISPR-Cas9 screening on immortalized cell lines from Syrian Hamster (BHK-21) and human (K562) for key genes that are associated with cell survival during prolonged cold exposure. From these screenings, they focused on glutathione peroxidase 4 (GPX4). Using genetic modifications or pharmacological interventions, and multiple cell models including primary cells from various mammalian species, they showed that GPX4 proteins are likely to retain their activities at 4 {degree sign}C, functioning to prevent cold-induced cell ferroptosis.

Strengths:

(1) This paper is neatly written and hence easy to follow.

(2) Experiments are well designed.

(3) The data showing the overall good cell survival after a prolonged cold exposure or repeated cold-warm cycles are helpful to show the advantages of the experimental instruments and methods the authors used, and hence the validity of their results.

(4) The CRISPR-Cas9 screening is a great attempt.

(5) Multiple cell types from hibernating mammals (cold tolerant) and cold-intolerant species are used to test their findings.

(6) Although some may argue that other labs have published works with different approaches that have pointed out the importance of GPX4 and ferroptosis in hamster cell survival from anoxia-reoxygenation or cold exposure models, hence hurting the novelty of this work, this reviewer thinks that it is highly valuable to have independent research groups and different methods/systems to validate an important concept.

Weaknesses:

(1) Only cell death was robustly surveyed; though cell proliferation was evaluated too in some experiments, other cellular functions, such as mitochondrial ATP production vs. glycolysis, and the extent of lipid peroxidation, could have been measured to reflect cellular physiology.

Validations on complex tissues or in vivo systems would have further strengthened the work and its impact.

CRISPR-Cas9 screening may have technical limitations as knock-out of some essential genes/pathways may lead to cell lethality during screening, and hence the relevance of these genes/pathways to cell cold tolerance may not be noted. From the data presented in this study, this reviewer thinks that the GPX4 pathway is likely a conserved mechanism for long-term cold survival, but not for cold sensitivity or acute cell death from cold exposure. In line with my such speculation, their CRISPR-Cas9 screening revealed genes in the GPX4 pathway from a relatively cold-sensitive human cell line, but the endogenous GPX4 pathway is seemingly operational in this cold-sensitive cell line. Also, these cells are viable after GPX4 knock-out. Dead cells from the acute cold exposure phase may detached, or their genomic DNAs have been severely damaged by the time of sample collection, hence not giving any meaningful sequencing reads. Crippling other factors/pathways such as FOXO1 (PMID: 38570500) or 5-aminolevulinic acid (ALA) metabolism (PMID: 35401816) have been shown to severely aggravate cold-induced cell death, including TUNEL-revealed DNA damage, within a much shorter time scale, whilst loss-function knockouts of FOXO1 or ALA Synthase 1 (ALAS1) are usually cell lethal. Thus, they and other possible essential genes may not be screenable from the current experimental protocol. These important points need to be taken into consideration by the authors.

Reviewer #3 (Public review):

Summary:

The manuscript by Chang et al. aims to investigate how the behavioral relevance of auditory and visual stimuli influences the way in which the primary auditory cortex encodes auditory, visual, and audiovisual information. The main result is that behavioral training induces an increase in the encoding of auditory and visual information and in multisensory enhancement that is mainly related to the choice located contralaterally with respect to the recorded hemisphere.

Strengths:

The manuscript reports the results of an elegant and well-planned experiment meant to investigate if the auditory cortex encodes visual information and how learning shapes visual responsiveness in the auditory cortex. Analyses are typically well done and properly address the questions raised

Weaknesses:

Major

(1) The authors apparently primarily focus their analyses of sensory-evoked responses in approximately the first 100 ms following stimulus onset. Even if I could not find an indication of which precise temporal range the authors used for analysis in the manuscript, this is the range where sensory-evoked responses are shown to occur in the manuscript figures. While this is a reasonable range for auditory evoked responses, the same cannot be said for visual responses, which commonly peak around 100-120 ms, in V1. In fact, the latency and overall shape of visual responses are quite different from typical visual responses, that are commonly shown to display a delay of up to 100 ms with respect to auditory responses. All traces that the authors show, instead, display visual responses strikingly overlapping with auditory ones, which is not in line with what one would expect based on our physiological understanding of cortical visually-evoked responses. Similarly, the fact that the onset of decoding accuracy (Figure 2j) anticipates during multisensory compared to auditory-only trials is hard to reconcile with the fact that visual responses have a later onset latency compared to auditory ones. The authors thus need to provide unequivocal evidence that the results they observe are truly visual in origin. This is especially important in view of the ever-growing literature showing that sensory cortices encode signals representing spontaneous motor actions, but also other forms of non-sensory information that can be taken prima facie to be of sensory origin. This is a problem that only now we realize has affected a lot of early literature, especially - but not only - in the field of multisensory processing. It is thus imperative that the authors provide evidence supporting the true visual nature of the activity reported during auditory and multisensory conditions, in both trained, free-choice, and anesthetised conditions. This could for example be achieved causally (e.g. via optogenetics) to provide the strongest evidence about the visual nature of the reported results, but it's up to the authors to identify a viable solution. This also applies to the enhancement of matched stimuli, that could potentially be explained in terms of spontaneous motor activity and/or pre-motor influences. In the absence of this evidence, I would discourage the author from drawing any conclusion about the visual nature of the observed activity in the auditory cortex.

(2) The finding that AC neurons in trained mice preferentially respond - and enhance - auditory and visual responses pertaining to the contralateral choice is interesting, but the study does not show evidence for the functional relevance of this phenomenon. As has become more and more evident over the past few years (see e.g. the literature on mouse PPC), correlated neural activity is not an indication of functional role. Therefore, in the absence of causal evidence, the functional role of the reported AC correlates should not be overstated by the authors. My opinion is that, starting from the title, the authors need to much more carefully discuss the implications of their findings.

MINOR:

(1) The manuscript is lacking what pertains to the revised interpretation of most studies about audiovisual interactions in primary sensory cortices following the recent studies revealing that most of what was considered to be crossmodal actually reflects motor aspects. In particular, recent evidence suggests that sensory-induced spontaneous motor responses may have a surprisingly fast latency (within 40 ms; Clayton et al. 2024). Such responses might also underlie the contralaterally-tuned responses observed by the authors if one assumes that mice learn a stereotypical response that is primed by the upcoming goal-directed, learned response. Given that a full exploration of this issue would require high-speed tracking of orofacial and body motions, the authors should at least revise the discussion and the possible interpretation of their results not just on the basis of the literature, but after carefully revising the literature in view of the most recent findings, that challenge earlier interpretations of experimental results.

(2) The methods section is a bit lacking in details. For instance, information about the temporal window of analysis for sensory-evoked responses is lacking. Another example: for the spike sorting procedure, limited details are given about inclusion/exclusion criteria. This makes it hard to navigate the manuscript and fully understand the experimental paradigm. I would recommend critically revising and expanding the methods section.

Reviewer #3 (Public Review):

Summary:

This important paper provides the best-to-date characterization of chirping in weakly electric fish using a large number of variables. These include environment (free vs divided fish, with or without clutter), breeding state, gender, intruder vs resident, social status, locomotion state and social and environmental experience, without and with playback experiments. It applies state-of-the-art methods for reducing the dimensionality of the data and finding patterns of correlation between different kinds of variables (factor analysis, K-means). The strength of the evidence, collated from a large number of trials with many controls, leads to the conclusion that the traditionally assumed communication function of chirps may be secondary to its role in environmental assessment and exploration that takes social context into account. Based on their extensive analyses, the authors suggest that chirps are mainly used as probes that help detect beats caused by other fish as well as objects.

Strengths:

The work is based on completely novel recordings using interaction chambers. The amount of new data and associated analyses is simply staggering, and yet, well organized in presentation. The study further evaluates the electric field strength around a fish (via modelling with the boundary element method) and how its decay parallels the chirp rate, thereby relating the above variables to electric field geometry. The BEM modelling also convincingly predicts how the electric image of a receiver conspecific on a sending fish is enhanced by a chirp.

The main conclusions are that the lack of any significant behavioural correlates for chirping, and the lack of temporal patterning in chirp time series, cast doubt on a primary communication goal for most chirps. Rather, the key determinants of chirping are the difference in frequency between two interacting conspecifics as well as individual subjects' environmental and social experience. The paper concludes that there is a lack of evidence for stereotyped temporal patterning of chirp time series, as well as of sender-receiver chirp transitions beyond the known increase in chirp frequency during an interaction. The authors carefully submit that the new putative echolocation function of chirps is not mutually exclusive with a possible communication function.

These conclusions by themselves will be very useful to the field. They will also allow scientists working on other "communication" systems to perhaps reconsider and expand the goals of the probes used in those senses. A lot of data are summarized in this paper, with thorough referencing to past work.

The alternative hypotheses that arise from the work are that chirps are mainly used as environmental probes for better beat detection and processing and object localization, and in this sense are self-directed signals. This led to their prediction that environmental complexity ("clutter") should increase chirp rate, which is fact was revealed by their new experiments. The authors also argue that waveform EODs have less power across high spatial frequencies compared to pulse-type fish, with a resulting relatively impoverished power of resolution. Chirping in wave-type fish could temporarily compensate for the lower frequency resolution while still being able to resolve EOD perturbations with a good temporal definition (which pulse-type fish lack due to low pulse rates).

The authors also advance the interesting idea that the sinusoidal frequency modulations caused by chirps are the electric fish's solution to the minute (and undetectable by neural wetware) echo-delays available to it, due to the propagation of electric fields at the speed of light in water. The paper provides a number of experimental avenues to pursue in order to validate the non-communication role of chirps.

Reviewer #3 (Public review):

Summary:

This study investigated the expression of Osterix (Osx) not only in osteoblasts but also significantly in osteocytes. Through Osx knockout, the osteocytic dendritic network was damaged, leading to communication disruption. This study investigated the regulatory role of Osx on osteoblast dendrites through Cx43.

Strengths:

This paper provides a good explanation of the role of Osx in osteocyte synapse and cell communication, enriching the understanding of Osx's functional significance. The results of the experiment support the conclusions of the study. This is an interesting study with a clear logical structure.

Weaknesses:

Some experimental results need to be supplemented, and there are still some details and errors in the text that need to be revised.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors used structural biology approaches to determine the molecular mechanism underlying the inactivation of the PIEZO1 ion channel. To this end, the authors presented structures of human PIEZO1 and its slow-inactivating mutants. The authors also determined the structures of these PIEZO1 constructs in complexes with the auxiliary subunit MDFIC, which substantially slows down PIEZO1 inactivation. From these structures, the authors observed a unique feature of human PIEZO1 in which the lipid molecules plugged the channel pore in fast-inactivating constructs. The authors proposed that these lipid molecules prevent ion permeation and underlie the molecular mechanism of human PIEZO1 inactivation.

Strengths:

Notedly, this manuscript reported the first structures of a human PIEZO1 channel, its channelopathy mutants, and their complexes with MDFIC. The proposed role of pore lipids in modulating PIEZO1 ion permeation is interesting.

Weaknesses:

The authors' conclusion regarding the role of pore lipids in PIEZO inactivation is based on the assumption that all structures of human PIEZO1 resolved in this work represent comparable functional states relevant to channel inactivation. The authors should at least acknowledge that this is a critical assumption that is difficult to validate. The fitting of the lipid molecule to cryo-EM density could be improved.

Comments on revisions:

Upon revision, the authors substantially weakened the statement regarding the correlation between curvature and inactivation. The authors also toned down the statement regarding the role of pore lipids in channel inactivation. However, I have a few additional comments.

(1) As I have stated above, the assumption here is that all structures presented in this work represent comparable functional states relevant to channel inactivation. However, this assumption could be invalid. For example, the WT channel could be in the closed conformation, whereas the mutant could be stabilized in a different functional state. I understand that this is very difficult to test structurally and functionally. Therefore, I think the authors should at least acknowledge this limitation/assumption.<br /> (2) This time, I reviewed the coordinates and the map of the PIEZO1 structures. For example, in the WT channel, the fitting of the lipid to the cryo-EM density is questionable and I personally wouldn't model this lipid in this pose.

Reviewer #3 (Public review):

Summary:

Boffi and colleagues sought to quantify the single-trial, azimuthal information in the dorsal cortex of the inferior colliculus (DCIC), a relatively understudied subnucleus of the auditory midbrain. They accomplished this by using two complementary recording methods while mice passively listened to sounds at different locations: calcium imaging that recorded large neuronal populations but with poor temporal precision and multi-contact electrode arrays that recorded smaller neuronal populations with exact temporal precision. DCIC neurons respond variably, with inconsistent activity to sound onset and complex azimuthal tuning. Some of this variably was explained by ongoing head movements. The authors used a naïve Bayes decoder to probe the azimuthal information contained in the response of DCIC neurons on single trials. The decoder failed to classify sound location better than chance when using the raw population responses but performed significantly better than chance when using the top principal components of the population. Units with the most azimuthal tuning were distributed throughout the DCIC, possessed contralateral bias, and positively correlated responses. Interestingly, inter-trial shuffling decreased decoding performance, indicating that noise correlations contributed to decoder performance. Overall, Boffi and colleagues, quantified the azimuthal information available in the DCIC while mice passively listened to sounds, a first step in evaluating if and how the DCIC could contribute to sound localization.

Strengths:

The authors should be commended for collection of this dataset. When done in isolation (which is typical), calcium imaging and linear array recordings have intrinsic weaknesses. However, those weaknesses are alleviated when done in conjunction - especially when the data is consistent. This data set is extremely rich and will be of use for those interested in auditory midbrain responses to variable sound locations, correlations with head movements, and neural coding.

The DCIC neural responses are complex with variable responses to sound onset, complex azimuthal tuning and large inter-sound interval responses. Nonetheless, the authors do a decent job in wrangling these complex responses: finding non-canonical ways of determining dependence on azimuth and using interpretable decoders to extract information from the population.

Weaknesses:

The decoding results are a bit strange, likely because the population response is quite noisy on any given trial. Raw population responses failed to provide sufficient information concerning azimuth for significant decoding. Importantly, the decoder performed better than chance when certain principal components or top ranked units contributed but did not saturate with the addition of components or top ranked units. So, although there is azimuthal information in the recorded DCIC populations - azimuthal information appears somewhat difficult to extract.

Although necessary given the challenges associated with sampling many conditions with technically difficult recording methods, the limited number of stimulus repeats precludes interpretable characterization of the heterogeneity across the population. Nevertheless, the dataset is public so those interested can explore the diversity of the responses.

The observations from Boffi and colleagues raises the question: what drives neurons in the DCIC to respond? Sound azimuth appears to be a small aspect of the DCIC response. For example, the first 20 principal components which explain roughly 80% of the response variance are insufficient input for the decoder to predict sound azimuth above chance. Furthermore, snout and ear movements correlate with the population response in the DCIC (the ear movements are particularly peculiar given they seem to predict sound presentation). Other movements may be of particular interest to control for (e.g. eye movements are known to interact with IC responses in the primate). These observations, along with reported variance to sound onsets and inter-sound intervals, question the impact of azimuthal information emerging from DCIC responses. This is certainly out of scope for any one singular study to answer, but, hopefully, future work will elucidate the dominant signals in the DCIC population. It may be intuitive that engagement in a sound localization task may push azimuthal signals to the forefront of DCIC response, but azimuthal information could also easily be overtaken by other signals (e.g. movement, learning).

Boffi and colleagues set out to parse the azimuthal information available in the DCIC on a single trial. They largely accomplish this goal and are able to extract this information when allowing the units that contain more information about sound location to contribute to their decoding (e.g., through PCA or decoding on their activity specifically). Interestingly, they also found that positive noise correlations between units with similar azimuthal preferences facilitate this decoding - which is unusual given that this is typically thought to limit information. The dataset will be of value to those interested in the DCIC and to anyone interested in the role of noise correlations in population coding. Although this work is first step into parsing the information available in the DCIC, it remains difficult to interpret if/how this azimuthal information is used in localization behaviors of engaged mice.

Reviewer #3 (Public Review):

Summary:

The authors use docking and molecular dynamics (MD) simulations to investigate transient conformations that are otherwise difficult to resolve experimentally. The docking and simulations suggest an interesting series of events whereby agonists initially bind to the low affinity site and then flip 180 degrees as the site contracts to its high affinity conformation. This work will be of interest to the ion channel community and to biophysical studies of pentameric ligand-gated channels.

Strengths:

I find the premise for the simulations to be good, starting with an antagonist bound structure as an estimate of the low affinity binding site conformation, then docking agonists into the site and using MD to allow the site to relax to a higher affinity conformation that is similar to structures in complex with agonists. The predictions are interesting and provide a view into what a transient conformation that is difficult to observe experimentally might be like.

Weaknesses:

A weakness is that the relevance of the initial docked low affinity orientations depend solely on in silco results, for which simulated vs experimental binding energies deviate substantially for two of the four ligands tested. This raises some doubt as to the validity of the simulations. I acknowledge that the calculated binding energies for two of the ligands were closer to experiment, and simulated efficiencies were a good representation of experimental measures, which gives some support to the relevance of the in silico observations. Regardless, some of the reviewers comments regarding the simulation methodology were not seriously addressed.

Reviewer #3 (Public review):

Summary:

This manuscript represents a technology development- specifically an micrococcal nuclease chromatin capture approach, termed MChIP-C to identify promoter centered chromatin interactions at single nucleosome resolution via a specific protein, similar to HiChIP, ChIA-PET, etc.. In general the manuscript is technically well done.

Strengths:

Methods appear to hold promise to improve both the sensitivity and resolution of protein-centered chromatin capture approaches.

Weaknesses:

Downsampling analysis gives a better idea of the strengths of the approach, especially related to individual loci. While this method does outperform other approaches, it remains technically sophisticated and for some labs may not be worth the additional effort for the increase in information. Also, until tested and proven by other groups, it is difficult to know how impactful this approach will be.

Reviewer #3 (Public review):

Summary:

Krwawicz et al., present evidence that expression of DNMTs in E. coli results in (1) introduction of alkylation damage that is repaired by AlkB; (2) confers hypersensitivity to alkylating agents such as MMS (and exacerbated by loss of AlkB); (3) confers hypersensitivity to oxidative stress (H2O2 exposure); (4) results in a modest increase in ROS in the absence of exogenous H2O2 exposure; and (5) results in the production of oxidation products of 5mC, namely 5hmC and 5fC, leading to cellular toxicity. The findings reported here have interesting implications for the concept that such genotoxic and potentially mutagenic consequences of DNMT expression (resulting in 5mC) could be selectively disadvantageous for certain organisms. The other aspect of this work which is important for understanding the biological endpoints of genotoxic stress is the notion that DNA damage per se somehow induces elevated levels of ROS.

Strengths:

The manuscript is well-written, and the experiments have been carefully executed providing data that support the authors' proposed model presented in Fig. 7 (Discussion, sources of DNA damage due to DNMT expression).

Weaknesses:

(1) The authors have established an informative system relying on expression of DNMTs to gauge the effects of such expression and subsequent induction of 3mC and 5mC on cell survival and sensitivity to an alkylating agent (MMS) and exogenous oxidative stress (H2O2 exposure). The authors state (p4) that Fig. 2 shows that "Cells expressing either M.SssI or M.MpeI showed increased sensitivity to MMS treatment compared to WT C2523, supporting the conclusion that the expression of DNMTs increased the levels of alkylation damage." This is a confusing statement and requires revision as Fig. 2 does ALL cells shown in Fig. 2 are expressing DNMTs and have been treated with MMS. It is the absence of AlkB and the expression of DNMTs that that causes the MMS sensitivity.

(2) It would be important to know whether the increased sensitivity (toxicity) to DNMT expression and MMS is also accompanied by substantial increases in mutagenicity. The authors should explain in the text why mutation frequencies were not also measured in these experiments.

(3) Materials and Methods. ROS production monitoring. The "Total Reactive Oxygen Species (ROS) Assay Kit" has not been adequately described. Who is the Vendor? What is the nature of the ROS probes employed in this assay? Which specific ROS correspond to "total ROS"?

(4) The demonstration (Fig. 4) that DNMT expression results in elevated ROS and its further synergistic increase when cells are also exposed to H2O2 is the basis for the authors' discussion of DNA damage-induced increases in cellular ROS. S. cerevisiae does not possess DNMTs/5mC, yet exposure to MMS also results in substantial increases in intracellular ROS (Rowe et al, (2008) Free Rad. Biol. Med. 45:1167-1177. PMC2643028). The authors should be aware of previous studies that have linked DNA damage to intracellular increases in ROS in other organisms and should comment on this in the text.

Reviewer #3 (Public review):

Summary:

The authors performed a detailed single-cell analysis of the early embryonic cranial neural plate with unprecedented temporal resolution between embryonic days 7.5 and 8.75. They employed diffusion analysis to identify genes that correspond to different temporal and spatial locations within the embryo. Finally, they also examined the global response of cranial tissue to a Smoothened agonist.

Strengths:

Overall, this is an impressive resource, well-validated against sets of genes with known temporal and spatial patterns of expression. It will be of great value to investigators examining the early stages of neural plate patterning, neural progenitor diversity, and the roles of signaling molecules and gene regulatory networks controlling the regionalization and diversification of the neural plate.

Weaknesses:

The manuscript should be considered a resource. Experimental manipulation is limited to the analysis of neural plate cells that were cultured in vitro for 12 hours with SAG. Besides the identification of a significant set of previously unreported genes that are differentially expressed in the cranial neural plate, there is little new biological insight emerging from this study. Some additional analyses might help to highlight novel hypotheses arising from this remarkable resource.

Reviewer #3 (Public Review):

The work by Dar et al. examines RNA metabolism under cellular stress, focusing on stress-granule-dependent RNA decay. It employs direct RNA sequencing with a Nanopore-based method, revealing that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy but is independent of the shortening of the poly(A) tail. This decay, however, is dependent on XRN1 and enriched in the stress granule transcriptome. Notably, inhibiting stress granule formation in G3BP1/2-null cells restores the RNA length to the same level as wild-type. It suppresses stress-induced decay, identifying RNA decay as a critical determinant of RNA metabolism during cellular stress and highlighting its dependence on stress-granule formation. This is an exciting and novel discovery utilizing innovative sequencing methods to studying mRNA decay.

Reviewer #3 (Public review):

The aim of the study was to map, a) whether different tissues exhibit different metabolic profiles (this is known already), what differences are found between female and male mice and how the profiles changes with age. In particular, the study recorded the activity of respirasomes, i.e. the concerted activity of mitochondrial respiratory complex chains consisting of CI+CIII2+CIV, CII+CIII2+CIV or CIV alone.

The strength is certainly the atlas of oxidative metabolism in the whole mouse body, the inclusion of the two different sexes and the comparison between young and old mice. The measurement was performed on frozen tissue, which is possible as already shown (Acin-Perez et al, EMBO J, 2020).

Weakness: The assay reveals the maximum capacity of enzyme activity, which is an artificial situation and may differ from in vivo respiration, as the authors themselves discuss. The material used was a very crude preparation of cells containing mitochondria and other cytosolic compounds and organelles. Thus, the conditions are not well defined and the respiratory chain activity was certainly uncoupled from ATP synthesis. Preparation of more pure mitochondria and testing for coupling would allow evaluation of additional parameters: P/O ratios, feedback mechanism, basal respiration, and ATP-coupled respiration, which reflect in vivo conditions much better. The discussion is rather descriptive and cautious and could lead to some speculations about what could cause the differences in respiration and also what consequences these could have, or what certain changes imply.<br /> Nevertheless, this study is an important step towards this kind of analysis.

Comments on the second revision:

I believe this is an important and interesting area of study, although I recognise that the assay which measures maximal enzyme activity under unphysiological conditions has its limitations. Nevertheless, it does seem possible to get a first glance of the respiratory situation in the respective tissue. There is a typo in the source data (Fig. xC) for skeletal muscle.

Reviewer #3 (Public review):

This paper seeks to understand the role of alveolar myofibroblasts in the abnormal lung development after saccular stage injury.

Strengths:

(1) Multiple models of neonatal injury are used, hyperoxia and transgenic models that target alveolar myofibroblasts.

(2) The authors integrate their data with prior published single-cell data from neonatal hyperoxia injury models and demonstrate concordant findings.

Weaknesses:

(1) As the authors acknowledge in the discussion, there are no spatial and temporal validation data of the single-cell findings. As the ductal myofibroblasts has many overlapping genes, localizing and quantifying the loss of these cells in injury as a plausible mechanistic driver would greatly strengthen the conclusion.

(2) As they note in their response, this proved to be technically difficult and current Pdgfra-lineage trace tools are not without their own limitations.

Summary:

Taken together, this manuscript provides a rich data set from a model of irreversible neonatal lung injury. The single-cell analysis methods are well-articulated and the limitations are acknowledged, allowing this paper to provide a foundation for future work to spatially and temporally validate these claims.

Reviewer #3 (Public review):

The paper addresses pivotal questions concerning the multifaceted functions of oyster hemocytes by integrating single-cell RNA sequencing (scRNA-seq) data with analyses of cell morphology, transcriptional profiles, and immune functions. In addition to investigating granulocyte cells, the study delves into the potential roles of blast and hyalinocyte cells. A key discovery highlighted in this research is the identification of cell types engaged in antimicrobial activities, encompassing processes such as phagocytosis, intracellular copper accumulation, oxidative bursts, and antimicrobial peptide synthesis.

A particularly intriguing aspect of the study lies in the exploration of hemocyte lineages, warranting further investigation, such as employing scRNA-seq on embryos at various developmental stages.

In the opinion of this reviewer, the discussion should compare and contrast the transcriptome characteristics of hemocytes, particularly granule cells, across the three species of bivalves, aligning with the published scRNA-seq studies in this field to elucidate the uniformities and variances in bivalve hemocytes.

Reviewer #3 (Public review):

Summary:

In multicellular organisms, autophagosomes are formed throughout the cytosol, while late endosomes/lysosomes are relatively confined in the perinuclear region. It is known that autophagosomes gain access to the lysosome-enriched region by microtubule-based trafficking. The mechanism by which autophagosomes move along microtubules remains incompletely understood. In this manuscript, Péter Lőrincz and colleagues investigated the mechanism driving the movement of nascent autophagosomes along the microtubule towards the non-centrosomal microtubule organizing center (ncMTOC) using the fly fat body as a model system. The authors took an approach whereby they examined autophagosome positioning in cells where autophagosome-lysosome fusion was inhibited by knocking down the HOPS subunit Vps16A. Despite being generated at random positions in the cytosol, autophagosomes accumulate around the nucleus when Vps16A is depleted. They then performed an RNA interference screen to identify the factors involved in autophagosome positioning. They found that the dynein-dynactin complex is required for the trafficking of autophagosomes toward ncMTOC. Dynein loss leads to the peripheral relocation of autophagosomes. They further revealed that a pair of small GTPases and their effectors, Rab7-Epg5 and Rab39-ema, are required for bidirectional autophagosome transport. Knockdown of these factors in Vps16a RNAi cells causes the scattering of autophagosomes throughout the cytosol.

Strengths:

The data presented in this study help us to understand the mechanism underlying the trafficking and positioning of autophagosomes.

Weaknesses:

Major concerns:

(1) The localization of EPG5 should be determined. The authors showed that EPG5 colocalizes with endogenous Rab7. Rab7 labels late endosomes and lysosomes. Previous studies in mammalian cells have shown that EPG5 is targeted to late endosomes/lysosomes by interacting with Rab7. EPG5 promotes the fusion of autophagosomes with late endosomes/lysosomes by directly recognizing LC3 on autophagosomes and also by facilitating the assembly of the SNARE complex for fusion. In Figure 5I, the EPG5/Rab7-colocalized vesicles are large and they are likely to be lysosomes/autolysosomes.

(2) The experiments were performed in Vps16A RNAi KD cells. Vps16A knockdown blocks fusion of vesicles derived from the endolysosomal compartments such as fusion between lysosomes. The pleiotropic effect of Vps16A RNAi may complicate the interpretation. The authors need to verify their findings in Stx17 KO cells, as it has a relatively specific effect on the fusion of autophagosomes with late endosomes/lysosomes.

(3) Quantification should be performed in many places such as in Figure S4D for the number of FYVE-GFP labeled endosomes and in Figures S4H and S4I for the number and size of lysosomes.

(4) In this study, the transport of autophagosomes is investigated in fly fat cells. In fat cells, a large number of large lipid droplets accumulate and the endomembrane systems are distinct from that in other cell types. The knowledge gained from this study may not apply to other cell types. This needs to be discussed.

Minor concerns:

(5) Data in some panels are of low quality. For example, the mCherry-Atg8a signal in Figure 5C is hard to see; the input bands of Dhc64c in Figure 5L are smeared.

(6) In this study, both 3xmCherry-Atg8a and mCherry-Atg8a were used. Different reporters make it difficult to compare the results presented in different figures.

(7) The small autophagosomes presented in Figures such as in Figure 1D and 1E are not clear. Enlarged images should be presented.

(8) The authors showed that Epg5-9xHA coprecipitates with the endogenous dynein motor Dhc64C. Is Rab7 required for the interaction?

(9) The perinuclear lysosome localization in Epg5 KD cells has no indication that Epg5 is an autophagosome-specific adaptor.

Reviewer #3 (Public review):

Summary:

Bennion et al. investigate how semantic relatedness proactively benefits the learning of new word pairs. The authors draw predictions from Osgood (1949), which posits that the degree of proactive interference (PI) and proactive facilitation (PF) of previously learned items on to-be-learned items depends on the semantic relationships between the old and new information. In the current study, participants subjects learn a set of word pairs ( "supplemental pairs"), followed by a second set of pairs ("base pairs"), in which the cue, target or both words are changed, or the pair was identical. Pairs were drawn from either a narrower or wider stimulus set and were tested after either a 5 minute or 48 hour delay. The results show that semantic relatedness overwhelmingly produces PF and greater memory interdependence between base and supplemental pairs, except in the case of unrelated pairs in a wider stimulus set after a short delay, which produced PI. In their final analyses, the authors compare their current results to previous work from their group studying the analogous retroactive effects of semantic relatedness on memory. These comparisons show generally similar, if slightly weaker, patterns of results. The authors interpret their results in the framework of recursive reminders (Hintzman, 2011), which posits that the semantic relationships between new and old word pairs promotes reminders of the old information during the learning of the new to-be-learned information. These reminders help to integrate the old and new information and result in additional retrieval practice opportunities that in turn improve later recall.

Strengths:

Overall, I thought that the analyses were thorough and well-thought-out and the results were incredibly well-situated in the literature, especially with the additional clarification and framing that the authors have made in response to reviewer comments. In particular, I found that the large sample size, inclusion of a wide range of semantic relatedness across the two stimulus sets, variable delays and the ability to directly compare the current results to their prior results on the retroactive effects of semantic relatedness were particular strengths of the authors' approach and make this an impressive contribution to the existing literature. I thought that their interpretations and conclusions were mostly reasonable and included appropriate caveats (where applicable).

Weaknesses:

The changes and additional analyses that the authors have made have addressed my concerns about their analyses. Including the additional Fig 1- Supp 1, panel C greatly helps with the interpretability across stimulus sets, and the additional analyses the authors have performed teasing apart whether cue and target similarity separately influence memorability and interdependence seem to support the rest of their conclusions.

Reviewer #3 (Public review):

Summary:

This important study combines in vitro and in vivo recording to determine how the firing of cortical and striatal neurons changes during a fever range temperature rise (37-40 oC). The authors found that certain neurons will start, stop, or maintain firing during these body temperature changes. The authors further suggested that the TRPV3 channel plays a role in maintaining cortical activity during fever.

Strengths:

The topic of how the firing pattern of neurons changes during fever is unique and interesting. The authors carefully used in vitro electrophysiology assays to study this interesting topic.

Weaknesses:

(1) In vivo recording is a strength of this study. However, data from in vivo recording is only shown in Figures 5A,B. This reviewer suggests the authors further expand on the analysis of the in vivo Neuropixels recording. For example, to show single spike waveforms and raster plots to provide more information on the recording. The authors can also separate the recording based on brain regions (cortex vs striatum) using the depth of the probe as a landmark to study the specific firing of cortical neurons and striatal neurons. It is also possible to use published parameters to separate the recording based on spike waveform to identify regular principal neurons vs fast-spiking interneurons. Since the authors studied E/I balance in brain slices, it would be very interesting to see whether the "E/I balance" based on the firing of excitatory neurons vs fast-spiking interneurons might be changed or not in the in vivo condition.

(2) The author should propose a potential mechanism for how TRPV3 helps to maintain cortical activity during fever. Would calcium influx-mediated change of membrane potential be the possible reason? Making a summary figure to put all the findings into perspective and propose a possible mechanism would also be appreciated.

(3) The author studied P7-8, P12-14, and P20-26 mice. How do these ages correspond to the human ages? it would be nice to provide a comparison to help the reader understand the context better.

Reviewer #3 (Public review):

Summary:

This study presents a useful computational tool, termed FLiSimBA. The MATLAB-based FLiSimBA simulations allow users to examine the effects of various noise factors (such as autofluorescence, afterpulse of the photomultiplier tube detector, and other background signals) and varying sensor expression levels. Under the conditions explored, the simulations unveiled how these factors affect the observed lifetime measurements, thereby providing useful guidelines for experimental designs. Further simulations with two distinct fluorophores uncovered conditions in which two different lifetime signals could be distinguished, indicating multiplexed dynamic imaging may be possible.

Strengths:

The simulations and their analyses were done systematically and rigorously. FliSimba can be useful for guiding and validating fluorescence lifetime imaging studies. The simulations could define useful parameters such as the minimum number of photons required to detect a specific lifetime, how sensor protein expression level may affect the lifetime data, the conditions under which the lifetime would be insensitive to the sensor expression levels, and whether certain multiplexing could be feasible.

Weaknesses:

The analyses have relied on a key premise that the fluorescence lifetime in the system can be described as two-component discrete exponential decay. This means that the experimenter should ensure that this is the right model for their fluorophores a priori and should keep in mind that the fluorescence lifetime of the fluorophores may not be perfectly described by a two-component discrete exponential (for which alternative algorithms have been implemented: e.g., Steinbach, P. J. Anal. Biochem. 427, 102-105, (2012)). In this regard, I also couldn't find how good the fits were for each simulation and experimental data to the given fitting equation (Equation 2, for example, for Figure 2C data).

Also, in Figure 2C, the 'sensor only' simulation without accounting for autofluorescence (as seen in Sensor + autoF) or afterpulse and background fluorescence (as seen in Final simulated data) seems to recapitulate the experimental data reasonably well. So, at least in this particular case where experimental data is limited by its broad spread with limited data points, being able to incorporate the additional noise factors into the simulation tool didn't seem to matter too much.

Reviewer #3 (Public review):

Summary:

This study reveals that sound exposure enhances drug delivery to the cochlea through the non-selective action of outer hair cells. The efficiency of sound-facilitated drug delivery is reduced when outer hair cell motility is inhibited. Additionally, low-frequency tones were found to be more effective than broadband noise for targeting substances to the cochlear apex. Computational model simulations support these findings.

Strengths:

The study provides compelling evidence that the broad action of outer hair cells is crucial for cochlear fluid circulation, offering a novel perspective on their function beyond frequency-selective amplification. Furthermore, these results could offer potential strategies for targeting and optimizing drug delivery throughout the cochlear spiral.

Weaknesses:

The primary weakness of this paper lies in the surgical procedure used for drug administration through the round window. Opening the cochlea can alter intracochlear pressure and disrupt the traveling wave from sound, a key factor influencing outer hair cell activity. However, the authors do not provide sufficient details on how they managed this issue during surgery. Additionally, the introduction section needs further development to better explain the background and emphasize the significance of the work.

Comments on revisions:

Thank you for addressing the comments and concerns. The author has responded to all points thoroughly and clarified them well. However, please include the key points from the responses to the comments (Introduction ((3), (5)) and Results ((5)) into the manuscript. While the explanations in the response letter are reasonable, the current descriptions in the manuscript may limit the reader's understanding. Expanding on these points in the Introduction, Results, or Discussion sections would enhance clarity and comprehensiveness.

Reviewer #3 (Public review):

Summary:

In their study, the authors combine developmental and comparative transcriptomics to identify candidate genes with plastic, canalized, or lineage-specific (i.e., divergent) expression patterns associated with an unusual overwintering phenomenon (Dehnel's phenomenon - seasonal size plasticity) in the Eurasian shrew. Their focus is on the shrinkage and regrowth of the hypothalamus, a brain region that undergoes significant seasonal size changes in shrews and plays a key role in regulating metabolic homeostasis. Through combined transcriptomic analysis, they identify genes showing derived (lineage-specific), plastic (seasonally regulated), and canalized (both lineage-specific and plastic) expression patterns. The authors hypothesize that genes involved in pathways such as the blood-brain barrier, metabolic state sensing, and ion-dependent signaling will be enriched among those with notable transcriptomic patterns. They complement their transcriptomic findings with a cell culture-based functional assessment of a candidate gene believed to reduce apoptosis.

Strengths:

The study's rationale and its integration of developmental and comparative transcriptomics are well-articulated and represent an advancement in the field. The transcriptome, known for its dynamic and plastic nature, is also influenced by evolutionary history. The authors effectively demonstrate how multiple signals-evolutionary, constitutive, and plastic-can be extracted, quantified, and interpreted. The chosen phenotype and study system are particularly compelling, as it not only exemplifies an extreme case of Dehnel's phenotype, but the metabolic requirements of the shrew suggest that genes regulating metabolic homeostasis are under strong selection.

Weaknesses:

(1) In a number of places (described in detail below), the motivation for the experimental, analytical, or visualization approach is unclear and may obscure or prevent discoveries.

(2) Temporal Expression - Figure 1 and Supplemental Figure 2 and associated text:<br /> - It is unclear whether quantitative criteria were used to distinguish "developmental shift" clusters from "season shift" clusters. A visual inspection of Supplemental Figure 2 suggests that some clusters (e.g., clusters 2, 8, and to a lesser extent 12) show seasonal variation, not just developmental differences between stages 1 and 2. While clustering helps to visualize expression patterns, it may not be the most appropriate filter in this case, particularly since all "season shift" clusters are later combined in KEGG pathway and GO analyses (Figure 1B).<br /> - The authors do not indicate whether they perform cluster-specific GO or KEGG pathway enrichment analyses. The current analysis picks up relevant pathways for hypothalamic control of homeostasis, which is a useful validation, but this approach might not fully address the study's key hypotheses.

(3) Differential expression between shrinkage (stage 2) and regrowth (stage 4) and cell culture targets<br /> - The rationale for selecting BCL2L1 for cell culture experiments should be clarified. While it is part of the apoptosis pathway, several other apoptosis-related genes were identified in the differential gene expression (DGE) analysis, some showing stronger differential expression or shrew-specific branch shifts. Why was BCL2L1 prioritized over these other candidates?<br /> - The authors mention maintaining (or at least attempting to maintain) a 1:1 sex ratio for the comparative analysis, but it is unclear if this was also done for the S. araneus analysis. If not, why? If so, was sex included as a covariate (e.g., a random effect) in the differential expression analysis? Sex-specific expression elevates with group variation and could impact the discovery of differentially expressed genes.

(4) Discussion: The term "adaptive" is used frequently and liberally throughout the discussion. The interpretation of seasonal changes in gene expression as indicators of adaptive evolution should be done cautiously as such changes do not necessarily imply causal or adaptive associations.

Reviewer #3 (Public review):

Summary:

Chen et al. identify endophilin A1 as a novel component of the inhibitory postsynaptic scaffold. Their data show impaired evoked inhibitory synaptic transmission in CA1 neurons of mice lacking endophilin A1, and an increased susceptibility to seizures. Endophilin can interact with the postsynaptic scaffold protein gephyrin and promote assembly of the inhibitory postsynaptic element. Endophilin A1 is known to play a role in presynaptic terminals and in dendritic spines, but a role for endophilin A1 at inhibitory postsynaptic densities has not yet been described.

Strengths:

The authors used a broad array of experimental approaches to investigate this, including tests of seizure susceptibility, electrophysiology, biochemistry, neuronal culture, and image analysis.

Weaknesses:

Many results are difficult to interpret, and the data quality is not always convincing, unfortunately. The basic premise of the study, that gephyrin and endophilin A1 interact, requires a more robust analysis to be convincing.

Reviewer #3 (Public review):

Summary:

The manuscript by Wu D. et al. explores an innovative approach to immunometabolism and obesity by investigating the potential of targeting macrophage Inositol-requiring enzyme 1α (IRE1α) in cases of overnutrition. Their findings suggest that pharmacological inhibition of IRE1α could influence key aspects such as adipose tissue inflammation, insulin resistance, and thermogenesis. Notable discoveries include the identification of High-Fat Diet (HFD)-induced CD9+ Trem2+ macrophages and the reversal of metabolically active macrophages' activity with IRE1α inhibition using STF. These insights could significantly impact future obesity treatments.

Strengths:

The study's key strengths lie in its identification of specific macrophage subsets and the demonstration that inhibiting IRE1α can reverse the activity of these macrophages. This provides a potential new avenue for developing obesity treatments and contributes valuable knowledge to the field.

Weaknesses:

The research lacks an in-depth exploration of the broader metabolic mechanisms involved in controlling diet-induced obesity (DIO). Addressing this gap would strengthen the understanding of how targeting IRE1α might fit into the larger metabolic landscape.

Impact and Utility:

The findings have the potential to advance the field of obesity treatment by offering a novel target for intervention. However, further research is needed to fully elucidate the metabolic pathways involved and to confirm the long-term efficacy and safety of this approach. The methods and data presented are useful, but additional context and exploration are required for broader application and understanding.

Reviewer #3 (Public review):

This study investigates how two cortical regions that are central to the study of rodent motor control (rostral forelimb area, RFA, and caudal forelimb area, CFA) interact during directional forelimb reaching in mice. The authors investigate this interaction using<br /> (1) optogenetic manipulations in one area while recording extracellularly from the other,<br /> (2) statistical analyses of simultaneous CFA/RFA extracellular recordings, and<br /> (3) network modeling.<br /> The authors provide solid evidence that asymmetry between RFA and CFA can be observed, although such asymmetry is only observed in certain experimental and analytical contexts.

The authors find asymmetry when applying optogenetic perturbations, reporting a greater impact of RFA inactivation on CFA activity than vice-versa. The authors then investigate asymmetry in endogenous activity during forelimb movements and find asymmetry with some analytical methods but not others. Asymmetry was observed in the onset timing of movement-related deviations of local latent components with RFA leading CFA (computed with PCA) and in a relatively higher proportion and importance of cross-area latent components with RFA leading than CFA leading (computed with DLAG). However, no asymmetry was observed using several other methods that compute cross-area latent dynamics, nor with methods computed on individual neuron pairs across regions. The authors follow up this experimental work by developing a two-area model with asymmetric dependence on cross-area input. This model is used to show that differences in local connectivity can drive asymmetry between two areas with equal amounts of across-region input.

Overall, this work provides a useful demonstration that different cross-area analysis methods result in different conclusions regarding asymmetric interactions between brain areas and suggests careful consideration of methods when analyzing such networks is critical. A deeper examination of why different analytical methods result in observed asymmetry or no asymmetry, analyses that specifically examine neural dynamics informative about details of the movement, or a biological investigation of the hypothesis provided by the model would provide greater clarity regarding the interaction between RFA and CFA.

Strengths:

The authors are rigorous in their experimental and analytical methods, carefully monitoring the impact of their perturbations with simultaneous recordings, and providing valid controls for their analytical methods. They cite relevant previous literature that largely agrees with the current work, highlighting the continued ambiguity regarding the extent to which there exists an asymmetry in endogenous activity between RFA and CFA.

A strength of the paper is the evidence for asymmetry provided by optogenetic manipulation. They show that RFA inactivation causes a greater absolute difference in muscle activity than CFA interaction (deviations begin 25-50 ms after laser onset, Figure 1) and that RFA inactivation causes a relatively larger decrease in CFA firing rate than CFA inactivation causes in RFA (deviations begin <25ms after laser onset, Figure 3). The timescales of these changes provide solid evidence for an asymmetry in the impact of inactivating RFA/CFA on the other region that could not be driven by differences in feedback from disrupted movement (which would appear with a ~50ms delay).

The authors also utilize a range of different analytical methods, showing an interesting difference between some population-based methods (PCA, DLAG) that observe asymmetry, and single neuron pair methods (granger causality, transfer entropy, and convergent cross mapping) that do not. Moreover, the modeling work presents an interesting potential cause of "hierarchy" or "asymmetry" between brain areas: local connectivity that impacts dependence on across-region input, rather than the amount of across-region input actually present.

Weaknesses:

There is no attempt to examine neural dynamics that are specifically relevant/informative about the details of the ongoing forelimb movement (e.g., kinematics, reach direction). Thus, it may be preemptive to claim that firing patterns alone do not reflect functional influence between RFA/CFA. For example, given evidence that the largest component of motor cortical activity doesn't reflect details of ongoing movement (reach direction or path; Kaufman, et al. PMID: 27761519) and that the analytical tools the authors use likely isolate this component (PCA, CCA), it may not be surprising that CFA and RFA do not show asymmetry if such asymmetry is related to the control of movement details. An asymmetry may still exist in the components of neural activity that encode information about movement details, and thus it may be necessary to isolate and examine the interaction of behaviorally-relevant dynamics (e.g., Sani, et al. PMID: 33169030).

The idea that local circuit dynamics play a central role in determining the asymmetry between RFA and CFA is not supported by experimental data in this paper. The plausibility of this hypothesis is supported by the model but is not explored in any analyses of the experimental data collected. Given the focus on this idea in the discussion, further experimental investigation is warranted.

Reviewer #3 (Public review):

Summary:

In this study, Harikrishnan Rajendran, Roi Weinberger, Ehud Fonio, and Ofer Feinerman measured the digging behaviours of queens and workers for the first 6 months of colony development, as well as groups of young or old ants. They also provide a quantitative model describing the digging behaviours and allowing predictions. They found that young ants dig more slanted tunnels, while older ants dig more vertically (straight down). This finding is important, as it describes a new form of age polyethism (a division of labour based on age). Age polyethism is described as a "yes or no" mechanism, where individuals perform or not a task according to their age (usually young individuals perform in-nest tasks, and older ones foraging). Here, the way of performing the task is modified, not only the propensity to carry it or not. This data therefore adds in an interesting way to the field of collective behaviours and division of labour.

The conclusions of the paper are well supported by the data. Measurements of the same individuals over time would have strengthened the claims.

Strengths:

I find that the measure of behaviour through development is of great value, as those studies are usually done at a specific time point with mature colonies. The description of a behaviour that is modified with age is a notable finding in the world of social insects. The sample sizes are adequate and all the information clearly provided either in the methods or supplementary.

Weaknesses:

I think the paper is failing to take into consideration or at least discuss the role of inter-individual variabilities. Tasks have been known to be undertaken by only a few hyper-active individuals for example. Comments on the choice to use averages and the potential roles of variations between individuals are in my opinion lacking. Throughout the paper wording should be modified to refer to the group and not the individuals, as it was the collective digging that was measured. Another issue I had was the use of "mature colony" for colonies with very few individuals and only 6 months of age. Comments on the low number of workers used compared to natural mature colonies would be welcome.

Reviewer #3 (Public review):

Summary:

In this study, the authors aimed to index individual variation in decision-making when decisions pit the interests of the self (gains in money, potential for electric shock) against the interests of an unknown stranger in another room (potential for unknown shock). In addition, the authors conducted an additional study in which male participants were either administered intranasal oxytocin or placebo before completing the task to identify the role of oxytocin in moderating task responses. Participants' choice data was analyzed using a harm aversion model in which choices were driven by the subjective value difference between the less and more painful options.

Strengths:

Overall I think this is a well-conducted, interesting, and novel set of research studies exploring decision-making that balances outcomes for the self versus a stranger, and the potential role of the hormone oxytocin (OT) in shaping these decisions. The pain component of the paradigm is well designed, as is the decision-making task, and overall the analyses were well suited to evaluating and interpreting the data. Advantages of the task design include the absence of deception, e.g., the use of a real study partner and real stakes, as a trial from the task was selected at random after the study and the choice the participant made was actually executed. 

Weaknesses:

The primary weakness of the paper concerns its framing. Although it purports to be measuring "hyper-altruism" it does not provide evidence to support why any of the behavior being measured is extreme enough to warrant the modifier "hyper" (and indeed throughout I believe the writing tends toward hyperbole, using, e.g., verbs like "obliterate" rather than "reduce"). More seriously, I do not believe that the task constitutes altruism, but rather the decision to engage, or not engage, in instrumental aggression.

I found it surprising that a paradigm that entails deciding to hurt or not hurt someone else for personal benefit (whether acquiring a financial gain or avoiding a loss) would be described as measuring "altruism." Deciding to hurt someone for personal benefit is the definition of instrumental aggression. I did not see that in any of the studies was there a possibility of acting to benefit the other participant in any condition. Altruism is not equivalent to refraining from engaging in instrumental aggression. True altruism would be to accept shocks to the self for the other's benefit (e.g., money).  The interpretation of this task as assessing instrumental aggression is supported by the fact that only the Instrumental Harm subscale of the OUS was associated with outcomes in the task, but not the Impartial Benevolence subscale. By contrast, the IB subscale is the one more consistently associated with altruism (e.g,. Kahane et al 2018; Amormino at al, 2022) I believe it is important for scientific accuracy for the paper, including the title, to be re-written to reflect what it is testing.

Relatedly: in the introduction I believe it would be important to discuss the non-symmetry of moral obligations related to help/harm--we have obligations not to harm strangers but no obligation to help strangers. This is another reason I do not think the term "hyper altruism" is a good description for this task--given it is typically viewed as morally obligatory not to harm strangers, choosing not to harm them is not "hyper" altruistic (and again, I do not view it as obviously altruism at all).

The framing of the role of OT also felt incomplete. In introducing the potential relevance of OT to behavior in this task, it is important to pull in evidence from non-human animals on origins of OT as a hormone selected for its role in maternal care and defense (including defensive aggression). The non-human animal literature regarding the effects of OT is on the whole much more robust and definitive than the human literature. The evidence is abundant that OT motivates the defensive care of offspring of all kinds. My read of the present OT findings is that they increase participants' willingness to refrain from shocking strangers even when incurring a loss (that is, in a context where the participant is weighing harm to themselves versus harm to the other). It will be important to explain why OT would be relevant to refraining from instrumental aggression, again, drawing on the non-human animal literature.

Another important limitation is the use of only male participants in Study 2. This was not an essential exclusion. It should be clear throughout sections of the manuscript that this study's effects can be generalized only to male participants.