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    1. Reviewer #1 (Public Review)

      This work challenges previously published results regarding the presence and abundance of 6mA in Drosophila genome, as well as the claim that the TET or DMAD enzyme serves as the "eraser" of this DNA methylation mark and its roles in development. This information is needed to clarify these questions in the field. Generally speaking, the methods for fly husbandry and treatment seem to be in accordance with those established ones in the field.

      Here are a couple of suggestions that could be discussed with the current work and addressed in the future, in order to better understand the roles of 6mA and TET.

      1. Regarding the estimated "200 to 400 methylated adenines per haplogenome", some insights regarding where they are enriched in the genome could inform the potential target sites regulated by 6mA.

      2. The TET-GFP and TET-CD-GFP knock-in lines give proper nuclear localization and could be used to identify genomic regions bound with full-length TET and TET-CD using anti-GFP for ChIP-seq or CUT&RUN (or CUT&TAG).

    1. Reviewer #1 (Public Review):


      Using chromaffin cells as powerful model systems for studying secretion, the authors study the regulatory role of complexin in secretion. Complexin is still enigmatic in its regulatory role, as it both provides inhibitory and facilitatory functions in release. The authors perform an extensive structure-function analysis of both the C- and N-terminal regions of complexin. There are several interesting findings that significantly advance our understanding of cpx/SNARe interactions in regulating release. C-terminal amphipathic helix interferes with SNARE complex assembly and thus clamps fusion. There are acidic residues in the C-term that may be seen as putative interaction partners for Synaptotagmin. The N-terminus of Complexin promoting role may be associated with an interaction with Syt1. In particular, the putative interaction with Syt1 is of high interest and supported by quite strong functional and biochemical evidence. The experimental approaches are state-of-the-art, and the results are of the highest quality and convincing throughout. They are adequately and intelligently discussed in the rich context of the standing literature. Whilst there are some concerns about whether the facilitatory actions of complexion have to be tightly linked to Syt1 interactions, the proposed model will significantly advance the field by providing new directions in future research.

      I have only minor comments related to the interpretation of the data:

      Fig 5 While the data very nicely show that CPX and Syt1 have interdependent interactions in the chromaffin neurons, this seems to be not the case in neurons, where the loss of complexins and synaptotagmins have additive effects, suggesting independent mechanisms (eg Xue et al., 2010). This would be a good opportunity to discuss some possible differences between secretion in endocrine cells vs neurons.

      Fig 8 Shows an apparent shift in Ca sensitivity in N-terminal mutants suggesting a modification of Ca sensitivity of Syt1. Could there be also an alternative mechanism, that explains this phenotype which is based on a role of the n-term lowering the energy barrier for fusion, that in turn shifts corresponding fusion rates to take place at lower Ca saturation levels?

    1. Reviewer #1 (Public Review):

      Summary:<br /> Yang et al combine high-speed video tracking of the limbs of freely moving mice with in vivo electrophysiology to demonstrate how striatal neurons encode single-limb gait. They also examine encoding other well-known aspects of locomotion, such as movement velocity and the initiation/termination of movement. The authors show that striatal neurons exhibit rhythmic firing phase-locked with mouse gait, while mice engage in spontaneous locomotion in an open field arena. Moreover, they describe gait deficits induced by severe unilateral dopamine neuron degeneration and associate these deficits with a relative strengthening of gait-modulation in the firing of D2-expressing MSNs. Although the source and function of this gait-modulation remain unclear, this manuscript uncovers an important physiological correlate of striatal activity with gait, which may have implications for gait deficits in Parkinson's Disease.

      Strengths:<br /> While some previous work has looked at the encoding of gait variables in the striatum and other basal ganglia nuclei, this paper uses more careful quantification of gait with video tracking. In addition, few if any papers do this in combination with optically-labeled recordings as were performed here.

      Weaknesses:<br /> The data collected has a great richness at the physiological and behavioral levels, and this is not fully described or explored in the manuscript. Additional analysis and display of data would greatly expand the interest and interpretability of the findings.

      There are also some caveats to the interpretation of the analyses presented here, including how to compare encoding of gait variables when animals have markedly different behaviors (eg comparing sham and unilaterally 6-OHDA treated mice), or how to interpret the loss of gait modulation when single unit activity is overall very low.

      1. The authors use circular analysis to quantify the degree to which striatal neurons are phase-locked to individual limbs during gait. The result of this analysis is shown as the proportion of units phase-locked to each limb, vector length, and vector angle (Fig 2H-K; Fig 4E-F; Fig 6E-F). Given that gait is a cyclic oscillation of the trajectories of all four limbs, one could expect that if one unit is phase-locked to one limb, it will also be phase-locked to the other three limbs but at a different phase. Therefore, it is not clear in the manuscript how the authors determine to which limb each unit is locked, and how some units are locked to more than one limb (Fig 2H). More methodological/analytical detail would be especially helpful.

      2. In Figures 2 and 3, the authors describe the modulation of striatal neurons by gait, velocity, and movement transitions (start/end), with most of their examples showing firing rates compatible with rates typical of striatal interneurons, not MSNs. In order to have a complete picture of the relationship between striatal activity and gait, a cell type-specific analysis should be performed. This could be achieved by classifying units into putative MSN, FS interneurons, and TANs using a spike waveform-based unit classification, as has been done in other papers using striatal single-unit electrophysiology. An example of each cell type's modulation with gait, as well as summary data on the % modulation, would be especially helpful.

      3. By normalizing limb trajectories to the nose-tail axis, the analysis ignores whether the mouse is walking straight, or making left/right turns. Is the gait-modulation of striatal activity shaped by ipsi- and contralateral turning? This would be especially important to understand changes in the unilateral disease model, given the imbalance in turning of 6-OHDA mice.

      4. It looks like the data presented in Figure 4 D-F comes from all opto-identified D1- and D2-MSNs. How many of these are gait-modulated? This information is missing (line 110). Pooling all units may dilute differences specific to gait-modulated units, therefore a similar analysis only on gait-modulated units should be performed.

      5. Since 6-OHDA lesions are on the right hemisphere, we would expect left limbs to be more affected than right limbs (although right limbs may also compensate). It is therefore surprising that RF and RR strides seem slightly shorter than LF and LR (Fig 5G), and no differences in other stride parameters (Fig 5H-J). Could the authors comment on that? It may be that this is due to rotational behavior. One interesting analysis would be to compare activity during similar movements in healthy and 6-OHDA mice, eg epochs in which mice are turning right (which should be present in both groups) or walking a few steps straight ahead (which are probably also present in both groups).

      6. Multiple publications have shown that firing rates of D1-MSN and D2-MSN are dramatically changed after dopamine neuron loss. Is it possible that changes observed in gait-modulation might be biased by changes in firing rates? For example, dMSNs have exceptionally low overall activity levels after dopamine depletion (eg Parker...Schnitzer, 2018; Ryan...Nelson, 2018; Maltese...Tritsch, 2021); this might reduce the ability to detect modulation in the firing of dMSNs as compared to iMSNs, which have similar or increased levels of activity in dopamine depleted mice. Does vector length correlate with firing rate? In addition, the normalization method used (dividing firing rate by minimum) may amplify very small changes in absolute rates, given that the firing rates for MSN are very low. The authors could show absolute values or Z-score firing rates (Figure 6 A, D).

      7. The analysis shown in Fig 3C should also be done for opto-identified D1- and D2-MSNs (and for waveform-based classified units as noted above).

      8. Discussion: the origin of the gait-modulation as well as the possible mechanisms driving the alterations observed in 6-OHDA mice should be discussed in more detail.

    1. Reviewer #1 (Public Review):

      The brain's code is not static. Neuronal activity patterns change as a result of learning, aging, and disease. Reliable tracking of activity from individual neurons across long time periods would enable detailed studies of these important dynamics. For this reason, the authors' efforts to track electrophysiological activity across days without relying on matching neural receptive fields (which can change due to learning, aging, and disease) are very important.

      By utilizing the tightly-spaced electrodes on Neuropixels probes, they are able to measure the physical distance and the waveform shape 'distance' between sorted units recorded on different days. To tune the matching algorithm and validate the results, they used the visual receptive fields of neurons in the mouse visual cortex (which tend to change little over time) as ground truth. Their approach performs quite well, with a high proportion of neurons accurately matched across multiple weeks. This suggests that the method may be useable in other cases where the receptive fields can't be used as ground truth to validate the tracking. This potential extendibility to tougher applications is where this approach holds the most promise.

      The main caveat (and disappointment) is that this paper does not address generalizability to other experimental conditions. Because it only looks at one brain area (visual cortex), in one species (mouse), using one type of spike sorter (Kilosort), and one type of behavioral prep (head-fixed), it is not clear if this approach is overfit to those conditions or if it will perform equally well in other conditions. Most importantly, in brain areas where neuronal receptive fields are more dynamic and can't be used as a ground truth diagnostic, it isn't clear how to apply the technique outlined in this study, since many of the parameters are tuned to a very specific set of conditions using visual receptive fields as ground truth.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The authors have nicely demonstrated the efficiency of the HCR v.3.0 using hr38 mRNA expression as a marker of neuronal activity. This is very important in the Drosophila neuroscience field as in situ hybridization in adult Drosophila brains has been so far very challenging to do and replicate. However, this method has been described before [Choi et al., (2018)] and, if I understand correctly, is now the property of the non-profit organization molecular Technologies, who are the ones responsible for designing the probes (the sequences are not provided). Here the authors present their work as a description of a new method, called HI-FISH. However, I do not consider this as a new method but rather an application, a "proof of principle" that HCR v.3.0 can be done even on challenging tissues such as the adult Drosophila brain. Hence, if HCR v3.0 is sensitive enough and powerful enough to be used as a marker of neuronal activity, we can use it, for other neurobiological purposes, using other gene probes.<br /> To demonstrate the efficiency of HI-FISH, the authors have addressed two biological questions. The first one addressed whether specific groups of neurons, known to trigger specific behaviours (here courtship and/or aggression) are indeed activated by the behavioural context they can promote. In other words: is the behavioural output of these neurons also a trigger for their activation? The second question addressed whether this method is powerful enough to distinguish two subgroups of a class of neurons called P1 known to be involved in the two behaviours considered. In other words, are the same P1 neurons that promote aggression and courtship?

      Strengths: The demonstration of the efficiency of the method is very convincing and well-performed. It gives the will for the reader to apply the method to their own subject.

      Weakness: The pictures provided for HI-FISH and catFISH do not corroborate with the quantification and therefore I am not convinced about the author's biological interpretation of their data. See below for details.

      Previously, using a split-gal4 line to restrict the Gal4 expression to a subset of P1 neurons, the authors have shown that these particular neurons when activated can trigger both aggressivity and courtship behaviour [Hoopfer et al., 2015]. The P1 neurons are composed of about 20 FruM neurons/hemibrain but are part of a bigger group that comprises about the same number of Fru- neurons that seem to be exclusively aggression-promoting neurons [Koganezawa et al., 2016]. Hence, this group of 40 neurons (pC1 neurons) contains aggressive-promoting neurons, courtship-promoting neurons, and perhaps neurons that can do both. Therefore, to address the first question, the authors compared hr38 expression in different groups of neurons, with a focus on subgroups, under different social contexts. While there is no ambiguity concerning the function of the Tk neurons as being exclusively aggressive-promoting neurons [Asahina et al., 2014], it is less clear when we look at the pC1 neurons. This is particularly evident for the P1a neurons which have been shown to be ambiguous as they can promote both aggression and courtship. For example, while optogenetic activation of these neurons promotes hr38 expression (Fig. 3D and fig sup. 4), it is less clear in the pictures to determine whether these specific P1a neurons labeled by the split-gal4 line are specifically activated by an aggressive behavioural context or a courtship behavioural context (Fig1, supp. 2 and fig. 4). Furthermore, the pictures chosen do not reflect the reality of the quantification (Fig. 2 B-D compared to sup. 2 or fig. 4C compared to fig. 4D) and therefore the authors conclusion. Because the drivers used are only expressed by a small subset of a larger population, I believe it would be more informative to separate in the quantification between the Gal4-expressing neurons and the non-expressing ones. Notably, based on the pictures provided, it looks like more P1 neurons (or rather pC1 neurons) are activated by a male-male encounter than by a male-female encounter. On the other hand, the splitGal4+ P1a seem to be more responsive to a courtship behavioural context (2/6 P1a neurons express hr38 in a courtship behavioural context while 0/9 _if we mentally abstract the increase of the background signal compared to the control picture_ seem to express hr38 in an aggression behavioural context). Hence, while activation of this P1asplit-Gal4 can promote both aggressive behaviour and courtship behaviour [Hoopfer et al., 2015], the authors didn't provide clear evidence (pictures not corroborating the quantification) that these specific small subpopulation of neurons are activated by one or the other or both behavioural conditions. Therefore my suggestion of differentiating in the quantification between the Gal4+ neurons from the others in the same local area.

      Fig. 3, suppl. 3: In this section the authors addressed the question of whether the HI-FISH can be used to identify the downstream targets of this subpopulation. As positive controls of known downstream targets, the authors looked at the pCd population which they recently published as being an indirect downstream target of the P1a neurons [Jung et al., Neuron 2020]. They identified the Kenyon cells and a group of dopaminergic neurons, the PAM neurons as being activated by the P1a neurons. To confirm the increase of hr38 expression is indeed the result of a neuronal response of these neurons to the P1a activation, the authors used a different strategy used by them and others before. Using Gcamp signal to monitor the neuronal response of the presumably downstream targets the authors activated the P1a neurons using optogenetic (chrimson). It is important to note that they have previously shown that depending on the frequency of the light pulses activation of the P1a neurons can trigger only aggression, both aggression and wing extension or only wing extension [Hoopfer et al., eLife 2015]. Here the authors use 50Hz which is a frequency that leads to wing extension during the stimulation and aggressive behaviour at the offset of the stimulus [Hoopfer et al., eLife 2015]. Interestingly, the Gcamp experiment shows activation of the Kenyon cells and the PAM neurons but this activity is not maintained when the stimulus is turned off, suggesting that these neurons are activated during a courtship context rather than an aggressive behavioural context. I think it would be nice to see in which behavioural context the Kenyon cells and PAM neurons are activated (hr38 expression in the different behavioural context using the corresponding Gal4).<br /> Fig.4 and supp.4: The demonstration that the catFISH can now be done in Drosophila brain with a new in situ method was nicely performed. Notably, the intronic Hr38 probe appears to be an excellent marker for recent neuronal activation. However, while the optogenetic activation of the P1a neurons used to quantify the time lapse for both probes nicely distinguishes between nuclear and cytoplasmic exonic hr38, it is very difficult to use the localization of this probe in the experimental setup the authors used. Also, With their setup, I would simply use the frequency of intronic hr38 as a marker of recent activation correlating or not with the frequency of exonic hr38 marker (present in both conditions first and second encounter). This is important as this experiment addresses the second biological question. Once again, the pictures chosen absolutely do not corroborate the quantification. For example, the picture of the double encounter with the same gender male-male context clearly shows a higher number of cells that are hr38INT positive (and therefore nuclear) than the picture of the female-female context (Fig. 4C), and thus even if we only considered the P1asplit-Gal4 positive cells. In the male-male picture, 5/6 P1a cells have the Hr38INT marker while the presence of this marker is debatable in the female-female context. Especially, in some of the cells these magenta dots appear to be localized in the cytoplasm, suggesting a non-specific signal. Therefore, I would suggest to quantify the percentage of Hr38INT positive cells as the only marker for recent activation and the relative level of Hr38EXN immunostaining, and this among the P1asplit-Gal4 positive cells and the gal4- ones. A high Hr38EXN level associated with the presence of hr38INT would indicate that the cell has been activated during both encounters, while a lower hr38EXN with no hr38INT would suggest only an activation during the 1st behavioural context. Finally, a lower hr38EXN associated with the presence of hr38INT would suggest the opposite, an activation only during the 2nd behaviour.<br /> Overall, by only looking at the pictures provided, I would conclude that the HCR applied with the hr38 probes seems to efficiently work and is usable to address the question of whether a specific group of neurons are indeed activated by a specific social behavioural context. However, I would also conclude that this technique nicely demonstrated that flies are not robots and that even in a "simple" organism model such as Drosophila melanogaster individual variability is present among a group of neurons. Hence, only the quantification of the gal4-expressing neurons in comparison with their neighbor neurons known to belong to the same functional group, would allow a conclusion toward a specificity of contextual response. Therefore, although activation of a small group of neurons can be enough to trigger a specific behaviour that shouldn't happen under a certain environmental context [Hoopfer et al., eLife 2015], the results presented here suggest that we should, using this method, considering the response of the neighbour cells of the Gal4+ ones. Although currently, the quantification of the author's data does not allow such analysis, to strengthen the author's argumentation, I would distinguish in their quantification between gal4+ from the others (Fig. 2 and 4). Furthermore, I am not certain that the distinction between cytoplasmic and nuclear hr38EXN is 100% feasible (based on the pictures provided). I would instead for the hr38EXN marker only use the relative intensity (Fig. 4D).

    1. Reviewer #1 (Public Review):

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

      Considering the following points would further strengthen the manuscript:


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

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

      3. Membrane potential measurements: It would be nice to see a calibration of the potential measurements; this would allow us to correlate the IV relationship with membrane potential. Without calibration, it is hard to compare unless the identical uptake of the dye is shown.

      Does paxilline or IbTx also induce depolarization?

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

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


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

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

    1. Reviewer #1 (Public Review):


      In this study, the authors showed that activation of RelA and Stat3 in hepatocytes of DSS-treated mice induced CYPs and thereby produced primary bile acids, particularly CDCA, which exacerbated intestinal inflammation.


      This study reveals the RelA/Stat3-dependent gene program in the liver influences intestinal homeostasis.


      Additional evidence will strengthen the conclusion.

      1. In Fig. 1C, photos show that phosphorylation of RelA and Stat3 was induced in only a few hepatocytes. The authors conclude that activation of both RelA and Stat3 induces inflammatory pathways. Therefore, the authors should show that phosphorylation of RelA and Stat3 is induced in the same hepatocytes during DSS treatment.

      2. In Fig. 5, the authors treated mice with CDCA intraperitoneally. In this experiment, the concentration of CDCA in the colon of CDCA-treated mice should be shown.

    1. Reviewer #1 (Public Review):


      Ravichandran et al investigate the regulatory panels that determine the polarization state of macrophages. They identify regulatory factors involved in M1 and M2 polarization states by using their network analysis pipeline. They demonstrate that a set of three regulatory factors (RFs) i.e., CEBPB, NFE2L2, and BCL3 can change macrophage polarization from the M1 state to the M2 state. They also show that siRNA-mediated knockdown of those 3-RF in THP1-derived M0 cells, in the presence of M1 stimulant increases the expression of M2 markers and showed decreased bactericidal effect. This study provides an elegant computational framework to explore the macrophage heterogeneity upon different external stimuli and adds an interesting approach to understanding the dynamics of macrophage phenotypes after pathogen challenge.


      This study identified new regulatory factors involved in M1 to M2 macrophage polarization. The authors used their own network analysis pipeline to analyze the available datasets. The authors showed 13 different clusters of macrophages that encounter different external stimuli, which is interesting and could be translationally relevant as in physiological conditions after pathogen challenge, the body shows dynamic changes in different cytokines/chemokines that could lead to different polarization states of macrophages. The authors validated their primary computational findings with in vitro assays by knocking down the three regulatory factors-NCB.


      One weakness of the paper is the insufficient analysis performed on all the clusters. They used macrophages treated with 28 distinct stimuli, which included a very interesting combination of pro- and anti-inflammatory cytokines/factors that can be very important in the context of in vivo pathogen challenge, but they did not characterize the full spectrum of clusters. Although they mentioned that their identified regulatory panels could determine the precise polarization state, they restricted their analysis to only the two well-established macrophage polarization states, M1 and M2. Analyzing the other states beyond M1 and M2 could substantially advance the field. They mentioned the regulatory factors involved in individual clusters but did not study the potential pathway involving the target genes of these regulatory factors, which can show the importance of different macrophage polarization states. Importantly, these findings were not validated in primary cells or using in vivo models.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this paper, the authors provide a characterisation of auditory responses (tones, noise, and amplitude-modulated sounds) and bimodal (somatosensory-auditory) responses and interactions in the higher-order lateral cortex (LC) of the inferior colliculus (IC) and compare these characteristics with the higher order dorsal cortex (DC) of the IC - in awake and anaesthetised mice. Dan Llano's group has previously identified gaba'ergic patches (modules) in the LC distinctly receiving inputs from somatosensory structures, surrounded by matrix regions receiving inputs from the auditory cortex. They here use 2P calcium imaging combined with an implanted prism to - for the first time - get functional optical access to these subregions (modules and matrix) in the lateral cortex of IC in vivo, in order to also characterise the functional difference in these subparts of LC. They find that both DC and LC of both awake and anaesthetised mice appear to be more responsive to more complex sounds (amplitude-modulated noise) compared to pure tones and that under anesthesia the matrix of LC is more modulated by specific frequency and temporal content compared to the gabaergic modules in LC. However, while both LC and DC appear to have low-frequency preferences, this preference for low frequencies is more pronounced in DC. Furthermore, in both awake and anesthetized mice, somatosensory inputs are capable of driving responses on their own in the modules of LC, but very little (possibly not at all) in the matrix. However, bimodal interactions may be different under awake and anesthesia in LC, which warrants deeper investigation by the authors: They find, under anesthesia, more bimodal enhancement in modules of LC compared to the matrix of LC and bimodal suppression dominating the matrix of LC. In contrast, under awake conditions bimodal enhancement is almost exclusively found in the matrix of LC, and bimodal suppression dominates both matrix and modules of LC.

      The paper provides new information about how subregions with different inputs and neurochemical profiles in the higher-order auditory midbrain process auditory and multisensory information, and is useful for the auditory and multisensory circuits neuroscience community.

      Strengths:<br /> The major strength of this study is undoubtedly the fact that the authors for the first time provide optical access to a subcortical region (the lateral cortex of the inferior colliculus (i.e. higher order auditory midbrain)) which we know (from previous work by the same group) have optically identifiable subdivisions with unique inputs and neurotransmitter release, and plays a central role in auditory and multisensory processing. A description of basic auditory and multisensory properties of this structure is therefore very useful for understanding auditory processing and multisensory interactions in subcortical circuits.

      Weaknesses:<br /> I have divided my comments about weaknesses and improvements into major and minor comments. All of which I believe are addressable by the reviewers to provide a more clear picture of their characterisation of the higher-order auditory midbrain.

      Major comment:<br /> 1. The differences between multisensory interactions in LC in anaesthetised and awake preparations appear to be qualitatively different, though the authors claim they are similar (see also minor comment related to figure 10H for further explanation of what I mean). However, the findings in awake and anaesthetised conditions are summarised differently, and plotting of similar findings in the awake figures and anaesthetised figures are different - and different statistics are used for the same comparisons. This makes it very difficult to assess how multisensory integration in LC is different under awake and anaesthetised conditions. I suggest that the authors plot (and test with similar statistics) the summary plots in Figure 8 (i.e. Figure 8H-K) for awake data in Figure 10, and also make similar plots to Figures 10G-H for anaesthetised data. This will help the readers understand the differences between bimodal stimulation effects on awake and anaesthetised preparations - which in its current form, looks very distinct. In general, it is unclear to me why the awake data related to Figures 9 and 10 is presented in a different way for similar comparisons. Please streamline the presentation of results for anaesthetised and awake results to aid the comparison of results in different states, and explicitly state and discuss differences under awake and anaesthetised conditions.

      2. The claim about the degree of tonotopy in LC and DC should be aided by summary statistics to understand the degree to which tonotopy is actually present. For example, the authors could demonstrate that it is not possible/or is possible to predict above chance a cell's BF based on the group of other cells in the area. This will help understand to what degree the tonotopy is topographic vs salt and pepper. Also, it would be good to know if the gaba'ergic modules have a higher propensity of particular BFs or tonotopic structure compared to matrix regions in LC, and also if general tuning properties (e.g. tuning width) are different from the matrix cells and the ones in DC.

      3. Throughout the paper more information needs to be given about the number of cells, sessions, and animals used in each panel, and what level was used as n in the statistical tests. For example, in Figure 4 I can't tell if the 4 mice shown for LC imaging are the only 4 mice imaged, and used in the Figure 4E summary or if these are just examples. In general, throughout the paper, it is currently not possible to assess how many cells, sessions, and animals the data shown comes from.

      4. Throughout the paper, to better understand the summary maps and plots, it would be helpful to see example responses of the different components investigated. For example, given that module cells appear to have more auditory offset responses, it would be helpful to see what the bimodal, sound-only, and somatosensory responses look like in example cells in LC modules. This also goes for just general examples of what the responses to auditory and somatosensory inputs look like in DC vs LC. In general example plots of what the responses actually look like are needed to better understand what is being summarised.

    1. Joint Public Review:

      This study sought to characterize the influence of acute stress on prosocial behavior, combining an effort-based task with neuroimaging, neuroendocrinological measures, and computational cognitive modeling. Two major results are reported: 1) Compared to controls, participants who experienced acute stress were less willing to exert effort for others, with more prominent effects for those who were more selfish; 2) More stressed participants exhibited an increase in activation in the dorsal anterior cingulate cortex and anterior insula, which are implicated in self-benefiting behavior. The approach is sophisticated and the findings are informative. Concerns regarding potential confounds and data reporting were addressed in a revised submission.

    1. Reviewer #1 (Public Review):

      This paper studies the effects of tACS on detection of silence gaps in an FM modulated noise stimulus. Both FM modulation of the sound and the tACS are at 2Hz, and the phase of the two is varied to determine possible interactions between the auditory and electric stimulation. Additionally, two different electrode montages are used to determine if variation in electric field distribution across the brain may be related to the effects of tACS on behavioral performance in individual subjects.

      Major strengths and weaknesses of the methods and results.

      The study appears to be well powered to detect modulation of behavioral performance with N=42 subjects. There is a clear and reproducible modulation of behavioral effects with the phase of the FM sound modulation. The study was also well designed and executed in terms of fMRI, current flow modeling, montage optimization targeting, and behavioral analysis. A particular merit of this study is to have repeated the sessions for most subjects in order to test repeat-reliability, which is so often missing in human experiments. The results and methods are generally well described and well conceived. The portion of the analysis related to behavior alone is excellent. The analysis of the tACS results are also generally well described, candidly highlighting how variable results are across subjects and sessions. The figures are all of high quality and clear. One weakness of the experimental design is that no effort was made to control for sensation effects. tACS at 2Hz causes prominent skin sensations which could have interacted with auditory perception and thus, detection performance.

      The central claim is that tACS modulates behavioral detection performance across the 0.5s  cycle of stimulation. Statistical analysis with randomize relative phase (between audio and tACS) show that detection performance is modulated by tACS. Neither the relative phase or the strength of this effect reproduces across subjects or sessions, which makes the interpretation of these results difficult. These result could be of interest to investigators in the field of tACS.

      The claim that the variation in the strength of the effect can be explained by variation of electric fields is not compelling.

      The following are more detailed comments to specific sections of the paper, including details on the concerns with the statistical analysis of the tACS effects.<br /> The introduction is well balanced, discussing the promise and limitations of previous results with tACS. The objectives are well defined.

      The analysis surrounding behavioral performance and its dependence on phase of the FM modulation (Figure 3) is masterfully executed and explained. It appears that it reproduces previous studies and points to a very robust behavioral task that may be of use in other studies.

      The definition of tACS(+) vs tACS(-) phase is adjusted to each subject/session, which seems unconventional.  For argument sake, let's assume the curves in Fig. 3E are random fluctuations. Then aligning them to best-fitting cosine will trivially generate a FM-amplitude fluctuation with cosine shape as shown in Fig. 4a. Selecting the positive and negative phase of that will trivially be larger and smaller than sham, respectively, as shown in Fig 4b.

      "Data from the optimal tACS lag and its opposite lag (corresponding trough) were excluded to avoid any artificial bias in estimating tACS effects induced by the alignment procedure (33)." The delay was found by fitting a cosine, so removing just the peaks of that cosine does little to avoid this problem.

      To demonstrate that this is not a trivial result of the definition, the analysis compares this to the same analysis but with a randomize alignment to the two stimuli (audio and tACS) in Figure 4d. Assuming this shuffle was done correctly, this shows that the modulation observed in 4b is not just a result of the analysis procedure.

      The authors are to be commended for analyzing the robustness of their observation across subjects and across sessions in Fig. 5. The lack of consistency in the optimal time delay between the two stimuli is hard to reconcile with the common theory that tACS entrains brain function.

      "To better understand what factors might be influencing inter-session variability in tACS effects, we estimated multiple linear models ..." "Inter-individual variability in the simulated E-field predicts tACS effects" Authors here are attempting to predict a property of the subjects that was just shown to not be a reliable property of the subject. Authors are picking 9 possible features for this, testing 33 possible models with N=34 data points. With these circumstances it is not hard to find something that correlates by chance. And some of the models tested had interaction terms, possibly further increasing the number of comparisons. In the absence of multiple comparison correction, what is happening here is that multiple models are fit to the data, and a statistical test is performed for the best model on the same (training) data. The corresponding claim that variations are explained by variations in electric field is not persuasive.

      "Can we reduce inter-individual variability in tACS effects ..." This section seems even more speculative and with mixed results.

      Given the concerns with the statistical analysis above, there are concerns about the following statements in the summary of the Discussion:

      "4) individual variability in tACS effect size was partially explained by two interactions: between the normal component of the E-field and the field focality, and between the normal component of the E-field and the distance between the peak of the electric field and the functional target ROIs."

      The complexity of this statement alone may be a good indication that this could be the result of false discovery due to multiple comparisons.

      For the same reason as stated above, the following statements in the Abstract do not appear to have adequate support in the data:

      "Inter-individual variability of tACS effects was best explained by the strength of the inward electric field, depending on the field focality and proximity to the target brain region. Although additional evidence is necessary, our results<br /> 42 also provided suggestive insights that spatially optimizing the electrode montage could be a promising tool to reduce inter-individual variability of tACS effects."

    1. Joint Public Review:

      This manuscript tackles an important question, namely how K+ affects substrate transport in the SLC6 family. K+ effects have previously been reported for DAT and SERT, but the prototypical SLC6-fold transporter LeuT was not known to be sensitive to the K+ concentration. In this manuscript, the authors demonstrate convincingly that K+ inhibits Na+ binding, and Na+-dependent amino acid binding at high concentrations, and that K+ inside of vesicles containing LeuT increases the transport rate. However, outside K+ apparently had very little effect. Uptake data are supplemented with binding data, using the scintillation proximity assay, and transition metal FRET, allowing the observation of the distribution of distinct conformational states of the transporter.

      Overall, the data are of high quality. I was initially concerned about the use of solutions of very high ionic strength (the Km for K+ is in the 200 mM range), however, the authors performed good controls with lower ionic strength solutions, suggesting that the K+ effect are specific and not caused by artifacts from the high salt concentrations.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this study, Nandy and colleagues examine neural and behavioral correlates of perceptual variability in monkeys performing a visual change detection task. They used a laminar probe to record from area V4 while two macaque monkeys detected a small change in stimulus orientation that occurred at a random time in one of two locations, focusing their analysis on stimulus conditions where the animal was equally likely to detect (hit) or not-detect (miss) a briefly presented orientation change (target). They discovered two behavioral measures that are significantly different between hit and miss trials - pupil size tends to be slightly larger on hits vs. misses, and monkeys are more likely to miss the target on trials in which they made a microsaccade shortly before target onset. They also examined multiple measures of neural activity across the cortical layers and found some measures that are significantly different between hits and misses.

      Strengths:<br /> Overall the study is well executed and the analyses are appropriate (though multiple issues do need to be addressed).

      Weaknesses:<br /> My main concern with this study is that with the exception of the pre-target microsaccades, the physiological and behavioral correlates of perceptual variability (differences between hits and misses) appear to be very weak and disconnected. Some of these measures rely on complex analyses that are not hypothesis-driven and where statistical significance is difficult to assess. The more intuitive analysis of the predictive power of trial outcomes based on the behavioral and neural measures is only discussed at the end of the paper. This analysis shows that some of the significant measures have no predictive power, while others cannot be examined using the predictive power analysis because these measures cannot be estimated in single trials. Given these weak and disconnected effects, my overall sense is that the current results do not significantly advance our understanding of the neural basis of perceptual variability.

    1. Reviewer #1 (Public Review):

      Zhu, et al present a genome-wide histone modification analysis comparing patients with schizophrenia (on or off antipsychotics) to non-psychiatric controls. The authors performed analyses across the dorsolateral prefrontal cortex and tested for enrichment of nearby genes and pathways. The authors performed an analysis measuring the effect of age on the epigenomic landscape as well. While this paper provides a unique resource around SCZ and its epigenetic correlates, and some potentially intriguing findings in the antipsychotic response dataset there were some potential missed opportunities - related to the integration of outside datasets and genotypes that could have strengthened the results and novelty of the paper.

      Major Comments

      1. Is there genotype data available for this cohort of donors or can it be generated? This would open several novel avenues of investigation for the authors. First the authors can test for enrichment of heritability for SCZ or even highly comorbid disorders such as bipolar. Second, it would allow the authors to directly measure the genetic regulation of histone markers by calculating QTLs (in this case histone hQTLs). The authors assert that although interesting, ATAC-seq approach does not provide the same chromatin state information as histone mods mapped by ChiP. Why do the authors not test this? There are several ATAC-seq datasets available for SCZ [https://pubmed.ncbi.nlm.nih.gov/30087329/]and an additional genomic overlap could help tease apart genetic regulation of the changes observed.

      2. Can the authors theorize why their analysis found significant effects for H3K27Ac for antipsychotic use when a recent epigenomic study of SCZ using a larger cohort of samples and including the same histone modifications did not [https://pubmed.ncbi.nlm.nih.gov/30038276/]? Given the lower n and lower number of cells in this group, it would be helpful if the authors could speculate on why they see this. Do the authors know if there is any overlap with the Girdhar study donors or if there are other phenotypic differences that could account for this?

      3. The reviewer is concerned about the low concordance between bulk nuclei RNA-seq and single-cell RNA-seq for SCZ (236 of 802 DEGs in NeuN+ and 63 of 1043 NEuN-). While it is not surprising for different cohorts to have different sets of DEGs these seem to be vastly different. Was there a particular cell type(s) that enriched for the authors' DEGs in the single-cell dataset? Do the authors know if any donors overlapped between these cohorts?

      4. Functional enrichment analyses: details are not provided by the authors and should be added. The authors need to consider a) providing a gene universe, ie only considering the sets of genes with nearby H3K4me3/ H3K27ac levels, to such pathway tools, and b) should take into account the fact that some genes have many more peaks with data. There are known biases in seemingly just using the best p-value per gene in other epigenetic analysis (ie. DNA methylation data) and software is available to run correct analyses: https://pubmed.ncbi.nlm.nih.gov/23732277.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The circuit mechanism underlying the formation of grid cell activity and the organization of grid cells in the medial entorhinal cortex (MEC) is still unclear. To understand the mechanism, the current study investigated synaptic interactions between stellate cells (SC) and PV+ interneurons (IN) in layer 2 of the MEC by combing optogenetic activations and paired patch-clamp recordings. The results convincingly demonstrated highly structured interactions between these neurons: specific and direct excitatory-inhibitory interactions existed at the scale of grid cell phase clusters, and indirect interactions occurred at the scale of grid modules.

      Strengths:<br /> Overall, the manuscript is very well written, the approaches used are clever, and the data were thoroughly analyzed. The study conveyed important information for understanding the circuit mechanism that shapes grid cell activity. It is important not only for the field of MEC and grid cells, but also for broader fields of continuous attractor networks and neural circuits.

      Weaknesses:<br /> (1) The study largely relies on the fact that ramp-like wide-field optogenetic stimulation and focal optogenetic activation both drove asynchronous action potentials in SCs, and therefore, if a pair of PV+ INs exhibited correlated activity, they should receive common inputs. However, it is unclear what criteria/thresholds were used to determine the level of activity asynchronization, and under these criteria, what percentage of cells actually showed synchronized or less asynchronized activity. A notable percentage of synchronized or less asynchronized SCs could complicate the results, i.e., PV+ INs with correlated activity could receive inputs from different SCs (different inputs), which had synchronized activity. More detailed information/statistics about the asynchronization of SC activity is necessary for interpreting the results.

      (2) The hypothesis about the "direct excitatory-inhibitory" synaptic interactions is made based on the GABAzine experiments in Figure 4. In the Figure 8 diagram, the direct interaction is illustrated between PV+ INs and SCs. However, the evidence supporting this "direct interaction" between these two cell types is missing. Is it possible that pyramidal cells are also involved in this interaction? Some pieces of evidence or discussions are necessary to further support the "direction interaction".

    1. Reviewer #1 (Public Review):

      The authors used mathematical models to explore the mechanism(s) underlying the process of polar tube extrusion and the transport of the sporoplasm and nucleus through this structure. They combined this with experimental observations of the structure of the tube during extrusion using serial block face EM providing 3 dimensional data on this process. They also examined the effect of hyperosmolar media on this process to evaluate which model fit the predicted observed behavior of the polar tube in these various media solutions. Overall, this work resulted in the authors arriving at a model of this process that fit the data (model 5, E-OE-PTPV-ExP). This model is consistent with other data in the literature and provides support for the concept that the polar tube functions by eversion (unfolding like a finger of a glove) and that the expanding polar vacuole is part of this process. Finally, the authors provide important new insights into the bucking of the spore wall (and possible cavitation) as providing force for the nucleus to be transported via the polar tube. This is an important observation that has not been in previous models of this process.

    1. Reviewer #1 (Public Review):

      In this study, the authors investigated the role of MAM and the Notch signalling pathway in the onset of the atrophic phenotype in both in vivo and in vitro models. The rationale used to obtain the data is one of the main strengths of the study. Already from the reading, the reasoning scheme used by the authors in setting up the study and evaluating the data obtained is clear. Using both cellular and mouse models in vivo consolidates the data obtained. The authors also methodologically described all the choices made in the supplementary section.

    1. Joint Public Review:

      The biogenesis of outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria is not fully understood, particularly client recognition and insertion by the conserved beta-assembly machinery (BAM), which is itself integrated in the outer membranes. So far, the last strand of an OMP, referred to as the beta-signal, has been known as a primary recognition motif by BAM. Here, authors have identified additional sequence motifs that are located in the upstream of the last strand.

      Here, authors carried out rigorous biochemical, biophysical, and genetic approaches to prove that the newly identified internal motifs are critical to the assembly of outer membrane proteins as well as to the interaction with the BAM complex. The identification of important regions on the substrates and Bam proteins during biogenesis is an important contribution that gives clues to the path substrates take en route to the membrane. Assessing the effect of the internal motifs in the assembly of model OMPs in the absence (in vitro) and presence (in vitro and in vivo) of BAM machinery aids a precise definition of the role of the motifs, solidifying the conclusions.

      The initial reviews raised several concerns:

      1. Strengthening the claim that the recognition of the internal signal by BAM is mediated by BamA and BamD via specific interactions.

      2. Justification of the rationale of the peptide inhibition assays as a primary tool to identify significant recognition motifs.

      3. More careful interpretation of the mutational effects on OMP assembly - that is, discerning the impairment of BAM-nascent polypeptide chain interaction from the impairment of intrinsic folding.

      4. Providing further clarification of the discrepancy between in vitro assay and in vivo assay regarding the effect of the mutation Y286A on OMP assembly.

      5. More elaboration on the rationale, interpretation, and representation of neutron refractory data.

      6. An explanation is lacking why the strain with BamD R197A does not display VCN sensitivity in contrast to the strain with BamD Y62A.

      Those concerns were well addressed in the revised manuscript in a rigorous manner.

      Overall, this study comprehensively addresses an important question in the field. The notion that additional signals assist in biogenesis is a novel concept that has been tested and verified at least for a subset of model OMPs in this study. The generalization of the conclusion awaits a further proof of the concept.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This manuscript examined the impact of prenatal alcohol exposure on genome-wide DNA methylation in the brain and liver, comparing ethanol-exposed mice to unexposed controls. They also investigated whether a high-methyl diet (HMD) could prevent the DNA methylation alterations caused by alcohol. Using bisulfite sequencing (n=4 per group), they identified 78 alcohol-associated differentially methylated regions (DMRs) in the brain and 759 DMRs in the liver, of which 85% and 84% were mitigated by the HMD group, respectively. The authors further validated 7 DMRs in humans using previously published data from a Canadian cohort of children with FASD.

      Overall, the findings from this study provide new insight into the impact of prenatal alcohol exposure, while also showing evidence for methyl-rich diets as an intervention to prevent the effects of alcohol on the epigenome. However, several methodological concerns limit the robustness of these results and should be addressed to further strengthen the conclusions of this study and its applicability to broader settings.

      Strengths:<br /> - The use of whole genome bisulfite sequencing allowed for the interrogation of the entire DNA methylome and DMR analysis, rather than a subset of CpGs.<br /> - The combination of data from animal models and humans allowed the authors to make stronger inferences regarding their findings.<br /> - The authors investigated a potential mechanism (high methyl diet) to buffer against the effects of prenatal alcohol exposure, which increases the relevance and applicability of this research.

      Weaknesses:<br /> - Mistakes and discontinuities in the reporting of results and methods made the manuscript difficult to follow. There was also some overuse of causal language and overinterpretation of differences.<br /> - The authors provide insufficient details to replicate their analyses, particularly for data quality control steps and statistical analyses.<br /> - The sample size was very small for the epigenetic analyses, which limits the robustness of the findings. This limitation is further emphasized by the cutoffs used to identify DMRs, which did not include multiple test corrections and used a delta cutoff that was not supported by the sequencing depth.<br /> - The authors do not account for potential confounders in their analyses, including birthweight, alcohol levels, and sex. This is a particular problem for the high-methyl diet analyses, in which the alcohol-exposed mice seemed to consume less alcohol than their non-diet counterparts.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In their manuscript, Massa and colleagues provide a map of the epigenetic landscape in podocytes and analyze the role of the transcription factor MafB in podocyte gene expression. They initially map the histone profile in adult podocytes of the mouse by assaying three different histone methylation marks, namely H3K4me3, H3K4me1, and H3K27me3 for active, primed, and repressed states. They then perform Wt1- and MafB-ChIP-Seq analysis to identify respective direct targets of those transcription factors. Subsequently, they employ an inducible MafB knockout model and show that homozygous knockout mice show proteinuria and FSGS, suggesting an important role for MafB in podocyte homeostasis. RNA-Seq analysis in mice two days after tamoxifen application identified direct and indirect MafB target genes. Finally, the authors turn to a constitutive MafB knockout model, carry out anti-H3K4me3 and anti-Wt1 ChIP experiments, and examine selected promoters. One main conclusion from this work is that MafB opens chromatin and thus facilitates the binding of other transcription factors like Wt1 to podocyte-specific genes.

      Strengths and weaknesses:<br /> The authors have performed an impressive number of experiments and generated very valuable data. They use state-of the-art technology and the data are presented well and are sound. This being said the manuscript contains significant novel data, but also experiments that are already available in some sort. The histone profile in adult mouse podocytes is novel and provides an interesting map of epigenetic marks in this particular cell type. It is maybe not too surprising that podocyte-differentiation genes have different chromatin accessibility than genes associated with general development. The Wt1-ChIP has been done before by several labs but is certainly an important control in this work. The MafB-ChIP is new. The inducible MafB knockout model including the identification of Tcf21 as a target gene has been published by others in 2020 (and is acknowledged by the authors). The experiments addressing the potential role of MafB in chromatin opening are new. I find that the data are certainly compatible with the model put forward by the authors, but they are not compelling.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The manuscript titled "Coevolution due to physical interactions is not a major driving force behind evolutionary rate covariation" by Little et al., explores the potential contribution of physical interaction between correlated evolutionary rates among gene pairs. The authors find that physical interaction is not the main driving of evolutionary rate covariation (ECR). This finding is similar to a previous report by Clark et al. (2012), Genome Research, wherein the authors stated that "direct physical interaction is not required to produce ERC." The previous study used 18 Saccharomycotina yeast species, whereas the present study used 332 Saccharomycotina yeast species and 11 outgroup taxa. As a result, the present study is better positioned to evaluate the interplay between physical interaction and ECR more robustly.

      Strengths & Weaknesses:<br /> Various analyses nicely support the authors' claims. Accordingly, I have only one significant comment and several minor comments that focus on wordsmithing - e.g., clarifying the interpretation of statistical results and requesting additional citations to support claims in the introduction.

    1. Reviewer #1 (Public Review):

      The study provides a complete comparative interactome analysis of α-arrestin in both humans and drosophila. The authors have presented interactomes of six humans and twelve Drosophila α-arrestins using affinity purification/mass spectrometry (AP/MS). The constructed interactomes helped to find α-arrestins binding partners through common protein motifs. The authors have used bioinformatic tools and experimental data in human cells to identify the roles of TXNIP and ARRDC5: TXNIP-HADC2 interaction and ARRDC5-V-type ATPase interaction. The study reveals the PPI network for α-arrestins and examines the functions of α-arrestins in both humans and Drosophila. The authors have carried out the necessary changes that were suggested.

      I would like to congratulate the authors and the corresponding authors of this manuscript for bringing together such an elaborate study on α-arrestin and conducting a comparative study in drosophila and humans.

    1. Reviewer #1 (Public Review):

      Zhou et al. have slightly expanded and improved their web tool from the previous submission, fixing some small issues and adding in additional sets of data from HMDP mice. Essentially, the authors have created a tool that facilitates the integrated analysis of omics datasets (particularly transcriptomics, but could be easily adapted to include proteomics) across tissues.

      The strength is that this is new; as far as I know, any other multi-tissue analysis software is relatively ad hoc and it is not easily supported by e.g. SRA/GEO, but rather you'd need to download the multiple datasets and DIY. The authors have now shown some statistically significant (albeit expected from literature) results created using their pipeline. Whether the method will be generally useful for the community depends on its further development and support, but of course whether a project is supported also depends on whether its first publication is accepted - somewhat of a Catch-22 for a reviewer. Right now, the results shown are a convincing proof-of-concept that would likely be of utility mostly to the hosting laboratory and their direct collaborators, but which, with continued development at a similar level of effort, could be more generally useful for the growing number of groups interested in cross-tissue analysis.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The CPC plays multiple essential roles in mitosis such as kinetochore-microtubule attachment regulation, kinetochore assembly, spindle assembly checkpoint activation, anaphase spindle stabilization, cytokinesis, and nuclear envelope formation, as it dynamically changes its mitotic localization: it is enriched at inner centromeres from prophase to metaphase but it is relocalized at the spindle midzone in anaphase. The business end of the CPC is Aurora B and its allosteric activation module IN-box, which is located at the C-terminal part of INCENP. In most well-studied eukaryotic species, Aurora B activity is locally controlled by the localization module of the CPC, Survivin, Borealin, and the N-terminal portion of INCENP. Survivin and Borealin, which bind the N terminus of INCENP, recognize histone residues that are specifically phosphorylated in mitosis, while anaphase spindle midzone localization is supported by the direct microtubule-binding capacity of the SAH (single alpha helix) domain of INCENP and other microtubule-binding proteins that specifically interact with INCENP during anaphase, which are under the regulation of CDK activity. One of these examples includes the kinesin-like protein MKLP2 in vertebrates.

      Trypanosoma is an evolutionarily interesting species to study mitosis since its kinetochore and centromere proteins do not show any similarity to other major branches of eukaryotes, while orthologs of Aurora B and INCENP have been identified. Combining molecular genetics, imaging, biochemistry, cross-linking IP-MS (IP-CLMS), and structural modeling, this manuscript reveals that two orphan kinesin-like proteins KIN-A and KIN-B act as localization modules of the CPC in Trypanosoma brucei. The IP-CLMS, AlphaFold2 structural predictions, and domain deletion analysis support the idea that (1) KIN-A and KIN-B form a heterodimer via their coiled-coil domain, (2) Two alpha helices of INCENP interact with the coiled-coil of the KIN-A-KIN-B heterodimer, (3) the conserved KIN-A C-terminal CD1 interacts with the heterodimeric KKT9-KKT11 complex, which is a submodule of the KKT7-KKT8 kinetochore complex unique to Trypanosoma, (4) KIN-A and KIN-B coiled-coil domains and the KKT7-KKT8 complex are required for CPC localization at the centromere, (5) CD1 and CD2 domains of KIN-A support its centromere localization. The authors further show that the ATPase activity of KIN-A is critical for spindle midzone enrichment of the CPC. The imaging data of the KIN-A rigor mutant suggest that dynamic KIN-A-microtubule interaction is required for metaphase alignment of the kinetochores and proliferation. Overall, the study reveals novel pathways of CPC localization regulation via KIN-A and KIN-B by multiple complementary approaches.

      Strengths:<br /> The major conclusion is collectively supported by multiple approaches, combining site-specific genome engineering, epistasis analysis of cellular localization, AlphaFold2 structure prediction of protein complexes, IP-CLMS, and biochemical reconstitution (the complex of KKT8, KKT9, KKT11, and KKT12).

      Weaknesses:<br /> - The predictions of direct interactions (e.g. INCENP with KIN-A/KIN-B, or KIN-A with KKT9-KKT11) have not yet been confirmed experimentally, e.g. by domain mutagenesis and interaction studies.

      - The criteria used to judge a failure of localization are not clearly explained (e.g., Figure 5F, G).

      - It remains to be shown that KIN-A has motor activity.

      - The authors imply that KIN-A, but not KIN-B, interacts with microtubules based on microtubule pelleting assay (Fig. S6), but the substantial insoluble fractions of 6HIS-KINA and 6HIS-KIN-B make it difficult to conclusively interpret the data. It is possible that these two proteins are not stable unless they form a heterodimer.

      - For broader context, some prior findings should be introduced, e.g. on the importance of the microtubule-binding capacity of the INCENP SAH domain and its regulation by mitotic phosphorylation (PMID 8408220, 26175154, 26166576, 28314740, 28314741, 21727193), since KIN-A and KIN-B may substitute for the function of the SAH domain.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this study, Faniyan and colleagues build on their recent finding that renal Glut2 knockout mice display normal fasting blood glucose levels despite massive glucosuria. Renal Glut2 knockout mice were found to exhibit increased endogenous glucose production along with decreased hepatic metabolites associated with glucose metabolism. Crh mRNA levels were higher in the hypothalamus while circulating ACTH and corticosterone were elevated in this model. While these mice were able to maintain normal fasting glucose levels, ablating afferent renal signals to the brain resulted in substantially lower blood glucose levels compared to wildtype mice. In addition, the higher CRH and higher corticosterone levels of the knockout mice were lost following this denervation. Finally, acute phase proteins were altered, plasma Gpx3 was lower, and major urinary protein MUP18 and its gene expression were higher in renal Glut2 knockout mice. Overall, the main conclusion that afferent signaling from the kidney is required for renal glut2 dependent increases in endogenous glucose production is well supported by these findings.

      Strengths:<br /> An important strength of the paper is the novelty of the identification of kidney-to-brain communication as being important for glucose homeostasis. Previous studies had focused on other functions of the kidney modulated by or modulating brain activity. This work is likely to promote interest in CNS pathways that respond to afferent renal signals and the response of the HPA axis to glucosuria. Additional strengths of this paper stem from the use of incisive techniques. Specifically, the authors use isotope-enabled measurement of endogenous glucose production by GC-MS/MS, capsaicin ablation of afferent renal nerves, and multifiber recording from the renal nerve. The authors also paid excellent attention to rigor in the design and performance of these studies. For example, they used appropriate surgical controls, confirmed denervation through renal pelvic CGRP measurement, and avoided the confounding effects of nerve regrowth over time. These factors strengthen confidence in their results. Finally, humans with glucose transporter mutations and those being treated with SGLT2 inhibitors show a compensatory increase in endogenous glucose production. Therefore, this study strengthens the case for using renal Glut2 knockout mice as a model for understanding the physiology of these patients.

      Weaknesses:<br /> A few weaknesses exist. Clarification of some aspects of the experimental design would improve the manuscript. However, most concerns relate to the interpretation of this study's findings. The authors state that loss of glucose in urine is sensed as a biological threat based on the HPA axis activation seen in this mouse model. This interpretation is understandable but speculative. Importantly, whether stress hormones mediate the increase in endogenous glucose production in this model and in humans with altered glucose transporter function remains to be demonstrated conclusively. For example, the paper found several other circulating and local factors that could be causal. In addition, the approach used in these studies cannot definitively determine whether renal glucose production or only hepatic glucose production was altered. This model is also unable to shed light on how elevated stress hormones might interact with insulin resistance, which is known to increase endogenous glucose production. That issue is of substantial clinical relevance for patients with T2D and metabolic disease. Finally, while findings from the Glut2 knockout mice are of scientific interest, it should be noted that the Glut2 receptor is critical to the function of pancreatic islets and as such is not a good candidate for pharmacological targeting.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In their study, Petersen et al. investigated the relationship between parameters of metabolic syndrome (MetS) and cortical thickness using partial least-squares correlation analysis (PLS) and performed subsequently a group comparison (sensitivity analysis). To do this, they utilized data from two large-scale population-based cohorts: the UK BioBank (UKB) and the Hamburg City Health Study (HCHS). They identified a latent variable that explained 77% of the shared variance, driven by several measures related to MetS, with obesity-related measures having the strongest contribution. Their results highlighted that higher cortical thickness in the orbitofrontal, lateral prefrontal, insular, anterior cingulate, and temporal areas is associated with lower MetS metric severity. Conversely, the opposite pattern was observed in the superior frontal, parietal, and occipital regions. A similar pattern was then observed in the sensitivity analysis when comparing two groups (MetS vs. matched controls) separately. They then mapped local cellular and network topological attributes to the observed cortical changes associated with MetS. This was achieved using cell-type-specific gene expressions from the Allen Human Brain Atlas and the group consensus functional and structural connectomes of the Human Connectome Project (HCP), respectively. This contextualization analysis allowed them to identify potential cellular contributions in these structures driven by endothelial cells, microglial cells, and excitatory neurons. It also indicated functional and structural interconnectedness of areas experiencing similar MetS effects.

      Strengths:<br /> The effects of metabolic syndrome on the brain are still incompletely understood, and such multi-scale analyses are important for the field. Despite the study's sole 'correlation-based' nature, it yields valuable results, including several scales of brain parameters (cortical thickness, cellular, and network-based). The results are robust and benefit from two 'large-scale' datasets, resulting in highly powered statistics.

      Weaknesses:<br /> However, some concerns arise regarding certain interpretations and claims made by the authors. In particular, it is not entirely convincing that the authors' results are relevant for studying insulin resistance as a clinical measure of MetS. This is due to the use of non-fasting glycemia as a metric, which the authors claim represents insulin resistance. While non-fasting blood glucose is a potential, albeit poor, indicator of insulin resistance, claiming a direct correlation between insulin resistance and cortical thickness does not seem entirely convincing. By doing so, the authors suggest that insulin resistance might have a weak contribution to cortical thickness abnormalities, with a rather low 'loading' of glycemia compared to the other MetS metrics, although this cannot be conclusively determined from these results.

    1. Reviewer #1 (Public Review):

      Smirnova et al. present a cryo-EM structure of a nucleosome-SIRT6 complex to understand how the histone deacetylase SIRT6 deacetylates the N-terminal tail of histone H3. The authors obtained the structure at sub-4 Å resolution and can visualize how interactions between the nucleosome and SIRT6 position SIRT6 to allow for H3 tail deacetylation. Through additional conformational analysis of their cryo-EM data, they reveal that SIRT6 positioning is flexible on the nucleosome surface, and this could accommodate the targeting of certain H3 tail residues. This work is significant as it represents the visualization of a histone deacetylase on its native nucleosomal target and reveals how substrate specificity is achieved. Importantly, it should be noted that recently two additional structures of the nucleosome-SIRT6 complex were already published. Therefore, Smirnova et al. confirm and complement these previous findings. Additionally, Smirnova et al. expand our understanding of the structural flexibility of SIRT6 on the nucleosome and clarify that SIRT6 also shows histone deacetylase activity on H3K27Ac.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This manuscript explores the impact of serotonin on olfactory coding in the antennal lobe of locusts and odor-evoked behavior. The authors use serotonin injections paired with an odor-evoked palp-opening response assay and bath application of serotonin with intracellular recordings of odor-evoked responses from projection neurons (PNs).

      Strengths:<br /> The authors make several interesting observations, including that serotonin enhances behavioral responses to appetitive odors in starved and fed animals, induces spontaneous bursting in PNs, and uniformly enhances PN responses to odors. Overall, I had no technical concerns.

      Weaknesses:<br /> While there are several interesting observations, the conclusions that serotonin enhanced sensitivity specifically and that serotonin had feeding-state-specific effects, were not supported by the evidence provided. Furthermore, there were other instances in which much more clarification was needed for me to follow the assumptions being made and inadequate statistical testing was reported.

      Major concerns.<br /> -To enhance olfactory sensitivity, the expected results would be that serotonin causes locusts to perceive each odor as being at a relatively higher concentration. The authors recapitulate a classic olfactory behavioral phenomenon where higher odor concentrations evoke weaker responses which is indicative of the odors becoming aversive. If serotonin enhanced the sensitivity to odors, then the dose-response curve should have shifted to the left, resulting in a more pronounced aversion to high odor concentrations. However, the authors show an increase in response magnitude across all odor concentrations. I don't think the authors can claim that serotonin enhances the behavioral sensitivity to odors because the locusts no longer show concentration-dependent aversion. Instead, I think the authors can claim that serotonin induces increased olfactory arousal.

      -The authors report that 5-HT causes PNs to change from tonic to bursting and conclude that this stems from a change in excitability. However, excitability tests (such as I/V plots) were not included, so it's difficult to disambiguate excitability changes from changes in synaptic input from other network components.

      -There is another explanation for the theoretical discrepancy between physiology and behavior, which is that odor coding is further processing in higher brain regions (ie. Other than the antennal lobe) not studied in the physiological component of this study. This should at least be discussed.

      -The authors cannot claim that serotonin underlies a hunger state-dependent modulation, only that serotonin impacts responses to appetitive odors. Serotonin enhanced PORs for starved and fed locusts, so the conclusion would be that serotonin enhances responses regardless of the hunger state. If the authors had antagonized 5-HT receptors and shown that feeding no longer impacts POR, then they could make the claim that serotonin underlies this effect. As it stands, these appear to be two independent phenomena.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Animals in natural environments need to identify predator-associated cues and respond with the appropriate behavioral response to survive. In rodents, some chemical cues produced by predators (e.g., cat saliva) are detected by chemosensory neurons in the vomeronasal organ (VNO). The VNO transmits predator-associated information to the accessory olfactory bulb, which in turn projects to the medial amygdala and the bed nucleus of the stria terminalis, two regions implicated in the initiation of antipredator defensive behaviors. A downstream area to these two regions is the ventromedial hypothalamus (VMH), which has been shown to control both active (i.e., flight) and passive (i.e, freezing) antipredator defensive responses via distinct efferent projections to the anterior hypothalamic nucleus or the periaqueductal gray, respectively. However, whether differences in predator-associated sensory information initially processed in the VNO and further conveyed to the VMH can trigger different types of behavioral responses remained unexplored. To address this question, here the authors investigated the behavioral responses of mice exposed to either fresh or old cat saliva, and further compared the underlying neural circuits that are activated by cat saliva with different freshness.

      The scientific question of the study is valid, the experiments were well-performed, and the statistical analyses are appropriate. However, there are some concerns that may directly affect the main interpretation of the results.

      Major Concerns:<br /> 1. An important point that the authors should clarify in this study is whether mice are detecting qualitative or quantitative differences between fresh and old cat saliva. Do the environmental conditions in which the old saliva was maintained cause degradation of Fel d 4, the main protein known for inducing a defensive response in rodents? (see Papes et al, 2010 again). If that is the case, one would expect that a lower concentration of Fel d 4 in the old saliva after protein degradation would result in reduced antipredator responses. Alternatively, if the authors believe that different proteins that are absent in the old saliva are contributing to the increased defensive responses observed with the fresh saliva, further protein quantification experiments should be performed. An important experiment to differentiate qualitative versus quantitative differences between the two types of saliva would be diluting the fresh saliva to verify if the amount of protein, rather than the type of protein, is the main factor regulating the behavioral differences.

      2. The authors claim that fresh saliva is recognized as an immediate danger by rodents, whereas old saliva is recognized as a trace of danger. However, the study lacks empirical tests to support this interpretation. With the current experimental tests, the behavioral differences between animals exposed to fresh vs. old saliva could be uniquely due to the reduced amount of the exact same protein (e.g., Fel d 4) in the two samples of saliva.

      3. In Figure 4H, the authors state that there were no significant differences in the number of cFos-positive cells between the two saliva-exposed groups. However, this result disagrees with the next result section showing that fresh and old saliva differentially activate the VMH. It is unclear why cFos quantification and behavioral correlations were not performed in other upstream areas that connect the VNO to the VMH (e.g., BNST, MeA, and PMCo). That would provide a better understanding of how brain activity correlates with the different types of behaviors reported with the fresh vs. old saliva.

      4. The interpretation that fresh and old saliva activates different subpopulations of neurons in the VMH based on the observation that cFos positively correlates with freezing responses only with the fresh saliva lacks empirical evidence. To address this question, the authors should use two neuronal activity markers to track the response of the same population of VHM cells within the same animals during exposure to fresh vs. old saliva. Alternatively, they could use single-cell electrophysiology or imaging tools to demonstrate that cat saliva of distinct freshness activates different subpopulations of cells in the VMH. Any interpretation without a direct within-subject comparison or the use of cell-type markers would become merely speculative. Furthermore, the authors assume that differential activations of mitral cells between fresh and old saliva result in the differential activation of VMH subpopulations (page 13, line 3). However, there are intermediate structures between the mitral cells and the VMH, which are completely ignored in this study (e.g., BNST, medial amygdala).

      5. The authors incorrectly cited the Papes et al., 2010 article on several occasions across the manuscript. In the introduction, the authors cited the Papes et al 2010 study to make reference to the response of rodents to chemical cues, but the Papes et al. study did not use any of the chemical cues listed by the authors (e.g., fox feces, snake skin, cat fur, and cat collars). Instead, the Papes et al. 2010 article used the same chemical cue as the present study: cat saliva. The Papes et al. 2010 article was miscited again in the results section where the authors cited the study to make reference to other sources of cat odor that differ from the cat saliva such as cat fur and cat collars. Because the Papes et al. 2010 article has previously shown the involvement of Trpc2 receptors in the VNO for the detection of cat saliva and the subsequent expression of defensive behaviors by using Trpc2-KO mice, the authors should properly cite this study in the introduction and across the manuscript when making reference to their findings.

      6. In the introduction, the authors hypothesized that the VNO detects predator cues and sends sensory signals to the VMH to trigger defensive behavioral decisions and stated that direct evidence to support this hypothesis is still missing. However, the evidence that cat saliva activates the VMH and that activity in the VMH is necessary for the expression of antipredator defensive response in rodents has been previously demonstrated in a study by Engelke et al., 2021 (PMID: 33947849), which was entirely omitted by the authors.

      7. In the discussion, the authors stated that their findings suggest that the induction of robust freezing behavior is mediated by a distinct subpopulation of VMH neurons. The authors should cite the study by Kennedy et al., 2020 (PMID: 32939094) that shows the involvement of VMH in the regulation of persistent internal states of fear, which may provide an alternative explanation for why distinct concentrations of saliva could result in different behavioral outcomes.

      8. The anatomical connectivity between the olfactory system and the ventromedial hypothalamus (VMH) in the abstract is unclear. The authors should clarify that the VMH does not receive direct inputs from the vomeronasal organ (VNO) nor the accessory olfactory bulb (AOB) as it seems in the current text.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this study, the authors investigated the mechanisms to repair DSBs induced in euchromatic (Eu) or heterochromatic (Het) contexts in Drosophila. They used a previously described reporter construct that can be used to differentiate between HR, SSA, and mutagenic end joining in response to an I-SceI-induced DSB. Different sub-pathways of end joining (NHEJ, MMEJ, and SD-MMEJ) could be further distinguished by DNA sequence analysis. The main findings of the study are: (1) HR repair is more frequent in Het than in the Eu context; (2) mutagenic EJ repair is more frequent than HR in both contexts; (3) sub-pathways of mutagenic EJ are variable even within the same chromatin domain; and (4) SD-MMEJ repair is associated with larger deletions in the Eu than within the Het compartment.

      Strengths:<br /> Overall, the study is well designed and the use of the Bam promoter to drive I-SceI removes some of the variability observed in previous studies. Importantly, the observation of different repair outcomes using the same reporter integrated at different genomic sites suggests that repair is influenced by chromatin state in addition to local DNA sequence context.

      Weaknesses:<br /> The main concern I have is the use of only one Eu site versus four for the Het insertions. Given the variability observed between the Het insertions, analysis of a second Eu insertion would give more confidence that the differences observed are significant. One puzzling finding is that HR is increased when the reporter is inserted within the Het domain relative to the Eu domain, suggesting more end resection, yet deletions are smaller for the Het insertions. Bright Ddc2/ATRIP focus formation at DSBs induced in the Het domain is consistent with extensive end resection in this compartment. The authors speculate that this finding could indicate differences in the density of RPA loading or recruitment of Pol theta near ends. I recognize that measuring RPA density on single-stranded DNA would be extremely challenging, but is it known if Pol theta is recruited to DSBs within the Het domain before they move to the periphery?

    1. Reviewer #1 (Public Review):

      The authors sought to resolve the coordinated functions of the two muscles that primarily power flight in birds (supracoracoideus and pectoralis), with particular focus on the pectoralis. Technology has limited the ability to resolve some details of pectoralis function, so the authors developed a model that can make accurate predictions about this muscle's function during flight. The authors first measured aerodynamic forces, wing shape changes, and pectoralis muscle activity in flying doves. They used cutting-edge techniques for the aerodynamic and wing shape measurements and they used well-established methods to measure activity and length of the pectoralis muscle. The authors then developed two mathematical models to estimate the instantaneous force vector produced by the pectoralis throughout the wing stroke. Finally, the authors applied their mathematical models to other-sized birds in order to compare muscle physiology across species.

      The strength of the methods is that they smoothly incorporate techniques from many complementary fields to generate a comprehensive model of pectoralis muscle function during flight. The high-speed structured-light technique for quantifying surface area during flight is novel and cutting-edge, as is the aerodynamic force platform used. These methods push the boundaries of what has historically been used to quantify their respective aspects of bird flight and their use here is exciting. The methods used for measuring muscle activation and length are standard in the field. Together, these provide both a strong conceptual foundation for the model and highlight its novelty. This model allows for estimations of muscle function that are not feasible to measure in live birds during flight at present. The weakness of this approach is that it relies heavily on a series of assumptions. While the research presented in this paper makes use of powerful methods from multiple fields, those methods each have assumptions inherent to them that simplify the biological system of study. This reduction in the complexity of phenomena allows specific measurements to be made. In joining the techniques of multiple fields to study greater complexity of the phenomenon of interest, the assumptions are all incorporated also. Furthermore, assumptions are inherent to mathematical modelling of biological phenomena. That being said, the authors acknowledge and justify their assumptions at each step and their model seems to be quite good at predicting muscle function.

      Indeed, the authors achieve their aims. They effectively integrate methods from multiple disciplines to explore the coordination and function of the pectoralis and supracoracoideus muscles during flight. The conclusions that the authors derive from their model address the intended research aim.

      The authors demonstrate the value of such interdisciplinary research, especially in studying complex behaviors that are difficult or infeasible to measure in living animals. Additionally, this work provides predictions for muscle function that can be tested empirically. These methods are certainly valuable for understanding flight, but also have implications for biologists studying movement and muscle function more generally.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This manuscript builds upon the authors' previous work on the cross-talk between transcription initiation and post-transcriptional events in yeast gene expression. These prior studies identified an mRNA 'imprinting' phenomenon linked to genes activated by the Rap1 transcription factor (TF), a surprising role for the Sfp1 TF in promoting RNA polymerase II (RNAPII) backtracking, and a role for the non-essential RNAPII subunits Rpb4/7 in the regulation of mRNA decay and translation. Here the authors aimed to extend these observations to provide a more coherent picture of the role of Sfp1 in transcription initiation and subsequent steps in gene expression. They provide evidence for (1) a physical interaction between Sfp1 and Rpb4, (2) Sfp1 binding and stabilization of mRNAs derived from genes whose promoters are bound by both Rap1 and Sfp1 and (3) an effect of Sfp1 on Rpb4 binding or conformation during transcription elongation.

      Strengths:<br /> This study provides evidence that a TF (yeast Sfp1), in addition to stimulating transcription initiation, can at some target genes interact with their mRNA transcripts and promote their stability. Sfp1 thus has a positive effect on two distinct regulatory steps. Furthermore, evidence is presented indicating that strong Sfp1 mRNA association requires both Rap1 and Sfp1 promoter binding and is increased at a sequence motif near the polyA track of many target mRNAs. Finally, they provide compelling evidence that Sfp1-bound mRNAs have higher levels of RNAPII backtracking and altered Rpb4 association or conformation compared to those not bound by Sfp1.

      Weaknesses:<br /> The Sfp1-Rpb4 association is supported only by a two-hybrid assay that is poorly described and lacks an important control. Furthermore, there is no evidence that this interaction is direct, nor are the interaction domains on either protein identified (or mutated to address function).

      The contention that Sfp1 nuclear export to the cytoplasm is transcription-dependent is not well supported by the experiments shown, which are not properly described in the text and are not accompanied by any primary data.<br /> The presence of Sfp1 in P-bodies is of unclear relevance and the authors do not ask whether Sfp1-bound mRNAs are also present in these condensates.

      Further analysis of Sfp1-bound mRNAs would be of interest, particularly to address the question of whether those from ribosomal protein genes and other growth-related genes that are known to display Sfp1 binding in their promoters are regulated (either stabilized or destabilized) by Sfp1.

      The authors need to discuss, and ideally address, the apparent paradox that their previous findings showed that Rap1 acts to destabilize its downstream transcripts, i.e. that it has the opposite effect of Sfp1 shown here.

      Finally, recent studies indicate that the drugs used here to measure mRNA stability induce a strong stress response accompanied by rapid and complex effects on transcription. Their relevance to mRNA stability in unstressed cells is questionable.

    1. Reviewer #1 (Public Review):

      Li et al. report here on the expression of a G-protein subunit Gng13 in ectopic tuft cells that develop after severe pulmonary injury in mice. By deleting this gene in ectopic tuft cells as they arise, the authors observed worsened lung injury and greater inflammation after influenza infection, as well as a decrease in the overall number of ectopic tuft cells. This was in stark contrast to the deletion of Trpm5, a cation channel generally thought to be required for all functional gustatory signaling in tuft cells, where no phenotype is observed. Strengths here include a thorough assessment of lung injury via a number of different techniques. Weaknesses are notable: confusingly, these findings are at odds with reports from other groups demonstrating no obvious phenotype upon influenza infection in mice lacking the transcription factor Pou2f3, which is essential for all tuft cell specification and development. The authors speculate that heterogeneity within nascent tuft cell populations, specifically the presence of pro- and anti-inflammatory tuft cells, may explain this difference, but they do not provide any data to support this idea.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this paper, Song, Shi, and Lin use an existing deep learning-based sequence model to derive a score for each haplotype within a genomic region, and then perform association tests between these scores and phenotypes of interest. The authors then perform some downstream analyses (fine-mapping, various enrichment analyses, and building polygenic scores) to ensure that these associations are meaningful. The authors find that their approach allows them to find additional associations, the associations have biologically interpretable enrichments in terms of tissues and pathways, and can slightly improve polygenic scores when combined with standard SNP-based PRS.

      Strengths:<br /> - I found the central idea of the paper to be conceptually straightforward and an appealing way to use the power of sequence models in an association testing framework.<br /> - The findings are largely biologically interpretable, and it seems like this could be a promising approach to boost power for some downstream applications.

      Weaknesses:<br /> - The methods used to generate polygenic scores were difficult to follow. In particular, a fully connected neural network with linear activations predicting a single output should be equivalent to linear regression (all intermediate layers of the network can be collapsed using matrix-multiplication, so the output is just the inner product of the input with some vector). Using the last hidden layer of such a network for downstream tasks should also be equivalent to projecting the input down to a lower dimensional space with some essentially randomly chosen projection. As such, I am surprised that the neural network approach performs so well, and it would be nice if the authors could compare it to other linear approaches (e.g., LASSO or ridge regression for prediction; PCA or an auto-encoder for converting the input to a lower dimensional representation).

      - A very interesting point of the paper was the low R^2 between the HFS scores in adjacent windows, but the explanation of this was unclear to me. Since the HFS scores are just deterministic functions of the SNPs, it feels like if the SNPs are in LD then the HFS scores should be and vice versa. It would be nice to compare the LD between adjacent windows to the average LD of pairs of SNPs from the two windows to see if this is driven by the fact that SNPs are being separated into windows, or if sei is somehow upweighting the importance of SNPs that are less linked to other SNPs (e.g., rare variants).

      - There were also a number of robustness checks that would have been good to include in the paper. For instance, do the findings change if the windows are shifted? Do the findings change if the sequence is reverse-complemented?

      - It was also difficult to contextualize the present work in terms of recent results showing that sequence models tend to not do very well at predicting cross-individual expression changes (and such results presumably hold for predicting cross-individual chromatin changes). In particular, it would be good for the authors to contrast their findings with the work of Alex Sasse and colleagues (https://www.biorxiv.org/content/10.1101/2023.03.16.532969.abstract) and Connie Huang and colleagues (https://www.biorxiv.org/content/10.1101/2023.06.30.547100.abstract).

    1. Reviewer #1 (Public Review):

      Summary:<br /> Shakhawat et al., investigated how enhancement of plasticity and impairment could result in the same behavioral phenotype. The authors tested the hypothesis that learning impairments result from saturation of plasticity mechanisms and had previously tested this hypothesis using mice lacking two class I major histocompatibility molecules. The current study extends this work by testing the saturation hypothesis in a Purkinje-cell (L7) specific Fmr1 knockout mouse mice, which have enhanced parallel fiber-Purkinje cell LTD. The authors found that L7-Fmr1 knockout mice are impaired on an oculomotor learning task and both pre-training, to reverse LTD, and diazepam, to suppress neural activity, eliminated the deficit when compared to controls.


      This study tests the "saturation hypothesis" to understand plasticity in learning using a well-known behavior task, VOR, and an additional genetic mouse line with a cerebellar cell-specific target, L7-Fmr1 KO. This hypothesis is of interest to the community as it evokes a novel inquisition into LTD that has not been examined previously.

      Utilizing a cell-specific mouse line that has been previously used as a genetic model to study Fragile X syndrome is a unique way to study the role of Purkinje cells and the Fmr1 gene. This increases the understanding in the field in regards to Fragile X syndrome and LTD.

      The VOR task is a classic behavior task that is well understood, therefore using this metric is very reliable for testing new animal models and treatment strategies. The effects of pretraining are clearly robust and this analysis technique could be applied across different behavior data sets.

      The rescue shown using diazepam is very interesting as this is a therapeutic that could be used in clinical populations as it is already approved.

      There was a proper use of controls and all animal information was described. The statistical analysis and figures are clear and well describe the results.

      Weaknesses:<br /> While the proposed hypothesis is tested using genetic animal models and the VOR task, LTD itself is not measured. This study would have benefited from a direct analysis of LTD in the cerebellar cortex in the proposed circuits.

      Diazepam was shown to rescue learning in L7-Fmr1 KO mice, but this drug is a benzodiazepine and can cause a physical dependence. While the concentrations used in this study were quite low and animals were dosed acutely, potential side-effects of the drug were not examined, including any possible withdrawal. This drug is not specific to Purkinje cells or cerebellar circuits, so the action of the drug on cerebellar circuitry is not well understood for the study presented.

      It was not mentioned if L7-Fmr1 KO mice have behavior impairments that worsen with age or if Purkinje cells and the cerebellar microcircuit are intact throughout the lifespan. Connections between Purkinje cells and interneurons could also influence the behavior results found.

      While males and females were both used for the current study, only 7 of each sex were analyzed, which could be underpowered. While it might be justified to combine sexes for this particular study, it would be worth understanding this model in more detail.

      Training was only shown up to 30 minutes and learning did not seem to plateau in most cases. What would happen if training continued beyond the 30 minutes? Would L7-Fmr1 KO mice catch-up to WT littermates?

      The pathway discussed as the main focus for VOR in this learning paradigm was connections between parallel fibers (PF) and Purkinje cells, but the possibility of other local or downstream circuitry being involved was not discussed. PF-Purkinje cell circuits were not directly analyzed, which makes this claim difficult to assess.

      The authors mostly achieved their aim and the results support their conclusion and proposed hypothesis. This work will be impactful on the field as it uses a new Purkinje-cell specific mouse model to study a classic cerebellar task. The use of diazepam could be further analyzed in other genetic models of neurodevelopmental disorders to understand if effects on LTD can rescue other pathways and behavior outcomes.

    1. Reviewer #1 (Public Review):


      Direction selectivity (DS) in the visual system is first observed in the radiating dendrites of starburst amacrine cells (SACs). Studies over the last two decades have aimed to understand the mechanisms that underlie these unique properties. Most recently, a 'space-time' model has garnered special attention. This model is based on two fundamental features of the circuit. First, distinct anatomical types of bipolar cells (BCs) are connected to proximal/distal regions of each of the SAC dendritic sectors (Kim et al., 2014). Second, that input across the length of the starburst is kinetically diverse, a hypothesis that has only recently gained some experimental support using iGluSnFR imaging (Srivastava et al., 2022). However, in these prior studies, the sustained/transient distinctions in BC input that are proposed to underlie direction selectivity were shown to be present mainly in responses to stationary stimuli. When BC receptive field properties are probed using white noise stimuli, the kinetic differences between proximal/distal BC input are relatively subtle or nonexistent (Gaynes et al., 2022; Strauss et al., 2022, Srivastava et al., 2022). Thus, if and how BCs contribute to direction selectivity driven by moving spots that are commonly used to probe the circuit remains to be clarified. To address this issue, Gaynes et al., combine evolutionary computational modeling (Ankri et al., 2020) with two-photon iGluSnFR imaging to address to what degree BCs contribute to the generation of direction selectivity in the starburst dendrites.


      Combining theoretical models and iGluSnFR imaging is a powerful approach as it first provides a basic intuition on what is required for the generation of robust DS, and then tests the extent to which the experimentally measured BC output meets these requirements.

      The conclusion of this study builds on the previous literature and comprehensively considers the diverse BC receptive field properties that may contribute to DS (e.g. size, lag, rise time, decay time).

      By 'evolving' bipolar inputs to produce robust DS in a model network, these authors provide a sound framework for understanding which kinetic properties could potentially be important for driving downstream DS. They suggest that response delay/decay kinetics, rather than the center/surround dynamics are likely to be most relevant (albeit the latter could generate asymmetric responses to radiating/looming stimuli).


      Finally, these authors report that the experimentally measured BC responses are far from optimal for generating DS. Thus, the BC-based DS mechanism does not appear to explain the robust DS observed experimentally (even with mutual inhibition blocked). Nevertheless, I feel the comprehensive description of BC kinetics and the solid assessment of the extent to which they may shape DS in SAC dendrites, is a significant advancement in the field.

    1. Reviewer #1 (Public Review):

      Summary: The current study examines the necessity of estrogen receptor alpha (ESR1) in GABA neurons located in the anteroventral and preoptic periventricular nuclei and the medial preoptic nucleus of hypothalamus. This brain area is implicated in regulating the pre-ovulatory LH surge in females, but the identity of the estrogen-sensitive neurons that are required remains unknown. The data indicate that approximately 70% knockdown of ESR1 in GABA neurons resulted in variable reproductive phenotypes. However, when the ESR1 knockdown also results in a decrease in kisspeptin expression by these cells, the females had disrupted LH surges, but no alterations in pulsatile LH release. These data support the hypothesis that kisspeptin cells in this region are critical for the pre-ovulatory LH surge in females.

      Strengths: The current study examined the efficacy of two guide RNAs to knockdown ESR1 in GABA neurons, resulting in an approximate 70% reduction in ESR1 in GABA neurons. The efficacy of this knockdown was confirmed in the brain via immunohistochemistry and the reproductive outcomes were analyzed several ways to account for differences in guide RNAs or the precise brain region with the ESR1 knockdown. The analysis was taken one step further by grouping mice based on kisspeptin expression following ESR1 knockdown and examining the reproductive phenotypes. Overall, the aims of the study were achieved, the methods were appropriate, and the data were analyzed extensively. This data supports the hypothesis that kisspeptin neurons in the anterior hypothalamus are critical for the preovulatory LH surge.

      Weaknesses: One minor weakness in this study is the conclusion that the two different guide RNAs didn't seem to have unique effects on GnRH cFos expression or the reproductive phenotypes. Though the data indicate a 60-70% knockdown for both gRNA2 and gRNA3, 3 of the 4 gRNA2 mice had no cFos expression in GnRH neurons during the time of the LH surge, whereas all mice receiving gRNA3 had at least some cFos/GnRH co-expression. In addition, when mice were re-categorized based on reduction (>75%) in kisspeptin expression, most of the mice in the unilateral or bilateral groups received gRNA2, whereas many of the mice that received gRNA3 were in the "normal" group with no disruption in kisspeptin expression. Whether these results occurred by chance or due to differences in the gRNAs remains unknown. Thus, additional experiments with increased sample sizes would be needed, even if the efficacy of the ESR1 knockdown was comparable, before concluding these 2 gRNAs don't have unique actions.

    1. Reviewer #1 (Public Review):

      Summary: The authors set out to clarify the molecular mechanism of endocytosis (re-uptake) of synaptic vesicle (SV) membrane in the presynaptic terminal following release. They have examined the role of presynaptic actin, and of the actin regulatory proteins diaphanous-related formins ( mDia1/3), and Rho and Rac GTPases in controlling the endocytosis. They successfully show that presynaptic membrane-associated actin is required for normal SV endocytosis in the presynaptic terminal and that the rate of endocytosis is increased by activation of mDia1/3. They show that RhoA activity and Rac1 activity act in a partially redundant and synergistic fashion together with mDia1/3 to regulate the rate of SV endocytosis. The work adds substantially to our understanding of the molecular mechanisms of SV endocytosis in the presynaptic terminal.

      Strengths: The authors use state-of-the-art optical recording of presynaptic endocytosis in primary hippocampal neurons, combined with well-executed genetic and pharmacological perturbations to document effects of alteration of actin polymerization on the rate of SV endocytosis. They show that removal of the short amino-terminal portion of mDia1 that associates with the membrane interrupts the association of mDia1 with membrane actin in the presynaptic terminal. They then use a wide variety of controlled perturbations, including genetic modification of the amount of mDia1/3 by knock-down and knockout, combined with inhibition of activity of RhoA and Rac1 by pharmacological agents, to document the quantitative importance of each agent and their synergistic relationship in regulation of endocytosis.<br /> The analysis is augmented by ultrastructural analyses that demonstrate the quantitative changes in numbers of synaptic vesicles and in uncoated membrane invaginations that are predicted by the optical recordings.<br /> The manuscript is well-written and the data are clearly explained. Statistical analysis of the data is strengthened by the very large number of data points analyzed for each experiment.

      Weaknesses: There are no major weaknesses. The optical images as first presented are small and it is recommended that the authors provide larger, higher-resolution images.

    1. Reviewer #1 (Public Review):

      Summary:<br /> O'Leary and colleagues present data identifying several procedures that alter discrimination between novel and familiar objects, including time, environmental enrichment, Rac-1, context reexposure, and brief reminders of the familiar object. This is complimented with an engram approach to quantify cells that are active during learning to examine how their activation is impacted following each of the above procedures at test. With this behavioral data, authors apply a modeling approach to understand the factors that contribute to good and poor object memory recall.

      Strengths:<br /> • Authors systematically test several factors that contribute to poor discrimination between novel and familiar objects. These results are extremely interesting and outline essential boundaries of incidental, nonaversive memory.<br /> • These results are further supported by engram-focused approaches to examine engram cells that are reactivated in states with poor and good object recognition recall.

      Weaknesses:<br /> • For the environmental enrichment, authors seem to suggest objects in the homecage are similar to (or reminiscent of) the familiar object. Thus, the effect of improved memory may not be related to enrichment per se as much as it may be related to the preservation of an object's memory through multiple retrievals, not the enriching experiences of the environment itself. This would be consistent with the brief retrieval figure. Authors should include a more thorough discussion of this.

      • Authors should justify the marginally increased number of engram cells in the non-enrichment group that did not show object discrimination at test, especially relative to other figures. More specific cell counting criteria may be helpful for this. For example, was the DG region counted for engram and cfos cells or only a subsection?

      • It is unclear why the authors chose a reactivation time point of 1hr prior to testing. While this may be outside of the effective time window for pharmacological interference with reconsolidation for most compounds, it is not necessarily outside of the structural and functional neuronal changes accompanied by reconsolidation-related manipulations.

      • Figure 5: Levels of exploration at test are inconsistent between manipulations. This is problematic, as context-only reexposures seem to increase exploration for objects overall in a manner that I'm unsure resembles 'forgetting'. Instead, cross-group comparisons would likely reveal increased exploration time for familiar and novel objects. While I understand 'forgetting' may be accompanied by greater exploration towards objects, this is inconsistent across and within the same figure. Further, this effect is within the period of time that rodents should show intact recognition. Instead, context-only exposures may form a competing (empty context) memory for the familiar object in that particular context.

      • I am concerned at the interpretation that a memory is 'forgotten' across figures, especially considering the brief reminder experiments. Typically, if a reminder session can trigger the original memory or there is rapid reacquisition, then this implies there is some savings for the original content of the memory. For instance, multiple context retrievals in the absence of an object reminder may be more consistent with procedures that create a distinct memory and subsequently recruit a distinct engram.

      • Authors state that spine density decreases over time. While that may be generally true, there is no evidence that mature mushroom spines are altered or that this is consistent across figures. Additionally, it's unclear if spine volume is consistently reduced in reactivated and non-reactivated engram cells across groups. This would provide evidence that there is a functionally distinct aspect of engram cells that is altered consistently in procedures resulting in poor recognition memory (e.g. increased spine density relative to spine density of non-reactivated engram cells and non-engram cells)

      • Authors should discuss how the enrichment-neurogenesis results here are compatible with other neurogenesis work that supports forgetting.

    1. Reviewer #1 (Public Review):

      This manuscript provides an important case study for in-depth research on the adaptability of vertebrates in deep-sea environments. Through analysis of the genomic data of the hadal snailfish, the authors found that this species may have entered and fully adapted to extreme environments only in the last few million years. Additionally, the study revealed the adaptive features of hadal snailfish in terms of perceptions, circadian rhythms and metabolisms, and the role of ferritin in high-hydrostatic pressure adaptation. Besides, the reads mapping method used to identify events such as gene loss and duplication avoids false positives caused by genome assembly and annotation. This ensures the reliability of the results presented in this manuscript. Overall, these findings provide important clues for a better understanding of deep-sea ecosystems and vertebrate evolution.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This valuable study analyzes the contribution of fungal and bacterial microbiota species to the growth and development of Drosophila. The authors use bacterial and fungal species associated with Drosophila in the wild to analyze their respective contributions in promoting larval growth in a decaying banana, mimicking the natural niche of fruit fly. They found that some fungal species and some fungus/bacteria combinations effectively promote growth by supplementing key branched amino acids in the food substratum. Production of these amino acids by Drosophila itself is not sufficient, and only fungal species that secrete these amino acids into the medium can sustain Drosophila growth. Thus, the authors clarify how facultative symbionts contribute to host growth in a natural setting by changing the food substratum in a dynamic manner.

      Strengths:<br /> The natural setting developed by the authors to analyze the impact of the microbiota is clearly valuable, as is the focus on the role of fungal microbiota species. This complements studies of Drosophila microbiota that have previously focused on bacterial species and used a lab setting.

      While there has been an extensive focus on obligate endosymbionts or gut symbionts, this study analyzes how facultative symbionts shape the food substratum and influence host growth.<br /> A last strength of this study is that it analyzes the contribution of Drosophila microbiota over a dynamic timeframe, analyzing how microbial species change in decaying fruit over time.

      Weaknesses:<br /> 1) The author should better review what we know of fungal Drosophila microbiota species as well as the ecology of rotting fruit. Are the microbiota species described in this article specific to their location/setting? It would have been interesting to know if similar species can be retrieved in other locations using other decaying fruits. The term 'core' in the title suggests that these species are generally found associated with Drosophila but this is not demonstrated. The paper is written in a way that implies the microbiota members they have found are universal. What is the evidence for this? Have the fungal species described in this paper been found in other studies? Even if this is not the case, the paper is interesting, but there should be a discussion of how generalizable the findings are.

      2) Can the author clearly demonstrate that the microbiota species that develop in the banana trap are derived from flies? Are these species found in flies in the wild? Did the authors check that the flies belong to the D. melanogaster species and not to the sister group D. simulans?

      3) Did the microarrays highlight a change in immune genes (ex. antibacterial peptide genes)? Whatever the answer, this would be worth mentioning. The authors described their microarray data in terms of fed/starved in relation to the Finke article. This is fine they should clarify if they observed significant differences between species (differences between species within bacteria or fungi, and more generally differences between bacteria versus fungi).

      4) The whole paper - and this is one of its merits - points to a role of the Drosophila larval microbiota in processing the fly food. Are these bacterial and fungal species found in the gut of larvae/adults? Are these species capable of establishing a niche in the cardia of adults as shown recently in by the Ludington lab (Dodge et al.,)? Previous studies have suggested that microbiota members stimulate the Imd pathway leading to an increase in digestive proteases (Erkosar/Leulier). Are the microbiota species studied here affecting gut signaling pathways beyond providing branched amino acids?

    1. Reviewer #1 (Public Review):


      This study explores the relationship between guanine-quadruplex (G4) structures and pathogenicity islands (PAIs) in 89 pathogenic strains. G4 structures were found to be non-randomly distributed within PAIs and conserved within the same strains. Positive correlations were observed between G4s and GC content across various genomic features, suggesting a link between G4 structures and GC-rich regions. Differences in GC content between PAIs and the core genome underscored the unique nature of PAIs. High-confidence G4 structures in Escherichia coli's regulatory regions were identified, influencing DNA integration within PAIs. These findings shed light on the molecular mechanisms of G4-PAI interactions, enhancing our understanding of bacterial pathogenicity and G4 structures in infectious diseases.


      The findings of this study hold significant implications for our understanding of bacterial pathogenicity and the role of guanine-quadruplex (G4) structures.

      Molecular Mechanisms of Pathogenicity: The study highlights that G4 structures are not randomly distributed within pathogenicity islands (PAIs), suggesting a potential role in regulating pathogenicity. This insight into the uneven distribution of G4s within PAIs provides a basis for further research into the molecular mechanisms underlying bacterial pathogenicity.

      Conservation of G4 Structures: The consistent conservation of G4 structures within the same pathogenic strains suggests that these structures might play a vital and possibly conserved role in the pathogenicity of these bacteria. This finding opens doors for exploring how G4s influence virulence across different pathogens.

      Unique Nature of PAIs: The differences in GC content between PAIs and the core genome underscore the unique nature of PAIs. This distinction suggests that factors such as DNA topology and G4 structures might contribute to the specialized functions and characteristics of PAIs, which are often associated with virulence genes.

      Regulatory Role of G4s: The identification of high-confidence G4 structures within regulatory regions of Escherichia coli implies that these structures could influence the efficiency or specificity of DNA integration events within PAIs. This finding provides a potential mechanism by which G4s can impact the pathogenicity of bacteria.


      No weaknesses were identified by this reviewer.

      Overall, the study provides fundamental insights into the pathogenicity island and conservation of G4 motifs.

    1. Reviewer #1 (Public Review):

      In this study, the authors examined the role of IBTK, a substrate-binding adaptor of the CRL3 ubiquitin ligase complex, in modulating the activity of the eiF4F translation initiation complex. They find that IBTK mediates the non-degradative ubiquitination of eiF4A1, promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and tumor cell growth. Correspondingly, phosphorylation of  IBTK by mTORC1/ S6K1 increases eIF4A1 ubiquitination and sustains oncogenic translation.


      This study utilizes multiple biochemical, proteomic, functional, and cell biology assays to substantiate their results.  Importantly, the work nominates IBTK as a unique substrate of mTORC1, and further validates eiF4A1 ( a crucial subunit of the ei44F complex) as a promising therapeutic target in cancer. Since IBTK interacts broadly with multiple members of the translational initial complex - it will be interesting to examine its role in eiF2alpha-mediated ER stress as well as eiF3-mediated translation. Additionally, since IBTK exerts pro-survival effects in multiple cell types, it will be of relevance to characterize the role of IBTK in mediating increased mTORC1 mediated translation in other tumor types, thus potentially impacting their treatment with eiF4F inhibitors.


      The findings are mostly well supported by data, but some areas need clarification and could potentially be enhanced with further experiments:

      1) Since eiF4A1 appears to function downstream of IBTK1, can the effects of IBTK1 KO/KD in reducing puromycin incorporation (in Fig 3A),  cap-dependent luciferase reporter activity (Fig 3G), reduced oncogene expression ( Fig 4A) or 2D growth/ invasion assays (Fig 4) be overcome or bypassed by overexpressing eiF4A1? These could potentially be tested in future studies. 

      2) The decrease in nascent protein synthesis in puromycin incorporation assays in Figure 3A suggest that the effects of IBTK KO are comparable to and additive with silvesterol. It would be of interest to examine whether silvesterol decreases nascent protein synthesis or increases stress granules in the IBTK KO cells stably expressing IBTK as well. 

      3) The data presented in Figure 5 regarding the role of mTORC1 in IBTK-mediated eiF4A1 ubiquitination needs further clarification on several points:

      - It is not clear if the experiments in Figure 5F with Phos-tag gels are using the FLAG-IBTK deletion mutant or the peptide containing the mTOR sites as it is mentioned on line 517, page 19 "To do so, we generated an IBTK deletion mutant (900-1150 aa) spanning the potential mTORC1-regulated phosphorylation sites" This needs further clarification.

      -It may be of benefit to repeat the Phos tag experiments with full-length FLAG-IBTK and/or endogenous IBTK with molecular weight markers indicating the size of migrated bands.

      -Additionally, torin or Lambda phosphatase treatment may be used to confirm the specificity of the band in separate experiments.

      -Phos-tag gels with the IBTK CRISPR KO line would also help confirm that the non-phosphorylated band is indeed IBTK. 

      -It is unclear why the lower, phosphorylated bands seem to be increasing (rather than decreasing) with AA starvation/ Rapa in Fig 5H.

    1. Reviewer #1 (Public Review):

      The manuscript investigates the binding of PHD-BD, a tandem of reader domains in the C-terminus of BPTF, to modified histone tail peptides and nucleosomes. It focuses on the differences in binding affinity between peptide and nucleosome substrates for BPTF PHD-BD. Using the dCypher approach, they find that multi-modified peptide substrates (both acetylation and methylation) do not increase PHD-BD binding affinity. They argue that histone peptide substrates do not support the histone code model, which champions that multivalent engagement by PHD-BD with a multi-modified substrate would lead to stronger binding when compared to the engagement of each domain alone. In contrast, when using nucleosome substrates, even though the overall affinity is reduced, the affinity for H3K4me3triac (double modification) is tighter than either modification on its own. This is consistent with the histone code model.

      A strength of the manuscript is that it further delineates the contribution of each domain by again using dCypher to compare peptide and nucleosome binding of the PHD and BD domains alone, as well as tandem domain constructs where each domain has been inactivated by a point mutation (W2891A for the PHD and N3007A for the BD). PHD alone had a lower affinity for nucleosomes than peptides overall. With peptide substrates, PHD had the highest affinity for H3K4me3 and reduced affinity for H3K4me3triac; while with nucleosomes this trend was reversed. BD alone showed an affinity for acetylated H3 and H4 peptides but surprisingly was unable to bind nucleosomes. PHD requires the combination of H3K4 methylation and H3 tail acetylation for binding, and when partnered with BD, which is not able to bind nucleosomes alone, interestingly confers specificity for K14ac and K18ac. The in vivo relevance is argued using CUT&RUN analysis.

      NMR spectroscopy is further used to show that PHD-BD binds acetylated H3 in a multivalent manner while forming a unique complex with H3K4me3triac. Deleting the N-terminal A1 region of H3 abolishes the binding of PHD-BD, implying its importance for recognition. The authors also discuss a "fuzzy complex" that forms between H3 and DNA, as well as H4 and DNA, which explains the occlusion of histone tail accessibility in the nucleosome. By changing the sidechain charge, such as with PTMs, this interaction can be weakened and allow PHD in this case to bind to the modified H3 tail. Comparisons between spectra of the H4 tail, H4 tail with DNA, and the H4 tail in the nucleosome are made and used to argue for H4-DNA interactions in the nucleosome.

      The conclusions of the manuscript are very well-supported by the data and reveal a lot of insight into how the two reader domains of BPTF interact with modified nucleosomes. In many places, however, the manuscript is written more generally as if the conclusions apply in all cases (e.g. the title, abstract, and introduction) and this remains to be determined. It is also overstated that there is a belief that peptides perfectly recapitulate nucleosomes. It should also be pointed out that the nucleosomes are multi-valent and the data cannot discriminate binding of a single PHD-BD to single or multiple tails, and that the work is limited as it is using a construct of BPTF and in fact, there is at least one other reader domain involved.

    1. Reviewer #1 (Public Review):

      This paper combines an array of techniques to study the role of cholecystokinin (CCK) in motor learning. Motor learning in a pellet reaching task is shown to depend on CCK, as both global and locally targeted CCK manipulations eliminate learning. This learning deficit is linked to reduced plasticity in the motor cortex, evidenced by both slice recordings and two-photon calcium imaging. Furthermore, CCK receptor agonists are shown to rescue motor cortex plasticity and learning in knockout mice. While the behavioral results are clear, the specific effects on learning are not directly tested, nor is the specificity pathway between rhinal CCK neurons and the motor cortex. In general, the results present interesting clues about the role of CCK in motor learning, though the specificity of the claims is not fully supported.

      Since all CCK manipulations were performed throughout learning, rather than after learning, it is not clear whether it is learning that is affected or if there is a more general motor deficit. Related to this point, Figure 1D appears to show a general reduction in reach distance in CCK-/- mice. A general motor deficit may be expected to produce decreased success on training day 1, which does not appear to be the case in Figure 1C and Figure 2B, but may be present to some degree in Figure 5B. Or, since the task is so difficult on day 1, a general motor deficit may not be observable. It is therefore inconclusive whether the behavioral effect is learning-specific.

      The paper implicates motor cortex-projecting CCK neurons in the rhinal cortex as being a key component in motor learning. However, the relative importance of this pathway in motor learning is not pinned down. The necessity of CCK in the motor cortex is tested by injecting CCK receptor antagonists into the contralateral motor cortex (Figure 2), though a control brain region is not tested (e.g. the ipsilateral motor cortex), so the specificity of the motor cortex is not demonstrated. The learning-related source of CCK in the motor cortex is also unclear, since even though it is demonstrated that CCK neurons in the rhinal cortex project to the motor cortex in Figure 4D, Figure 4C shows that there is also a high concentration of CCK neurons locally within the motor cortex. Likewise, the importance of the projection from the rhinal cortex to the motor cortex is not specifically tested, as rhinal CCK neurons targeted for inactivation in Figure 5 include all CCK cells rather than motor cortex-projecting cells specifically.

      CCK is suggested to play a role in producing reliable activity in the motor cortex through learning through two-photon imaging experiments. This is useful in demonstrating what looks like normal motor cortex activity in the presence of CCK receptor antagonist, indicating that the manipulations in Figure 2 are not merely shutting off the motor cortex. It is also notable that, as the paper points out, the activity appears less variable in the CCK manipulations (Figure 3G). However, this could be due to CCK manipulation mice having less-variable movements throughout training. The Hausdorff distance is used for quantification against this point in Figure 1E, though the use of the single largest distance between trajectories seems unlikely to give a robust measure of trajectory similarity, which is reinforced by the CCK-/- traces looking much less variable than WT traces in Figure 1D. The activity effects may therefore be expected from a general motor deficit if that deficit prevented the mice from normal exploratory movements and restricted the movement (and activity) to a consistently unsuccessful pattern.

      Finally, slice experiments are used to demonstrate the lack of LTP in the motor cortex following CCK knockout, which is rescued by CCK receptor agonists. This is a nice experiment with a clear result, though it is unclear why there are such striking short-term depression effects from high-frequency stimulation observed in Figure 6A that are not observed in Figure 1H. Also, relating to the specificity of the proposed rhinal-motor pathway, these experiments do not demonstrate the source of CCK in the motor cortex, which may for example originate locally.

    1. Reviewer #1 (Public Review):

      This is a review of the manuscript entitled "Pharmacologic hyperstabilisation of the HIV-1 capsid lattice induces capsid failure" by Faysal et al., in this manuscript the authors used an elegant single virion fluorescence assay based on TIRF to measure the stability of mature HIV cores. Virions were biotinylated and captured onto glass coverslips through specific Biotin-Avidin interactions. Immobilized virions were then introduced to the imaging buffer which contained the pore-forming protein DLY, and fluorescently labeled CypA. Mature virions were identified through the binding of CypA which had a red fluorescent tag allowing them to measure the dynamics of GFP trapped within the mature cores as well as the CypA bound outside the core. The authors show that the addition of LEN starting from about 50nM stabilized the mature cores even after cores have ruptured and released their internal GFP. Higher concentration of Len results in ultrastabilization of the cores and rapid rupture leading to the release of GFP at an earlier timepoint. A biochemical assembly assay was performed which showed uM quantities of Len synergized with IP6 to promote CA assembly. Purified mature virions were also treated with 700nM of Len and analyzed by CryoET, this analysis showed an increased representation of irregular cores within the Len-treated sample. Putting all of this together, the authors concluded that Len facilitates core rupture through hyperstabilization of HIV cores, as described in the title.

      While I have found this work technically well performed and well explained, I do not believe that the presented data supports the conclusions reached by the authors.

    1. Reviewer #1 (Public Review):<br /> <br /> This thorough study expands our understanding of BMP signaling, a conserved developmental pathway, involved in processes diverse such as body patterning and neurogenesis. The authors applied multiple, state-of-art strategies to the anthozoan Nematostella vectensis in order to first identify the direct BMP signaling targets - bound by the activated pSMAD1/5 protein - and then dissect the role of a novel pSMAD1/5 gradient modulator, zwim4-6. The list of target genes features multiple developmental regulators, many of which are bilaterally expressed, and which are notably shared between Drosophila and Xenopus. The analysis identified in particular zswim4-6 a novel nuclear modulator of the BMP pathway conserved also in vertebrates. A combination of both loss-of-function (injection of antisense morpholino oligonucleotide, CRISPR/Cas9 knockout, expression of dominant negative) and gain-of-function assays, and of transcriptome sequencing identified that zwim acts as a transcriptional repression of BMP signaling. Functional manipulation of zswim5 in zebrafish shows a conserved role in modulating BMP signaling in a vertebrate.<br /> The particular strength of the study lies in the careful and thorough analysis performed. This is solid developmental work, where one clear biological question is progressively dissected, with the most appropriate tools. The functional results are further validated by alternative approaches. Data is clearly presented and methods are detailed.

      I have a couple of comments.<br /> 1) I was intrigued - as the authors - by the fact that the ChiP-Seq did not identify any known BMP ligand bound by pSMAD1/5. Are these genes found in the published ChiP-Seq data of the other species used for the comparative analysis? One hypothesis could be that there is a change in the regulatory interactions and that the initial set-up of the gradient requires indeed a feedback loop, which is then turned off at later gastrula. In this case, immunoprecipitation at early gastrula, prior to the set-up of the pSMAD1/5 gradient, could reveal a different scenario. Alternately, the regulation could be indirect, for example, through RGM, an additional regulator of BMP signaling expressed on the side of lower BMP activity, which is among the targets of the ChiP-Seq. This aspect could be discussed. Additionally, even if this is perhaps outside the scope of this study, I think it would be informative to further assess the effect of ZSWIM manipulation on RGM (and vice versa).<br /> 2) I do not fully understand the rationale behind the choice of performing the comparative assays in zebrafish: as the conservation was initially identified in Xenopus, I would have expected the experiment to be performed in frog. Furthermore, reading the phylogeny (Figure 4A), it is not obvious to me why ZSWIM5 was chosen for the assay (over the other paralog ZSWIM6). Could the Authors comment on this experiment further?

    1. Reviewer #1 (Public Review):


      Through a series of psychophysical experiments, Merkel et al examined the process of feature-based resource allocation during parallel feature value tracking, where subjects are asked to simultaneously track changing but spatially inseparable color streams. They find that tracking precision is highly imbalanced between streams, and the tracking precision changes over time by alternating between streams at a rate of ~1Hz.


      The study addresses an intriguing research question that fills a gap in existing literature, and was carefully designed and well-executed, with a series of experiments and control experiments.


      1. My main concern is the null effect of precision estimation pattern between cued and un-cued trials. It is well established that relative to the un-cued stimuli, the cued stimuli obtain more attentional resource and this study claimed serial attentional resource allocation during parallel feature value tracking. However, all Experiments 3a-c did not find any difference in precision estimates between these two types of trials.<br /> 2. Results of Exp.1 in the main text were different from those in Figure.<br /> 3. It would be helpful to add more details for the assignation of response 1 and response 2 to target 1 and target 2, respectively, in all experiments.

    1. Reviewer #1 (Public Review):<br /> <br /> In several developmental systems, the core Planar Cell Polarity (PCP) pathway organises the dynamics of cellular behaviours underlying morphogenesis. During pupal stages, the Drosophila wing undergoes a complex morphogenetic process that results in the simultaneous elongation and narrowing of the wing blade along the proximal-distal and anterior-posterior axes, respectively. It was proposed that this dynamic process is driven by mechanical stress that results in cell deformations and cell rearrangements. However, prior work by Etournay et al. (eLife 2015) shows that mutants that reduce of mechanical stresses do not completely eliminate oriented cell rearrangements. Here, Piscitello-Gomez et al. use imaging techniques previously developed by them and others, combined with a computational analysis of a rheological model, to evaluate the role of the core-PCP pathway as a possible patterning cue that could orient cell rearrangements in this system. Surprisingly, the authors found that core-PCP mutants only affect an early retraction velocity upon laser ablation, but do not seem to drive overall morphogenesis in this system. Therefore, the original question of the work, namely, identifying the patterning cues that establish oriented cell rearrangements in this system, remains unanswered.

      The work exemplifies how the integration of mechanical perturbations, image analysis, and computational modelling could be used to investigate the contribution of a specific patterning cue in morphogenesis. While the conclusions of the manuscript are solid and the data support the conclusion that core-PCP pathway mutants do not display an altered cell dynamic or cell elongation phenotype relative to wild-type controls, one challenge of the approach is that the time-lapse imaging technique is done only in a handful of pupal wings. This does not permit to conclude whether subtle changes in cell elongation or cell rearrangements could account for observed changes in the shape of adult wings (that are more round in these mutants). Other patterning and polarity cues such as Fat-Daschous or Toll-like signalling are suggested by the authors, but their examination is left for future studies.

    1. Reviewer #1 (Public Review):

      Mohibi et al. utilized genetic approaches to determine the role of FDX1 in the regulation of development, oncogenesis, and metabolism. The strengths of the current study are the utilization of both in vivo and in vitro methods coupled to classical biochemical/molecular biology tools and lipidomic screening. The data provided is convincing demonstrating genetic loss of even one allele of FDX1 promotes premature death, increased incidence of adenocarcinoma, and dysregulated lipid metabolism. The authors provide further mechanistic evidence showing enhanced SREBP2 activation, which could potentially be underlying the altered lipid metabolism observed in their model. These findings are likely to provide a novel target for the amelioration to lipid metabolic disorders as the authors show genetic overexpression of FDX1 can reduce intracellular lipid accumulation.

  2. Dec 2023
    1. Reviewer #1 (Public Review):

      Weber et al. collect locus coeruleus (LC) tissue blocks from 5 neurotypical European men, dissect the dorsal pons around the LC, and prepare 2-3 tissue sections from each donor on a slide for 10X spatial transcriptomics. From three of these donors, they also prepared an additional section for 10x single nucleus sequencing. Overall, the results validate well-known marker genes for the LC (e.g. DBH, TH, SLC6A2), and generate a useful resource that lists genes that are enriched in LC neurons in humans, with either of these two techniques. A comparison with publicly available mouse and rat datasets identifies genes that show reliable LC enrichment across species. Their analyses also support recent rodent studies that have identified subgroups of interneurons in the region surrounding the LC, which show enrichment for different neuropeptides. In addition, the authors claim that some LC neurons co-express cholinergic markers and that a population of serotonin (5-HT) neurons is located within or near the LC. These last two claims must be taken with great caution, as several technological limitations restrict the interpretation of these results. Technical limitations currently limit the ability to integrate spatial and single-nucleus sequencing, yet the manuscript presents a valuable resource on the gene expression landscape of the human LC.

    1. Reviewer #1 (Public Review):

      The work by Ohigashi and colleagues addresses the developmental and lineage relationship of a newly characterized thymus epithelial cell (TEC) progenitor subset. The authors take advantage of an elegant and powerful set of experimental approaches to demonstrate that CCL21-expressing TECs appear early in thymus organogenesis and that these cells, which are centrally located, go on to give rise to medullary (m)TECs. What makes the findings intriguing is that these CCL21-expressing mTECs are a distinct subset, which do not express RANK or AIRE, and transcriptomic and lineage tracing approaches point to these cells as potential mTEC progenitor-like cells. Of note, using in vitro and in vivo precursor-product cell transfer experiments, the authors show that this subset has a developmental potential to give rise to AIRE+ self-antigen-displaying mTECs, revealing that CCL21-expressing mTECs can give rise to distinct mTEC subsets. This functional duality provides an attractive rationale for the necessary function of mTECs, which is to attract CCR7+ thymocytes that have just undergone positive selection in the thymus cortex to enter the medulla to undergo tolerance-induction against self-antigen-displaying mTECs. Overall, the work is well supported and offers new insights into the diverse functions of the medullary compartment, and how two distinct subsets of mTECs can achieve it.

    1. Reviewer #1 (Public Review):

      Summary: A description of a modern protocol for cervical screening that likely could be used in any country of the world, based on self-sampling, extended HPV genotypinng and AI-assisted visual inspection - which is probably the best available combination today.

      Strengths: Modern, optimised protocol, designed for global use. Innovative.

      Weaknesses: The protocol is not clear. I could not even find how many women were going to be enrolled, the timelines of the study, the statistical methods ("comparing" is not statistics) or the power calculations.<br /> Tables 2 and 3 are too schematic - surely the authors must have an approximate idea of what the actual numbers are behind the green, red and yellow colors.<br /> Figure 1 comparing screening and vaccination is somewhat misleading. They screen 20 birth cohorts but vaccinate only 5 birth cohorts. Furthermore, the theoretical gains of screening has not really been attained in any country in practise. Modelling can be a difficult task and the commentary does not provide any detail on how to evaluate what was done. It just seems unnecessary to attack vaccination as a motivation on why screening needs to be modernised.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The presented study focuses on the role of formin-like 2 (FMNL2) in oocyte meiosis. The authors assessed FMNL2 expression and localization in different meiotic stages and subsequently, by using siRNA, investigated the role of FMNL2 in spindle migration, polar body extrusion, and distribution of mitochondria and endoplasmic reticulum (ER) in mouse oocytes.

      Strengths:<br /> Novelty in assessing the role of formin-like 2 in oocyte meiosis.

      Weaknesses:<br /> Methods are not properly described.<br /> Overstating presented data.<br /> It is not clear what statistical tests were used.

      My main concern is that there are missing important details of how particular experiments and analyses were done. The material and methods section is not written in the way that presented experiments could be repeated - it is missing basic information (e.g., used mouse strain, timepoints of oocytes harvest for particular experiments, used culture media, image acquisition parameters, etc.). Some of the presented data are overstated and incorrectly interpreted. It is not clear to me how the analysis of ER and mitochondria distribution was done, which is an important part of the presented data interpretation. I'm also missing important information about the timing of particular stages of assessed oocytes because the localization of both ER and mitochondria differs at different stages of oocyte meiosis. The data interpretation needs to be justified by proper analysis based on valid parameters, as there is considerable variability in the ER and mitochondria structure and localization across oocytes based on their overall quality and stage.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Fukuhara, Maenaka, and colleagues report a crystal structure of the canine distemper virus (CDV) attachment hemagglutinin protein globular domain. The structure shows a dimeric organization of the viral protein and describes the detailed amino-acid side chain interactions between the two protomers. The authors also use their best judgement to comment on predicted sites for the two cellular receptors - Nectin-4 and SLAM - and thus speculate on the CDV host tropism. A complementary AFM study suggests a breathing movement at the hemagglutinin dimer interface.

      Strengths:<br /> The study of CDV and related Paramyxoviruses is significant for human/animal health and is very timely. The crystallographic data seem to be of good quality.

      Weaknesses:<br /> While the recent CDV hemagglutinin cryo-EM structure is mentioned, it is not compared to the present crystal structure, and thus the context of the present study is poorly justified. Additionally, the results of the AFM experiment are not unexpected. Indeed, other paramyxoviral RBP/G proteins also show movement at the protomer interface.

    1. Reviewer #1 (Public Review):

      In the manuscript, the authors tried to explore the molecular alterations of adipose tissue and skeletal muscle in PCOS by global proteomic and phosphorylation site analysis. In the study, the samples are valuable, while there are no repeats for MS and there are no functional studies for the indicted proteins, phosphorylation sites. The authors achieved their aims to some extent, but not enough.

    1. Reviewer #1 (Public Review):

      In this revised preprint the authors investigate whether a presumably allosteric P2RX7 activating compound that they previously discovered reduces fibrosis in a bleomycin mouse model. They chose this particular model as publicly available mRNA data indicate that the P2RX7 pathway is downregulated in idiopathic pulmonary fibrosis patients compared to control individuals. In their revised manuscript, the authors use three proxies of lung damage, Ashcroft score, collagen fibers, and CD140a+ cells, to assess lung damage following the administration of bleomycin. These metrics are significantly reduced on HEI3090 treatment. Additional data implicate specific immune cell infiltrates and cytokines, namely inflammatory macrophages and damped release of IL-17A, as potential mechanistic links between their compound and reduced fibrosis. Finally, the researchers transplant splenocytes from WT, NLRP3-KO, and IL-18-KO mice into animals lacking the P2RX7 receptor to specifically ascertain how the transplanted splenocytes, which are WT for P2RX7 receptor, respond to HEI3090 (a P2RX7 agonist). Based on these results, the authors conclude that HEI3090 enhanced IL-18 production through the P2RX7-NLRP3 inflammasome axis to dampen fibrosis.

      These findings could be interesting to the field, as there are conflicting results as to whether NLRP3 activation contributes to fibrosis and if so, at what stage(s) (e.g., acute damage phase versus progression). The revised manuscript is more convincing in that three orthogonal metrics for lung damage were quantified. However, major weaknesses of the study still include inconsistent and small effect sizes of HEI3090 treatment versus either batch effects from transplanted splenocytes or the effects of different genetic backgrounds. Moreover, the fundamental assumption that HEI3090 acts specifically and functionally through the P2RX7 pathway in this model cannot be directly tested, as the authors now provide results indicating that P2RX7 knockout mice do not establish lung fibrosis on bleomycin treatment.

      In order to provide clear evidence that HEI3090 functions through P2RX7, a different lung fibrosis model that does not require P2RX7 would be necessary. For example, in such a system the authors could demonstrate a lack of HEI3090-mediated therapeutic effect on P2RX7 knockout. Molecularly, additional evidence on specificity, such as thermal proteome profiling and direct biophysical binding experiments, would also enhance the authors' argument that the compound indeed binds P2RX7 directly and specifically. Since all small molecules have some degree of promiscuity, the absence of an additional P2RX7 modulator, or direct recombinant IL-18 administration (as suggested by another reviewer), is needed to orthogonally validate the functional importance of this pathway. Another way the authors could probe pathway specificity would involve co-administering α-IL-18 with HEI3090 in several key experiments (similar to Figure 4L).

    1. Reviewer #1 (Public Review):


      In this manuscript, the authors observed that miR-199b-5p is elevated in osteoarthritis (OA) patients. They also found that overexpression of miR-199b-5p induced OA-like pathological changes in normal mice and inhibiting miR-199b-5p alleviated symptoms in knee OA mice. They concluded that miR-199b-5p is not only a potential micro-target for knee OA but also provides a potential strategy for the future identification of new molecular drugs.


      The data are generated from both human patients and animal models.


      The data presented in this manuscript is not solid enough to support their conclusions. There are several questions that need to be addressed to improve the quality of this study.

      The following questions that need to be addressed to improve the quality of the study.

      1. Exosomes were characterized by electron microscopy and western blot analysis (for CD9, 264 CD63, and CD81). However, figure S1 only showed two sample WB results and there is no positive and negative control as well as the confused not clear WB figure.

      2. The sequencing of miRNAs in serum exosomes showed that 88 miRNAs were upregulated and 89 miRNAs were downregulated in KOA patients compared with the control group based on fold change > 1.5 and p < 0.05. Figure 2 legend did not clearly elucidate what those represent and why the authors chose those five miRNAs to further validate although they did mention it with several words in line 108 'based on the p-value and exosomal'.

      3. In Figure 3 legend and methods, the authors did not mention how they performed the cell viability assay. What cell had been used? How long were they treated and all the details? Other figure legends have the same problem without detailed information.

      4. The authors claimed that Gcnt2 and Fzd6 are two target genes of miR-199b-5p. However, there is no convincing evidence such as western blot to support their bioinformatics prediction.

      5. To verify the binding site on 3'UTR of two potential targets, the authors designed a mouse sequence for luciferase assay, but not sure if it is the same when using a human sequence.

  3. Nov 2023
    1. Reviewer #1 (Public Review):

      The Eph receptor tyrosine kinase family plays a critical function in multiple physiological and pathophysiological processes. Hence, understating the regulation of these receptors is a highly important question. Through extensive experiments in cell lines and cultured neurons Chang et.al show that the signaling hub protein, MYCBP2 positively regulates the overall stability of a specific member of the family, EPHB2, and by that the cellular response to ephrinBs.<br /> Overall, this work sheds light on the divergent in the regulatory mechanisms of the Eph receptors family. Although the physiological importance of this new regularly mechanism in mammals awaits to be discovered, the authors provide genetic evidence using C.elegans that it is evolutionarily conserved.

    1. Reviewer #1 (Public Review):

      The paper presents a novel approach to expand iPSC-derived pdx1+/nkx6.1+ pancreas progenitors, making them potentially suitable for GMP-compatible protocols. This advancement represents a significant breakthrough for diabetes cell replacement therapies, as one of the current bottlenecks is the inability of expanding PP without compromising their differentiation potential. The study employs a robust dataset and state-of-the-art methodology, unveiling crucial signaling pathways (eg TGF, Notch...) responsible for sustaining pancreas progenitors while preserving their differentiation potential in vitro.

      The current version of the paper has improved, increasing the clarity and providing clear explanations to the comments raised regarding quantifications, functionality of the cells in vivo etc...

      The discussion on challenges adds depth to the study and encourages future research to build upon these important findings

    1. Reviewer #1 (Public Review):


      This study demonstrated a novel exciting link between the conserved miRNA-target axis of miR-182-Lrp6 in liver metabolism which causatively contributes to type 2 diabetes and NAFLD in mice and, potentially, humans.


      The direct interaction and inhibition of Lrp6 by miR-182 are convincingly shown. The effects of miR-182-5p on insulin sensitivity are also credible for the in vivo and in vitro gain-of-function experiments.


      However, the DIO cohorts lack key assays for insulin sensitivity such as ITT or insulin-stimulated pAKT, as well as histological evidence to support their claims and strengthen the link between miR-182-5p and T2D or NAFLD. Besides, the lack of loss-of-function experiments limits its aptitude as a potential therapeutic target.

    1. Reviewer #1 (Public Review):

      The study presented in this manuscript presents very convincing evidence that purifying selection is the main force shaping the landscape of TE polymorphisms in B. distachyon, with only a few putatively adaptive variants detected, even though most conclusions are based on the 10% of polymorphisms contributed by retrotransposons. That first conclusion is not novel, however, as it had already been clearly established in natural A. thaliana strains (Baduel et al. Genome Biol 2021) and in experimental D. simulans lines (Langmüller et al. NAR 2023), two studies that the authors do not mention, or improperly mention. In contrast to the conclusions reached in A. thaliana, however, Horvath et al. report here a seemingly deleterious effect of TE insertions even very far away from genes (>5kb), a striking observation for a genome of relatively similar size. If confirmed, as a caveat of this study is the lack of benchmarking of the TE polymorphisms calls by a pipeline known for a high rate of false positives (see detailed Private Recommendations #1), this set of observations would make an important addition to the knowledge of TE dynamics in the wild and questioning our understanding of the main molecular mechanisms through which TEs can impact fitness.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The paper carries out an impressive and exhaustive non-sense mutagenesis using deep mutational scanning (DMS) of the gonadotropin-releasing hormone receptor for the WT protein and two single point mutations that I) influence TM insertion (V267T) and ii) influence protein stability (W107A), and then measures the effect of these mutants on correct plasma membrane expression (PME).

      Overall, most mutations decreased mGnRHR PME levels in all three backgrounds, indicating poor mutational tolerance under these conditions. The W107A variant wasn't really recoverable with low levels of plasma membrane localisation. For the V267T variant, most additional mutations were more deleterious than WT based on correct trafficking, indicating a synergistic effect. As one might expect, there was a higher degree of positive correlation between V267T/W107A mutants and other mutants located in TM regions, confirming that improper trafficking was a likely consequence of membrane protein co-translational folding. Nevertheless, context is important, as positive synergistic mutants in the V27T could be negative in the W107A background and vice versa. Taken together, this important study highlights the complexity of membrane protein folding in dissecting the mechanism-dependent impact of disease-causing mutations related to improper trafficking.

      Strengths:<br /> This is a novel and exhaustive approach to dissecting how receptor mutations under different mutational backgrounds related to co-translational folding, could influence membrane protein trafficking.

      Weaknesses:<br /> The premise for the study requires an in-depth understanding of how the single-point mutations analysed affect membrane protein folding, but the single-point mutants used seem to lack proper validation. Furthermore, plasma membrane expression has been used as a proxy for incorrect membrane protein folding, but this not necessarily be the case, as even correctly folded membrane proteins may not be trafficked correctly, at least, under heterologous expression conditions. In addition, mutations can affect trafficking and potential post-translational modifications, like glycosylation.

    1. Reviewer #1 (Public Review):

      The regulation of motor autoinhibition and activation is essential for efficient intracellular transport. This manuscript used biochemical approaches to explore two members in the kinesin-3 family. They found that releasing UNC-104 autoinhibition triggered its dimerization whereas unlocking KLP-6 autoinhibition is insufficient to activate its processive movement, which suggests that KLP-6 requires additional factors for activation, highlighting the common and diverse mechanisms underlying motor activation. They also identified a coiled-coil domain crucial for the dimerization and processive movement of UNC-104. Overall, these biochemical and single-molecule assays were well performed, and their data support their statements. The manuscript is also clearly written, and these results will be valuable to the field.

    1. Reviewer #1 (Public Review):

      The study examines how hemocytes control whole-body responses to oxidative stress. Using single cell sequencing they identify several transcriptionally distinct populations of hemocytes, including one subset that show altered immune and stress gene expression. They also find that knockdown of DNA Damage Response (DDR) genes in hemocytes increases expression of the immune cytokine, upd3, and that both upd3 overexpression in hemocytes and hemocyte knockdown of DDR genes leads to increased lethality upon oxidative stress. And they find that the PQ-induced lethality seen when the DDR is disrupted can be rescued in upd3 null background, suggesting links between proper regulation of DDR in hemocytes, modulation of systemic upd3 signaling, and the control of oxidative stress survival.

      The paper has two key strengths:

      1, The single cell analyses provide a clear description of how oxidative stress can cause distinct transcriptional changes in different populations of hemocytes. These results add to the emerging them in the field that there functionally different subpopulations of hemocytes that can control organismal responses to stress.<br /> 2, The discovery that DDR genes are required upon oxidative stress to modulate upd3 cytokine production and lethality provides interesting new insight into the DDR may play non-canonical roles in controlling organismal responses to stress.

    1. Reviewer #1 (Public Review):


      This study is one of several around the world to investigate how urban wildlife responded to changes in human activity during the lockdowns associated with the COVID-19 pandemic. Unlike several other studies on the topic that used observational data from citizen science programs, this project relied on passive acoustic monitoring to record bird vocalizations during and after stringent lockdown periods in an urban environment. The authors focused on three species that differ in their level of adaptation to human presence, providing an ecologically relevant comparison that highlights the importance of micro-habitats for species living in close proximity to humans.


      The element that sets this study apart from most others examining avian responses to COVID-19 lockdowns is the use of passive acoustic monitoring. As the authors describe, this method offers several advantages over other methods (though, it does come with some limitations on what questions can be addressed). Perhaps the most relevant advantage is that it offers the ability to concurrently measure anthropogenic noise in the environment, which is one of the most likely mechanisms for effects on wildlife from changes to human activity. These authors were, therefore, able to show local-scale differences in bird responses to human activity measured at the same scale. To my knowledge, only one other study (Derryberry et al. Science. 2020) has used recordings of vocalizations to examine the influence of COVID-19 lockdowns on a bird species.

      It was encouraging to see a study that focused on the local-scale impacts of lockdowns, with methods that could investigate effects within micro-habitats. Logistics prevented many other projects from operating at such fine scales, making the results from this study particularly useful for the examination of rapid changes in bird behavior. This does mean that comparisons between this study and others examining the effects of COVID-19 lockdowns on birds should be done with care, as the effects described here may have been the result of different processes, operating at different spatial and temporal scales. However, that also means this study fills an important gap in our knowledge of how wildlife reacts to human activity in urban spaces.


      One drawback of the approach is that the acoustic sampling only occurred during the pandemic: samples were taken during several lockdown periods in the early spring (March through early May) of 2020 and then for a period of 10 days after the end of the final lockdown period in late May of 2020. Unfortunately, this means that the interpretation of the effect of lockdowns could have been affected by any shifts in the birds' vocal behavior that resulted from unmodeled environmental factors or normal seasonal phenology during that three-month period. However, the authors chose focal species that would be less prone to seasonal changes in vocal behavior and their approach did account for several factors to minimize any such effects.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This important study nicely integrates a breadth of experimental and computational data to address fundamental aspects of RNA methylation by an important for biology and health RNA methyltransferases (MTases). 

      Strengths:<br /> The authors offer compelling and strong evidence, based on carefully performed work with appropriate and well-established techniques to shed light on aspects of the methyl transfer mechanism of the methyltransferase-like protein 3 (METTL3), which is part of the methyltransferase-like proteins 3 & 14 (METTL3-14) complex. 

      Weaknesses:<br /> 
The significance of this foundational work is somewhat diminished mostly due to mostly efficient communication of certain aspects of this work. Parts of the manuscript are somewhat uneven and don't quite mesh well with one another. The manuscript could be enhanced by careful revision and significant textual and figure edits. 

Examples of recommended edits that would improve clarity and allow accessibility to a broader audience are highlighted in some detail below.

    1. Reviewer #1 (Public Review):

      The manuscript by Dr. Shinkai and colleagues is about the posttranslational modification of a highly important protein, MT3, also known as the growth inhibitory factor. Authors postulate that MT3, or generally all MT isoforms, are sulfane sulfur binding proteins. The presence of sulfane sulfur at each Cys residue has, according to the authors, a critical impact on redox protein properties and almost does not affect zinc binding. They show a model in which 20 Cys residues with sulfane sulfur atoms can still bind seven zinc ions in the same clusters as unmodified protein. They also show that recombinant MT3 (but also MT1 and MT2) protein can react with HPE-IAM, an efficient trapping reagent of persulfides/polysulfides. This reaction performed in a new approach (high temperature and high reagent concentration) resulted in the formation of bis-S-HPE-AM product, which was quantitatively analyzed using LC-MS/MS. This analysis indicated that all Cys residues of MT proteins are modified by sulfane sulfur atoms. The authors performed a series of experiments showing that such protein can bind zinc, which dissociates in the reaction with hydrogen peroxide or SNAP. They also show that oxidized MT3 is reduced by thioredoxin. It gives a story about a new redox-dependent switching mechanism of zinc/persulfide cluster involving the formation of cystine tetrasulfide bridge.

      The whole story is hard to follow due to the lack of many essential explanations or full discussion. What needs to be clarified is the conclusion (or its lack) about MT3 modification proven by mass spectrometry. Figure 1B shows the FT-ICR-MALDI-TOF/MS spectrum of recombinant MT3. It clearly shows the presence of unmodified MT3 protein without zinc ions. Ions dissociate in acidic conditions used for MALDI sample preparation. If the protein contained all Cys residues modified, its molecular weight would be significantly higher. Then, they show the MS spectrum (low quality) of oxidized protein (Fig. 1C), in which new signals (besides reduced apo-MT3) are observed. They conclude that new signals come from protein oxidation and modification with one or two sulfur atoms. If the conclusion on Cys residue oxidation is reasonable, how this protein contains sulfur is unclear. What is the origin of the sulfur if apo-MT does not contain it? Oxidized protein was obtained by acidification of the protein, leading to zinc dissociation and subsequent neutralization and air oxidation. Authors should perform a detailed isotope analysis of the isotopic envelope to prove that sulfur is bound to the protein. They say that the +32 mass increase is not due to the appearance of two oxygen donors. They do not provide evidence. This protein is not a sulfane sulfur binding protein, or its minority is modified. Moreover, it is unacceptable to write that during MT3 oxidation are "released nine molecules of H2". How is hydrogen molecule produced? Moreover, zinc is not "released", it dissociates from protein in a chemical process.

      Another important point is a new approach to the HPE-IAM application. Zinc-binding MT3 was incubated with 5 mM reagent at 60oC for 36 h. Authors claim that high concentration was required because apoMT3 has stable conformation. Figure 2B shows that product concentration increases with higher temperature, but it is unclear why such a high temperature was used. Figure 1D shows that at 37oC, there is almost no reaction at 5 mM reagent. Changing parameters sounds reasonable only when the reaction is monitored by mass spectrometry. In conclusion, about 20 sulfane sulfur atoms present in MT3 would be clearly visible. Such evidence was not provided. Increased temperature and reagent concentration could cause modification of cysteinyl thiol/thiolates as well, not only persulfides/polysulfides. Therefore, it is highly possible that non-modified MT3 protein could react with HPE-IAM, giving false results. Besides mass spectrometry, which would clearly prove modifications of 20 Cys, authors should use very important control, which could be chemically synthesized beta- or alfa-domain of MT3 reconstituted with zinc (many protocols are present in the literature). Such models are commonly used to test any kind of chemistry of MTs. If a non-modified chemically obtained domain would undergo a reaction with HPE-IAM under such rigorous conditions, then my expectation would be right.

      - The remaining experiments provided in the manuscript can also be applied for non-modified protein (without sulfane sulfur modification) and do not provide worthwhile evidence. For instance, hydrogen peroxide or SNAP may interact with non-modified MTs. Zinc ions dissociate due to cysteine residue modification, and TCEP may reduce oxidized residue to rescue zinc binding. Again, mass spectrometry would provide nice evidence.

      - The same is thioredoxin (Fig. 7) and its reaction with oxidized MT3. Nonmodified and oxidized MT3 would react as well.

      - If HPE-IAM reacts with Cys residues with unmodified MT3, which is more likely the case under used conditions, the protein product of such reaction will not bind zinc. It could be an explanation of the cyanolysis experiment (Fig. 6).

      - Figure 4 shows the reactivity of (pol)sulfides with TCEP and HPE-IAM. What are redox potentials? Do they correlate with the obtained results?

      - Raman spectroscopy experiments would illustrate the presence of sulfane sulfur in MT3 only if all Cys were modified.

      - The modeling presented in this study is very interesting and confirms the flexibility of metallothioneins. MT domains are known to bind various metal ions of different diameters. They adopt in this way to larger size the ions. The same mechanism could be present from the protein site. The presence of 9 or 11 sulfur atoms in the beta or alfa domain would increase the size of the domains without changing the cluster structure.

      - Comment to authors. Apo-MT is not present in the cell. It exists as a partially metallated species. The term "apo-MT" was introduced to explain that MTs are not fully saturated by metals and function as a metal buffer system. Apo-MT comes from old ages when MT was considered to be present only in two forms: apo-form and fully saturated forms.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The authors sought to establish a biochemical strategy to study ESAT-6 and CFP-10 biochemistry. They established recombinant reagents to study these protein associations in vitro revealing an unexpected relationship at low pH. They next develop much-needed reagents to study these proteins in an infection context and reveal that treatment with an ESAT-6 nanobody enhances Mtb control.

      Strengths:<br /> The biochemical conclusions are supported by multiple configurations of the experiments. They combine multiple approaches to study a complex problem.

      Weaknesses:<br /> It would be valuable to understand if the nanobody is disrupting the formation of the ESAT6-CFP10 complex. It is unclear how the nanobody is functioning to enhance control in the infection context. More detail or speculation in the discussion would have been valuable. Where is the nanobody in the cell during infection?

    1. Reviewer #1 (Public Review):


      Exposure to cranial irradiation (IR) leads to cognitive deficits in the survivors of brain cancer. IR upregulates miR-206-3p, which in turn reduces the PAK3-LIMK1 axis leading to the loss of F and G-actin ratio and, thereby, mature dendritic spine loss. Silencing miR-206-3p reverses these degenerative consequences.

      Strengths:<br /> The authors show compelling data indicating a clear correlation between PAK3 knockdown and the loss of mature dendritic spine density. In contrast, overexpression of PAK3 in the irradiated neurons restored mature spine types and recovered the F/G ratio. These in vitro results support the authors' hypotheses that PAK3 and LIMK1-mediated downstream signaling impact neuronal structure and reorganization in vitro. These data were supported by similar experiments using differentiated human neurons. Importantly, silencing miR-206-30 using antagonist miR also reverses IR-induced downregulation of the PAK3-LIMK1 axis, preventing spine loss and cognitive deficits.


      All the miR-206-3p data are presented from in vitro cortical neurons or human stem cell-derived neuron cultures. This data (IR-induced elevation of miR-206-3p) should also be confirmed in vivo using an irradiated mouse brain to correlate the cognitive dysfunction timepoint.

      Antago-miR-206-3p reversed Ir-induced upregulation of miR-206 (in vitro), and prevent reductions in PAK3 and downstream markers. Importantly, it reversed cognitive deficits induced by IR. This data should be supported by in vivo staining for important dendritic markers, including cofillin, p-cofilin, PSD-95, F- and G-actin within the hippocampal and PFC regions.

      Other neuronal and non-neuronal targets of miR-206-3p should be discussed and looked into as a downstream impact of IR-induced functional and physiological impairments in the brain.

    1. Reviewer #1 (Public Review):

      In this study, authors performed multiple sets of mesoscale chromatin simulations at nucleosome resolution to study the effects of TF binding on chromatin structures. Through simulations at various conditions, authors performed systemically analysis to investigate how linker histone, tail acetylation, and linker DNA length can operate together with TFs to regulate chromatin architecture. Using gene Eed as one example, authors found that binding of Myc:Max could repress the gene expression by increasing fiber folding and compaction and this repression can be reversed by the linker histone. Understanding how transcription factors bind to regulatory DNA elements and modulate chromatin structure and accessibility is an essential question in epigenetics. Through modelling of TF binding to chromatin structures at nucleosome levels, authors demonstrated that TF binding could create microdomains that are visible in the ensemble-based contact maps and short DNA linkers prevent the formation microdomains. It has also been shown that tail acetylation and TF binding have opposite effects on chromatin compaction and linker histone can compete for the linker DNA with TF binding to impair the effect of TF binding. This study improves our knowledge on how TFs collaborate with different epigenetic marks and chromatin features to regulate chromatin structure and accessibility, which will be of broad interest to the community.

      For this reviewer, there were a few notable limitations. One was the implicit model of TF binding, which is modelled by adding harmonic restraints at two DNA beads. The model is very simple and it lacks kind of validation of how the results can be extended to many other TFs. In addition, the results of TF binding creating microdomains are very interesting but it requires further quantitative analysis of how microdomains was affected under different conditions. Also, some definitions and protocols demand further elucidation.

    1. Reviewer #1 (Public Review):


      This revised study follows up on previous work showing a female-specific enhancer region of PAX1 is associated with adolescent idiopathic scoliosis (AIS). This new analysis combines human GWAS analysis from multiple countries to identify a new AIS-associated coding variant in the COL11A1 gene (COL11A1P1335L). Using a Pax1 knockout mouse they go on to find that PAX1 and Collagen XI protein are expressed in the intervertebral discs (IVDs) and robustly in the growth plate, showing that COL11A1 expression is reduced in Pax1 mutant growth plate. Moreover, other AIS-associated genes, Gpr126 and Sox6, were also reduced in Pax1 mutant mice, suggesting a common pathway is involved in AIS.

      Using SV40 immortalized costal cartilage cells, derived from floxed Col11a1 mice primary rib cage cartilage, they go to show that removal of Col11a1 leads to reduction of Mmp3 expression. In this context, the expression of wild-type Col11a1 restored regular levels of Mmp3 expression, while expression of the AIS-associated Col11a1P1335L allele failed to restore normal Mmp3 expression. This supports a model that the AIS-associated Col11a1P1335L allele leads to the dysregulation of ECM in vivo.

      Using this culture system, they go on to test the role of the estrogen receptor ESR2, showing that loss of this receptor leads to reduced Mmp3 and Pax1 expression, and increased Col11a1 expression. They support this by showing similar gene expression changes and estrogen receptor function in Rat cartilage endplate cell culture.

      Altogether, this study nicely brings together an impressive number of human genetic data from multi-ethnic AIS cohorts and controls from across the globe and functionally tests these findings in cell culture and animal models. This study wonderfully integrates other findings from other human and mouse work in AIS and supports a new molecular mechanism by which estrogen can interact and synergize with COL11A1/PAX1/MMP3 signaling to change ECM development and dynamics, thus providing a tangible model for mutations and dysregulation of this pathway can increase the susceptibility of scoliosis.


      This work integrates a large cohort of human genetic data from AIS patient and control from diverse ethnic backgrounds, across the globe. This work attempts to functionally test their findings in vivio and by use of cell culture.


      Many of the main functional work was done in cell culture and not in vivo.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The study's abstract, introduction, and conclusions are not supported by the methods and results conducted. In fact, the results presented suggest that Arabidopsis could easily adapt to an extremely high CO2 environment.

      This study offers good evidence pointing to a genetic basis for Arabidopsis thaliana's response to elevated CO2 (eCO2) levels and its subsequent impact on the leaf ionome. The natural variation analyses in the study support the hypothesis that genetic factors, rather than local adaptation, guide the influence of eCO2 on the ionome of rosette leaves in Arabidopsis. However, the manuscript's claim regarding its role in "the development of biofortified crops adapted to a high-CO2 world" (line 23) is overstated, especially given the absence of any analysis on the influence of eCO2 on the seed ionome and Arabidopsis is a poor model for harvest index for any crop. The manuscript, in its current form, necessitates massive revisions, particularly in clarifying its broader implications and in providing more substantial evidence for some of its assertions.

      Major Drawbacks and Questions:

      1. Evidence for the Central Premise:<br /> The foundational premise of the study is the assertion that rising atmospheric CO2 levels result in a decline in plant mineral content. This phenomenon is primarily observed in C3 plants, with C4 plants seemingly less affected. The evidence provided on this topic is scant and, in some instances, contradicts the authors' own references. The potential reduction of certain minerals, especially in grains, can be debated. For instance, reduced nitrogen (N) and phosphorus (P) content in grains might not necessarily be detrimental for human and animal consumption. In fact, it could potentially mitigate issues like nitrogen emissions and phosphorus leaching. Labeling this as a "major threat to food security" (line 30) is exaggerated. While the case for microelements might be more compelling, the introduction fails to articulate this adequately. Furthermore, the introduction lacks any discussion on how eCO2 might influence nutrient allocation to grains, which would be crucial in substantiating the claim that eCO2 poses a threat to food security. A more comprehensive introduction that clearly delineates the adverse effects of eCO2 and its implications for food security would greatly enhance the manuscript.

      2. Exaggerated Concerns:<br /> The paper begins with the concern that carbon fertilization will lead to carbon dilution in our foods. While we indeed face numerous genuine threats in the coming decades, this particular issue is manageable. The increase in CO2 alone offers many opportunities for boosting yield. However, the heightened heat and increased evapotranspiration will pose massive challenges in many environments.

      Figure 4 in fact suggests that 43% of the REGMAP panel (cluster 3) is already pre-adapted to very high CO2 levels. This suggests annual species could adapt very rapidly.

      3. Assumptions on CO2 Levels:<br /> The assumption of 900ppm seems to be based on a very extreme climate change scenario. Most people believe we will overshoot the 1.5{degree sign}C scenario, however, it seems plausible that 2.5 to 3{degree sign}C scenarios are more likely. This would correspond to around 500ppm of CO2. https://www.nature.com/articles/s41597-022-01196-7/tables/4

      4. Focus on Real Challenges:<br /> We have numerous real challenges, such as extreme heat and inconsistent rainfall, to address in the context of climate change. However, testing under extreme CO2 conditions and then asserting that carbon dilution will negatively impact nutrition is exaggerated.

      In contrast, the FACE experiments are fundamental and are conducted at more realistic eCO2 levels. Understanding the interaction between a 20% increase in CO2 and new precipitation patterns is key for global carbon flux prediction.

      As I look at the literature on commercial greenhouse tomato production, 1000ppm of eCO2 is common, but it also looks like the breeders and growers have already solved for flavor and nutrition under these conditions.

      Conclusion:<br /> While the study provides valuable insights into the genetic underpinnings of Arabidopsis thaliana's response to elevated CO2 levels, it requires an entirely revised writeup, especially in its abstract, broader claims and implications. The manuscript would benefit from a more thorough introduction, a clearer definition of its scope, and a clear focus on the limits of this study.

    1. Reviewer #1 (Public Review):

      Peng et al develop a computational method to predict/rank transcription factors (TFs) according to their likelihood of being pioneer transcription factors--factors that are capable of binding nucleosomes--using ChIP-seq for 225 human transcription factors, MNase-seq and DNase-seq data from five cell lines. The authors developed relatively straightforward, easy to interpret computational methods that leverage the potential for MNase-seq to enable relatively precise identification of the nucleosome dyad. Using an established smoothing approach and local peak identification methods to estimate positions together with identification of ChIP-seq peaks and motifs within those peaks which they referred to as "ChIP-seq motifs", they were able to quantify "motif profiles" and their density in nucleosome regions (NRs) and nucleosome depleted regions (NDRs) relative to their estimated nucleosome dyad positions. Using these profiles, they arrived at an odd-ratio based motif enrichment score along with a Fisher's exact test to assess the odds and significance that a given transcription factor's ChIP-seq motifs are enriched in NRs compared to NDRs, hence, its potential to be a pioneer transcription factor. They showed that known pioneer transcription factors had among the highest enrichment scores, and they could identify a number of relatively novel pioneer TFs with high enrichment scores and relatively high expression in their corresponding cell line. They used multiple validation approaches including (1) calculating the ROC-AUC and Matthews correlation coefficient (MCC) and generating ROC and precision-recall curves associated with their enrichment score based on 32 known pioneer TFs among their 225 TFs which they used as positives and the remaining TFs (among the 225) as negatives; (2) use of the literature to note that known pioneer TFs that acted as key regulators of embryonic stem cell differentiation had a highest enrichment scores; (3) comparison of their enrichment scores to three classes of TFs defined by protein microarray and electromobility shift assays (1. strong binder to free and nucleosomal DNA, 2. weak binder to free and nucleosomal DNA, 3. strong binding to free but not nucleosomal DNA); and (4) correlation between their calculated TF motif nucleosome end/dyad binding ratio and relevant data from an NCAP-SELEX experiment. They also characterize the spatial distribution of TF motif binding relative to the dyad by (1) correlating TF motif density and nucleosome occupancy and (2) clustering TF motif binding profiles relative to their distance from the dyad and identifying 6 clusters.

      The strengths of this paper are the use of MNase-seq data to define relatively precise dyad positions and ChIP-seq data together with motif analysis to arrive at relatively accurate TF binding profiles relative to dyad positions in NRs as well as in NDRs. This allowed them to use a relatively simple odds ratio based enrichment score which performs well in identifying known pioneer TFs. Moreover, their validation approaches either produced highly significant or reasonable, trending results.

      The weaknesses of the paper are relatively minor, and the authors do a good job describing the limitations of the data and approach.

    1. Reviewer #1 (Public Review):


      Mainali and colleagues provide evidence for Itaconate stabilising Cpt1a via a decrease in ubiquitination. This in turn likely regulates fatty acid oxidation which in turn would appear to be involved in thermoregulation in the context of sepsis.


      These findings add to our knowledge of the role of Itaconate in sepsis and its rather complex effects on metabolism, specifically lipid metabolism.


      1. This is a complex paper and would benefit from a schematic depicting the key findings.

      2. The paper would benefit from additional supporting evidence. Would it be possible to measure fatty acid oxidation by metabolic tracing here, in IRG-deficient cells or in response to 4-OI? Although changes in protein level for Cpt1A are seen, this is correlated with fatty acid oxidation rather than direct demonstration. This may be challenging but would strengthen the manuscript.

      3. The aspect concerning body temperature regulation is confusing. Would Itaconate not promote fatty acid oxidation to increase or maintain body temperature? Itaconate must therefore not be involved in the hypothermic response? Bringing UCP1 into the finding is confusing and needs to be better explained. Again a diagram would help, but enhanced BAT fatty acid oxidation and UCP1 expression appear linked here, with both being affected by Itaconate. This needs clarifying.

    1. Reviewer #1 (Public Review):

      The association of vitamin D supplementation in reducing Asthma risk is well studied, although the mechanistic basis for this remains unanswered. In the presented study, Kilic and co-authors aim to dissect the pathway of Vitamin D-mediated amelioration of allergic airway inflammation. They use initial leads from bioinformatic approaches, which they then associate with results from a clinical trial (VDAART) and then validate them using experimental approaches in murine models. The authors identify a role of VDR in inducing the expression of the key regulator Ikzf3, which possibly suppresses the IL-2/STAT5 axis, consequently blunting the Th2 response and mitigating allergic airway inflammation.

      The major strength of the paper lies in its interdisciplinary approach, right from hypothesis generation, and linkage with clinical data, as well as in the use of extensive ex vivo experiments and in vivo approaches using knock-out mice. The study presents some interesting findings including an inducible baseline absence/minimal expression of VDR in lymphocytes, which could have physiological implications and needs to be explored in future studies.<br /> However, the study presents a potential for further dissection of relevant pathophysiological parameters using additional techniques, to explain certain seemingly associative results, and allow for a more effective translation.

      Several results in the study suggest multiple factors and pathways influencing the phenotype seen, which remain unexplored. The inferences of this study also need to be read in the context of the different sub-phenotypes and endotypes of Asthma, where the Th2 response may not be predominant. While this does not undermine the importance of this elegant study, it is essential to emphasise a holistic picture while interpreting the results.

    1. Reviewer #1 (Public Review):

      The manuscript by Geurrero and colleagues introduces two new metrics that extend the concept of "druggability"- loosely speaking, the potential suitability of a particular drug, target, or drug-target interaction for pharmacological intervention-to collections of drugs and genetic variants. The study draws on previously measured growth rates across a combinatoriality complete mutational landscape involving 4 variants of the TEM-50 (beta lactamase) enzyme, which confers resistance to commonly used beta-lactam antibiotics. To quantify how growth rate - in this case, a proxy for evolutionary fitness - is distributed across allelic variants and drugs, they introduce two concepts: "variant vulnerability" and "drug applicability".

      Variant vulnerability is the mean vulnerability (1-normalized growth rate) of a particular variant to a library of drugs, while drug applicability measures the mean across the collection of genetic variants for a given drug. The authors rank the drugs and variants according to these metrics. They show that the variant vulnerability of a particular mutant is uncorrelated with the vulnerability of its one-step neighbors, and analyze how higher-order combinations of single variants (SNPs) contribute to changes in growth rate in different drug environments.

      The work addresses an interesting topic and underscores the need for evolution-based metrics to identify candidate pharmacological interventions for treating infections. The authors are clear about the limitations of their approach - they are not looking for immediate clinical applicability - and provide simple new measures of druggability that incorporate an evolutionary perspective, an important complement to the orthodoxy of aggressive, kill-now design principles.

      As I said in my initial review, I think the work could be improved with additional analysis that tie the new metrics to evolutionary outcomes. Without this evidence, or some other type of empirical or theoretical support for the utility of these metrics, I am not fully convinced that these concepts have substantial impact. The new metrics could indeed be useful--and they have intuitive appeal--but the current revisions stop short of demonstrating that these intuitive notions hold up under "realistic" conditions (whether in simulation, theory, or experiment).

    1. Reviewer #1 (Public Review):

      Summary:<br /> The study follows the role of yeast eIF2A protein as a potential translation initiation factor engaged in the non-canonical translation initiation under stress conditions and as a substitute for eIF2. Using ribosome profiling, RNA-Seq and reporter-based assays authors evaluated the role of eIF2A protein under regular or stress conditions (cells starved for branched amino acids). The authors found that yeast cells depleted of eIF2A protein do not change significantly their translation initiation, or translation in general. In contrast to previously reported data for human homolog, yeast eIF2A does not significantly contribute to the regulation of the uORFs, regardless of whether they start with canonical AUG or near cognate start codons. eIF2A is not involved in the repression of IRES element in the URE2 gene or has a role in purine biosynthesis. It appears that in yeast eIF2A contributes to the regulation of a very limited number of mRNAs (32 with significant changes in translation efficiency), where only 17 of such messages indeed are consistent with eIF2A deletion, and single mRNA (HKR1) could be validated in reporter assay.

      Strengths:<br /> The main strength of the manuscript is a complete analysis and unbiased approach using genomic analysis methods (ribosome profiling and RNA-seq) as well as reporter validation studies. Additional strengths of the manuscript are scientific rigor and statistics associated with data analyses, clear data presentation, and discussion of the results in the context of the previous studies and results.

      Weaknesses:<br /> No weaknesses were noted by this reviewer.

    1. Reviewer #1 (Public Review):

      Summary and Strengths:

      Zhang et al. conducted a study in which they isolated and characterized a Marburg virus (MARV) glycoprotein-specific antibody, AF-03. The antibody was obtained from a phage-display library. The study shows that AF-03 competes with the previously characterized MARV-neutralizing antibody MR78, which binds to the virus's receptor binding site. The authors also performed GP mutagenesis experiments to confirm that AF-03 binds near the receptor binding site. In addition, the study confirmed that AF-03, like MR78, can neutralize Ebola viruses with cleaved glycoproteins. Finally, the authors demonstrated that NPC2-fused AF-03 was effective in neutralizing several filovirus species.


      1. The main premise of this study is unclear. Flyak et al. in 2015 described the isolation and characterization of a large panel of neutralizing antibodies from a Marburg survivor (Flyak et al., Cell, 2015). Based on biochemical and structural characterization, Flyak proposed that the Marburg neutralizing antibodies bind to the NPC1 receptor binding side. In the same study, it has been shown that several MARV-neutralizing antibodies can bind to cleaved Ebola glycoproteins that were enzymatically treated to remove the mucin-like domain and glycan cap. In the following study, it has been shown that the bispecific-antibody strategy can be used to deliver Marburg-specific antibodies into the endosome, where they can neutralize Ebola viruses (Wec et al., Science 2016). Finally, the use of lysosome-resident protein NPC2 to deliver antibody cargos to late endosomes has been previously described (Wirchnianski et al., Front. Immunol, 2021)

      The above-mentioned studies are not referenced in the introduction. The authors state that "there is no licensed treatment or vaccine for Marburg [virus] infection." While this is true, there are human antibodies that recognize neutralizing epitopes - that information can't be excluded while providing the rationale for the study. Furthermore, the authors use the word "novel" to describe the AF-03 antibody. How novel is AF-03 if multiple Marburg-neutralizing antibodies were previously characterized in multiple studies? Since AF-03 competes with previously characterized MR78, it binds to the same antigenic region as MR78. AF-03 also has comparable neutralization potency as MR78.

      2. Without the AF-03-MARV GP crystal structure, it's unclear how van der Waals interactions, H-bonds, and polar and electrostatic interactions can be evaluated. While authors use computer-guided homology modeling, this technique can't be used to determine critical interactions. Furthermore, Flyak et al. reported that binding to the NPC1 receptor binding site is the main mechanism of Marburg virus neutralization by human monoclonal antibodies. Since both AF-03 (this study) and MR78 (Flyak study) competed with each other, that information alone was sufficient for GP mutagenesis experiments that identified the NPC1 receptor binding site as the main region for mutagenesis.

      3. The AF-03-GP affinity measurements were performed using bivalent IgG molecules and trimeric GP molecules. This format does not allow accurate measurements of affinity due to the avidity effect. The reported KD value is abnormally low due to avidity effects. The authors need to repeat the affinity experiments by immobilizing trimeric GPs and then adding monovalent AF-03 Fab.

    1. Reviewer #1 (Public Review):


      Mandal et al build upon their earlier work in CD 4 T cells to address the role of WASP in cytotoxic T cell mechanosensing. As shown previously by this group and others, the authors present evidence that tumour cell lysis is stiffness dependent and requires CTL WASP expression. They proceed to show that CTLs engaging targets form actin-rich foci, that the formation of these structures is dependent upon tumour cell stiffness and WASP dependent actin nucleation. Traction force measurements show that WASP is involved in force generation, and evidence that WASP plays a role in mechanosensing comes from studies showing that stiffness dependent phosphorylation of early TCR signalling intermediates (but not the later stages of T cell activation) is WASP dependent, as is phosphorylation of the tension sensor CasL. Finally, the authors provide in vivo data that WASP-deficient T cells kill tumours inefficiently.


      The paper is well-written and brings together a range of well-established techniques for measuring T cell stiffness responses, force production, signalling, and effector function. Although some of the findings are necessarily correlative, the authors have largely achieved their aims. One particularly interesting observation is that stiffness dependent phosphorylation of ZAP70 requires WASP expression. Evidence that ZAP70 phosphorylation is WASP dependent is important, as it suggests that forces exerted by WASP are needed for some of the earliest stages of TCR signalling, perhaps TCR deformation itself. This observation, made in CD8 T cells, is particularly interesting given that previous work from this group [Kumari et al eLife 2015] showed that ZAP70 phosphorylation was intact in WASP-/- CD4 T cell blasts. In that study, the first clear differences in TCR signaling were seen at the level of PLCγ phosphorylation. This could represent an interesting difference between CD4 and CD8 T cells, but supplemental data from Figure S2 also show WASP dependence for CD3ζ and ZAP70 phosphorylation in naïve CD4 T cells. Unfortunately, this interesting issue was not discussed or pursued experimentally.


      While the study is well executed, it is rather limited in scope, and many of the observations have been reported previously in other systems. These weaknesses limit the impact of the study. In particular, the authors have previously shown in CD4 T cells that the nucleation promoting activity of WASP is responsible for the formation of actin foci, for early TCR signalling events associated with T cell activation, for traction force generation and for CasL phosphorylation [Kumari et al eLife 2015, Kumari et al EMBO J 2020]. It could be argued that this paper extends findings made originally in CD4 cells to include CD8 T cells. But the authors did not make this clear, and the advance is rather incremental. Moreover, similar studies have been done in CD8 T cells by other labs. Most notably, the Huse group has conducted highly relevant work investigating the mechanobiology of CTL function in vitro and in vivo [Basu et al Cell 2016, Wang et al Nat Comms 2022, Tamzalit et al Sci Immunol 2019, Tello-Lafoz et al Immunity 2021, de Jesus et al bioRxiv Preprint 2023]. Indeed, one study showed that WASP depletion impairs the formation of protrusions that deform the target cell surface and promote target lysis [Tamzalit et al Sci Immunol 2019]. Mandal et al cite this work and argue that what they show differs from the mechanopotentiation shown in Tamzalit et al, but they don't explore the issue further. They also fail to cite work from Tello-Lafoz et al showing that regulated changes in target cell stiffness contribute to CTL vulnerability. Finally, Mandal et al. fail to deal with evidence that WASP participates in many phases of the CTL response, including adhesion, migration, granule release, and serial killing. All of these are likely contributors to the in vivo phenotypes shown in Figure 4.

    1. Reviewer #1 (Public Review):

      The authors investigate the function of the PTB domain containing adaptor protein Numb in skeletal muscle structure and function. In particular, the effects of reduced Numb expression in aging muscle is proposed as a mechanism for reduced contractile function associated with sarcopenia. Using ex-vivo analysis of conditional Numb and Numblike knockout muscle the authors demonstrate that loss of Numb but not the related Numblike gene expression perturbs muscle force generation. In order to explore the molecular mechanisms involved, Numb interacting proteins were identified in C2C12 cell cultured myotubes by immunoprecipitation and LC-MS/MS. The authors identify Septin 7 as well as Septin 2, 9 and 10 as a Numb binding proteins and demonstrate that loss of Numb/Numblike in myofibers causes changes in Septin 7 subcellular localization. Of note, whether additional septins form a complex or are also disrupted by Numb/Numblike loss remains an interesting area for further investigation. Additional investigation of the specificity and mapping of the Numb-Septin 7 (or another Septin) interaction would be of interest and provide an approach for future studies to demonstrate the biological relevance and specificity of the Numb-Septin 7 interaction in skeletal muscle

    1. Reviewer #1 (Public Review):


      Perampalam et al. describe novel methods for genome-wide CRISPR screening to identify and validate genes essential for HGSOC spheroid viability. In this study, they report that Netrin signaling is essential for maintaining disseminated cancer spheroid survival, wherein overexpression of Netrin pathway genes increases tumor burden in a xenograft model of ovarian cancer. They also show that high netrin expression correlates with poor survival outcomes in ovarian cancer patients. The study provides insights into the biology of netrin signaling in DTC cluster survival and warrants development of therapies to block netrin signaling for treating serous ovarian cancer.


      - The study identifies Netrin signaling to be important in disseminated cancer spheroid survival<br /> - A Novel GO-CRISPR methodology was used to find key genes and pathways essential for disseminated cancer cell survival


      - The term dormancy is not fully validated and requires additional confirmation to claim the importance of Netrin signaling in "dormant" cancer survival.<br /> - Findings shown in the study largely relate to cancer dissemination and DTS survival rather than cancer dormancy.

    1. Reviewer #1 (Public Review):


      This manuscript presents the development of a new microscope method termed "open-top two-photon light sheet microscopy (OT-TP-LSM)". While the key aspects of the new approach (open-top LSM and Two-photon microscopy) have been demonstrated separately, this is the first system of integrating the two. The integration provides better imaging depth than a single-photon excitation OT-LSM.


      - The use of liquid prism to minimize the aberration induced by index mismatching is interesting and potentially helpful to other researchers in the field.<br /> - The use of propidium iodide (PI) provided a deeper imaging depth.


      - Details are lacking on imaging time, data size, the processing time to generate large-area en face images, and inference time to generate pseudo H&E images. This makes it difficult to assess how applicable the new microscope approach might be in various pathology applications.

    1. Joint Public Review:

      In countries endemic for P vivax the need to administer a primaquine (PQ) course adequate to prevent relapse in G6PD deficient persons poses a real dilemma. On one hand PQ will cause haemolysis; on the other hand, without PQ the chance of relapse is very high. As a result, out of fear of severe haemolysis, PQ has been under-used.

      In view of the above, the authors have investigated in well-informed volunteers, who were kept under close medical supervision in hospital throughout the study, two different schedules of PQ administration: (1) escalating doses (to a total of 5-7 mg/kg); (2) single 45 mg dose (0.75 mg/kg).

      It is shown convincingly that regimen (1) can be used successfully to deliver within 3 weeks, under hospital conditions, the dose of PQ required to prevent P vivax relapse.

      As expected, with both regimens acute haemolytic anaemia (AHA) developed in all cases. With regimen (2), not surprisingly, the fall in Hb was less, although it was abrupt. With regimen (1) the average fall in Hb was about 4 G. Only in one subject the fall in Hb mandated termination of the study.

      Since the data from the Chicago group some sixty years ago, there has been no paper reporting a systematic daily analysis of AHA in so many closely monitored subjects with G6PD deficiency. The individual patient data in the Supplementary material are most informative and more than precious.

      Comments on the revised version:

      In my view this important paper is further improved in this revised version (R2), particularly with respect to clarity in the discussion. All the points I had previously raised have been tackled.

    1. Reviewer #1 (Public Review):

      Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to the loss of innervation of skeletal muscles, caused by the dysfunction and eventual death of lower motor neurons. A variety of approaches have been taken to treat this disease. With the exception of three drugs that modestly slow progression, most therapeutics have failed to provide benefit. Replacing lost motor neurons in the spinal cord with healthy cells is plagued by a number of challenges, including the toxic environment, inhibitory cues that prevent axon outgrowth to the periphery, and proper targeting of the axons to the correct muscle groups. These challenges seem to be well beyond our current technological approaches. Avoiding these challenges altogether, Bryson et al. seek to transplant the replacement motor neurons into the peripheral nerves, closer to their targets. The current manuscript addresses some of the challenges that will need to be overcome, such as immune rejection of the allograft and optimizing maturation of the neuromuscular junction.

    1. Reviewer #1 (Public Review):


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


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


      One of the limitations of the study is the small number of samples employed, which is insufficient to make associations with outcomes with confidence. Although the authors discuss the potential heterogeneity of primitive stem, they do not directly address the heterogeneity of hematopoietic potential or response to TKI treatment in the results presented. Another limitation is that the BCR-ABL + versus BCR-ABL- status of cells was not confirmed by direct sequencing for BCR-ABL. The BCR-ABL status of cells sorted based on CD26 and CD35 was evaluated in only two samples. We also note that the surface markers identified were previously reported by the same authors using different single-cell approaches, which limits the novelty of the findings. It will be important to determine whether the GEP and surface markers identified here are able to distinguish BCR-ABL+ and BCR-ABL- primitive stem cells later in the course of TKI treatment. Finally, although the authors do describe differential gene expression between CML and normal, BCR:ABL+ and BCR:ABL-, primitive stem cells they have not as yet taken the opportunity to use these findings to address questions regarding biological mechanisms related to CML LSC that impact on TKI response and outcomes.

    1. Reviewer #1 (Public Review):


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


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


      There are two weaknesses.

      1. When the number of features is reduced the precision drops to ~0.8.<br /> 2. How applicable is the method for other non-human datasets?

    1. Reviewer #1 (Public Review):


      Liang et. al., uses a previously devised full isotope labeling of peptidoglycan followed by mass spec to study the kinetics of Lpp tethering to PG and the hydrolysis of this bond by YafK.


      -The labeling and mass spec analysis technique works very well to discern differentially labelled Tri-KR muropeptide containing new and old Lpp and PG.


      -Only one line of experimentation using mass spec based analysis of labeled PG-Lpp is used to make all conclusions in the paper. The evidence is also not enough to fully deleanate the role of YafK.<br /> -Only one mutant (YafK) is used to make the conclusion.<br /> -The paper makes a lot of 'implications' with minimal proof to support their hypothesis. Other lines of experimentations must be added to fully delineate their claims.<br /> -Time points to analyse Tri-KR isotopologues in Wt (0,10,20,40,60 min) and yafK mutant (0,15, 25, 40, 60 min) are not the same.<br /> -Experiments to define physiological role of YafK are also missing.

    1. Reviewer #1 (Public Review):

      Guan et al. explored the mechanisms responsible for the development, maintenance, and functional properties of a specific subset of unconventional T cells expressing a Va3.2 T cell receptor that recognizes a peptide, QFL, presented by the class Ib protein Qa-1. Prior studies from this group showed that cells from mice deficient in the ER protease ERAAP elicit responses in wild-type animals enriched for Qa-1-restricted CD8 T cells. They further showed that a significant proportion of these responses were directed against the QFL peptide derived from a conserved protein with incompletely understood functions. Many of these so-called QFL T cells expressed Va3.2-Ja21, were present in the spleen of wild-type mice, and exhibited a memory-like phenotype. Due to their relatively low frequency and weak staining with Qa-1 tetramers, analyzing QFL T cells has been challenging. Therefore, the authors generated dextramers, which permitted them to more rigorously identify these cells. They confirmed some of their previous findings and further showed that Va3.2+ and Va3.2- QFL T cells were present in the intestinal epithelium, where they also express CD8alpha homodimers, a characteristic of most small intestinal intraepithelial lymphocytes (siIELs), and most similar to the so-called natural siIELs that acquire their innate functions in the thymus. The authors show that TAP but not Qa-1 or ERAAP expression are required for the development of these cells, and both Qa-1 and ERAAP are required for the natural siIEL phenotype. Some of these findings were confirmed using a new TCR transgenic mouse expressing the QFL TCR. They further show that retention but not homing of QFL T cells to the intestinal epithelium involves commensal microorganisms, and using in silico approaches, they identify a commensal that contains a peptide similar to QFL that can activate QFL T cells. Finally, they show that this organism, P. pentosaceus, can promote gut retention of QFL T cells when it is introduced into germ-free mice. From these findings, the authors conclude that the microbiota influence the maintenance of Qa-1-restricted T cells.


      1. The authors employ a number of new reagents and elegant approaches to explore the development, maintenance and functional properties of QFL T cells.<br /> 2. Generally, conclusions made are well supported by the data presented.<br /> 3. One limitation of the work is that the immunological functions of QFL T cells remain unclear.<br /> 4. In their revised manuscript, the authors present additional data that have appropriately addressed the reviewer comments.

    1. Reviewer #1 (Public Review):


      Khan et. al., investigated the functional redundancy of the non-canonical L-cysteine synthases of M. tuberculosis, CysM and CysK2, focussing on their role in mitigating the effects of host-derived stress. They found that while deletion mutants of the two synthases (Rv∆cysM, Rv∆cysK2) have similar transcriptomes under standard conditions, their transcriptional response to oxidative stress is distinct. The impact of deleting the synthases also differentially affected the pools of L-cysteine-derived metabolites. They show that the mutants (Rv∆cysM, Rv∆cysK2) have impaired survival in peritoneal macrophages and in a mouse model of infection. Importantly, they show that the survival of the mutants increases when the host is defective in producing reactive oxygen and nitrogen species, linking the phenotype to a defect in combating host-derived stress. Finally, they show that compounds inhibiting L-cysteine synthases reduce the intracellular survival of M. tuberculosis.


      1. The distinct transcriptome of the Rv∆cysM and Rv∆cysK2 mutants in the presence of oxidative stress provides solid evidence that these mutants are distinct in their response to oxidative stress, and suggests that they are not functionally redundant.<br /> 2. The use of macrophages from phox-/- and INF-/- mice and an iNOS inhibitor for the intracellular survival assays provides solid evidence that the survival defect seen for the Rv∆cysM and Rv∆cysK2 mutants is related to their reduced ability to combat host-derive oxidative and nitrosative stress. This is further supported by the infection studies in phox-/- and INF-/- mice.


      1. There are several previous studies looking at the transcriptional response of M. tuberculosis to host-derived stress, however, the authors do not discuss initial RNA-seq data in the context of these studies. Furthermore, while several of the genes in sulfur assimilation and L-cysteine biosynthetic pathway genes are upregulated by more than one stress condition, the data does not support the statement that it is the "most commonly upregulated pathway in Mtb exposed to multiple host-like stresses".<br /> 2. For the quantification of the metabolites, it isn't clear how the abundance was calculated (e.g., were standards for each metabolite used? How was abundance normalised between samples?), and this information should be included to strengthen the data. Furthermore, labelling with L-methionine was performed to determine the rate of synthesis of the L-cysteine-derived metabolites. L-cysteine is produced from L-methionine via the transsulfuration pathway, which is independent of CysM and CysK2. It is therefore difficult to interpret this experiment, as the impact of deleting CysM and CysK2 on the transsulfuration pathway is likely indirect.

      3. The ability of L-cysteine to rescue the survival defect of the Rv∆cysM and Rv∆cysK2 mutants in macrophages is interpreted as exogenous L-cysteine being able to compensate for reduced intracellular levels. However, there is no evidence that L-cysteine is being taken up by the mutants and an alternate explanation is that L-cysteine functions as an antioxidant within cells i.e., it reduces intracellular ROS.

      The authors sought to investigate the functional redundancy of the non-canonical L-cysteine synthases CysM and CysK2. While their distinct transcriptional response to oxidative stress suggests distinct physiological roles, the study did not explore these differences and therefore provides only preliminary insight into the underlying reasons for this observation. In the context of drug development, this work suggests that while L-cysteine synthase inhibitors do not have high potency for killing intracellular M. tuberculosis, they have the potential to decrease the pathogen's survival in the presence of host-derive stress.

    1. Reviewer #1 (Public Review):

      Summary and Strengths:

      The manuscript presents novel results on the regulation of Drosophila wing growth by the protocadherins Ds and Fat. The manuscript performs a more careful analysis of disc volume, larval size, and the relationship between the two, in normal and mutant larvae, and after localized knockdown or overexpression of Fat and Ds. Not all of the results are equally surprising given the previous work on Fat, Ds, and their regulation of disc growth, pupariation, and the Hippo pathway, but the presentation and detail of the presented data is new. The most novel results concern the scaling of gradients of Fat and Ds protein during development, a largely unstudied gradient of Fat protein, and using overexpression of Ds to argue that changes in the Ds gradient do not underlie the slowing and halting of cell divisions during development.


      Below I list questions and suggestions about the methodology, the presentation, and the interpretation of the data.

      1) Pouch growth: division or recruitment? The study chooses to examine growth only in the prospective wing blade (the "pouch") rather than the wing disc as a whole. This can create biases, as fat and ds manipulations often cause stronger effects on growth, and on Hippo signaling targets, in the adjacent hinge regions of the disc. So I am curious about this choice.

      The limitation to the wing region also creates some problems for the measurements themselves. The division between wing and pouch is not a strict lineage boundary, and thus cells can join or leave this region, creating two different reasons for changes in wing pouch size; growth of cells already in the region, or recruitment of cells into or out of the region. The authors do not discuss the second mechanism.

      It is not at all clear that the markers for the pouch used by the authors are stable during development. One of these is Vg expression, or the Vg quadrant enhancer. But the Vg-expressing region is thought to increase by recruitment over late second and third instar through a feed-forward mechanism by which Vg-expressing cells induce Vg expression in adjacent cells. In fact, this process is thought to be driven in part by Fat and Ds (Zecca et al 2010). So when the authors manipulate Fat and Ds are they increasing growth or simply increasing Vg recruitment? I would prefer that this limitation be addressed.

      The second pouch marker the authors use is epithelial folding, but this also has problems, as Fat and Ds manipulations change folding. Even in wild type, the folding patterns are complex. For instance, to make folding fit the Vg-QE pattern at late third the authors appear to be jumping in the dorsal pouch between two different sets of folds (Fig 1S2A). The authors also do not show how they use folding patterns in younger, less folded discs, nor provide evidence that the location of the folds are the same and do not shift relative to the cells. They also do not explain how they use folds and measure at later wpp and bpp stages, as the discs unfold and evert, exposing cells that were previously hidden in the folds.

      Finally, the authors limit their measurements to cells with exposed apical faces and thus a measurable area but apparently ignore the cells inside the folds. At late third, however, a substantial amount of the prospective wing blade is found within the folds, especially where they are deepest near the A/P compartment boundary. Using the third vein sensory organ precursors as markers, the L3-2 sensillum is found just distal to the fold, the L3-1 and the ACV sensilla are within the fold, and the GSR of the distal hinge is found just proximal to the fold. That puts the proximal half of the central wing blade in the fold, and apparently uncounted in their assays. These cells will however be exposed at wpp and especially bpp stages. How are the authors adjusting for this?

      2) Stabilizing and destabilizing interactions between Fat and Ds- The authors describe a distal accumulation of Fat protein in the wing, and show that this is unlikely to be through Fat transcription. They further try to test whether the distal accumulation depends on destabilization of proximal Fat by proximal Ds by looking at Fat in ds mutant discs.

      However, the authors do not describe how they take into account the stabilizing effects of heterophilic binding between the extracellular domains (ECDs) of Fat and Ds; without one, the junctional levels and stability of the other is reduced (Ma et al., 2003; Hale et al. 2015). So when they show that the A-P gradient of Fat is reduced in a ds mutant, is this because of the loss of a destabilizing effect of Ds on Fat, as they assume, or is it because all junctional Fat has been destabilized by loss of extracelluarlar binding to Ds? The description of the Fat gradient in Ds mutants is also confusing (see note 6 below), making this section difficult for the reader to follow.

      The authors do not propose or test a mechanism for the proposed destabilization. Fat and Ds bind not only through their ECDs, but binding has now also been demonstrated through their ICDs (Fulford et al. 2023)

      3) Ds gradient scales by volume, rather than cell number - This is an intriguing result, but the authors do not discuss possible mechanisms.

      4) Autonomous effects on growth- Fat and Ds are already known to have autonomous effects on growth and Hippo signaling from clonal analyses and localized knockdowns. One novelty here is showing that localized knockdown does not delay pupariation in the way that whole animal knockdown does, although the mechanism is not investigated. Another novelty is that the authors find stronger wing pouch overgrowth after localized ds RNAi or whole disc loss of fat than after localized fat RNAi, the latter being only 11% larger. The fat RNAi result would have strengthened by testing different fat RNAi stocks, which vary in their strength and are commonly weaker than null mutations, or stronger drivers such as the ap-gal4 they used for some of their ds-RNAi experiments or use of UAS-dcr2. Another reason for caution is that Garoia (2005) found much stronger overgrowth in fat mutant clones, which were about 75% larger than control clones.

      5) Flattening of Ds gradients does not slow growth. One model suggests that the flattening of the Ds gradient, and thus polarized Ds-Fat binding, account for slowed growth in older discs. The difficulty in the past has been that two ways of flattening the Ds gradient, either removing Ds or overexpressing Ds uniformly, give opposite results; the first increases growth, while the latter slows it. Both experiments have the problem of not just flattening the gradient, but also altering overall levels of Ds-Fat binding, which will likely alter growth independent of the gradients. Here, the authors instead use overexpression to create a strong Ds gradient (albeit a reversely oriented one) that does not flatten, and show that this does not prevent growth from slowing and arresting.

      To make sure that this is not some effect caused by using a reverse gradient, one might instead induce a more permanent normally oriented Ds gradient and see if this also does not alter growth; there is a ds Trojan gal4 line available that might work for this, and several other proximal drivers.

      Another possible problem is that, unlike previous studies, the authors have not blocked the Four-jointed gradient; Fj alters Fat-Ds binding and might regulate polarity independently of Ds expression. A definitive test would be to perform the tests above in four-joined mutant discs.

      The Discussion of these data should be improved. The authors state in the Discussion "The significance of these dynamics is unclear, but the flattening of the Fat gradient is not a trigger for growth cessation." While the Discussion mentions the effects of Ds on Fat distribution in some detail, this is the only phrase that discusses growth, which is surprising given how often the gradient model of growth control is mentioned elsewhere. The reader would be helped if details are given about what experiment supports this conclusion, the effect on not only growth cessation but cell cycle time, and why the result differs from those of Rogjula 2008 and Willecke 2008 using Ds and Fj overexpression.

      6) Discussion of Dpp. The authors spend much of the discussion speculating on the possibility that Fat and Ds control growth by changing the wing's sensitivity to the BMP Dpp. As the manuscript contains no new data on Dpp, this is somewhat surprising. The discussion also ignores Schwank (2011), who argues that Fat and Dpp are relatively independent. There have also been studies showing genetic interactions between Fat and signaling pathways such as Wg (Cho and Irvine 2004) and EGF (Garoia 2005).

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this paper by Zhang, the authors build a physical framework to probe the mechanisms that underlie the exchange of molecules between coexisting dense and dilute liquid-like phases of condensates. They first propose a continuum model, in the context of a FRAP-like experiment where the fluorescently labeled molecules inside the condensate are bleached at t=0 and the recovery of fluorescence is measured. Through this model, they identify how the key timescales of internal molecular mixing, replenishment from dilute phase, and interface transfer contribute to molecular exchange timescale. Motivated by a recent experiment reported by some of the co-authors previously (Brangwynne et al. in 2019) finding strong interfacial resistance in in-vitro protein droplets of LAF-1, they seek to understand the microscopic features contributing to the interfacial conductance (inversely proportional to the resistance). To check, they perform coarse-grained MD simulations of sticker-spacer self-associative polymers and report how conductance varies significantly even across the few explored sequences. Further, by looking at individual trajectories, they postulate that "bouncing" - i.e., molecules that approach the interface but are not successfully absorbed - is a strong contributor to this mass transfer limitation. Consistent with their predictions, sequences that have more free unbound stickers (i.e., for example through imbalance sequence sticker stoichiometries) have higher conductances and they show a simple linear scaling between the number of unbound stickers and conductance. Finally, they predict a droplet-size-dependent transition in recovery time behavior.

      Strengths:<br /> 1. This paper is well-written overall and clear to understand.

      2. By combining coarse-grained simulations, continuum modeling, and comparison to published data, the authors provide a solid picture of how their proposed framework relates to molecular exchange mechanisms that are dominated by interface resistance and LAF-1 droplets.

      3. The choice of different ways to estimate conductance from simulation and reported data are thoughtful and convincing in their near agreement (although a little discussion of why and when they differ would be merited as well).

      Weaknesses:<br /> 1. Almost the entirety of this paper is motivated by a previously reported FRAP experiment on a particular LAF-1 droplet in vitro. There are a few major concerns I have with how the original data is used, how these results may generalize, and the lack of connection of predictions with any other experiments (published or new).

      a. The mean values of cdense, cdilute, diffusivities, etc. are taken from Taylor et al. to rule in the importance of interfacial mass transfer limits. While this may be true, the values originally inferred (in the 2019 paper that this paper is strongly built off) report extremely large confidence intervals/inferred standard errors. The authors should accordingly report all their inferences with correct standardized errors or confidence intervals, which in turn, allow us to better understand these data.

      b. The generalizability of this model is hard to gauge when all comparisons are made to a single experiment reported in a previous paper.<br /> i. Conceptually, the model is limited to single-component sticker-spacer polymers undergoing phase separation which is already a very simplified model of condensates - for e.g., LAF1 droplets in the cell have no perceptible interfacial mass limitations, also reported in Taylor et al. 2019 - so how these mechanisms relate to living systems as opposed to specific biochemistry experiments. So the authors need to discuss the implications and limitations of their model in the living context where there are multiple species, finite-size effects, and active processes at play.

      ii. Second, can the authors connect their model to make predictions of the impact of perturbations to LAF-1 on exchange timescales? For example, are mutants (which change the number or positioning of "stickers") expected to show particular trends in conductances or FRAP timescales? Since LAF-1 is a relatively well-studied protein in vitro, can the authors further contrast their expectations with already published datasets that explore these perturbations, even if they don't generate new data?

      iii. A key prediction of the interface limitation model is the size-dependent crossover in FRAP dynamics. Can the authors reanalyze published data on LAF-1 (albeit of different-size droplets) to check their predictions? At the least, is the crossover radius within experimentally testable limits?

      c. The authors nicely relate the exchange timescale to various model parameters. Is LAF-1 the only protein for which the various dilute/dense concentrations/diffusivities are known? Given the large number of FRAP and other related studies, can the authors report on a few other model condensate protein systems? This will help broaden the reach of this model in the context of other previously reported data. If such data are lacking, a discussion of this would be important.

      2. The reported sticker-spacer simulations, while interesting, represent a very small portion of the parameter space. Can the authors - through a combination of simulation, analyses, or physical reasoning, comment on how the features of their underlying microscopic model (sequence length, implicit linker length, relative stoichiometry of A/B for a given length, overall concentration, sequence pattern properties like correlation length) connect to conductance? This will provide more compelling evidence relating their studies beyond the cursory examination of handpicked sequences. A more verbose description of some of the methods would be appreciated as well, including specifically how to (a) calculate the bond lifetime of isolated A-B pair, and (b) how equilibration/convergence of MD simulations is established.

      3. A lot of the main text repeats previously published models (continuum ones in Taylor et al. 2019 and Hubsatch et al., 2021, amongst others) and the idea of interface resistance being limiting was already explored quantitatively in Taylor 2019 (including approximate estimates of mass transfer limitations) - this is fine in context. While the authors do a good job of referring to past work in context, the main results of this paper, in my reading, are:<br /> - a simplified physical form relating conductance timescales.<br /> - sticker-spacer simulations probing microscopic origins.<br /> - analysis of size-dependent FRAP scaling.

      I am stating this not as a major weakness, but, rather - I would recommend summarizing and categorizing the sections to make the distinctions between previously reported work and current advances sufficiently clear.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This manuscript explores the importance of food type on virus infection dynamics using a nematode virus as a model system. The authors demonstrate that susceptibility to viral infection can change by several orders of magnitude based on the type of bacterial food that potential hosts consume. They go on to show that, for the bacterial food source that reduces susceptibility, the effect is modulated by quorum sensing molecules that the bacteria produce.

      Strengths:<br /> This manuscript shows convincingly that nematode susceptibility to viral infection changes by several orders of magnitude (i.e. doses must be increased by several orders of magnitude to infect the same fraction of the population) depending on the bacterial food source on which hosts are reared. The authors then focus on the bacteria that reduce host susceptibility to viral infection and demonstrate that certain bacterial quorum-sensing compounds are required to see this effect of reduced susceptibility. Overall, sample sizes are large, methods are generally rigorous, experiments are repeated, and patterns are clear.

      Weaknesses:<br /> Although the molecular correlate of reduced susceptibility is identified (i.e. quorum sensing compounds) the mechanisms underlying this effect are missing. For example, there are changes in susceptibility due to altered nutrition, host condition, the microbiome, feeding rate, mortality of infected hosts, etc. In addition, the authors focus almost entirely on the reduction in susceptibility even though I personally find the increased susceptibility generated when reared on Ochrobactrum to be much more exciting.

      I was a bit surprised that there was no data on basic factors that could have led to reductions in susceptibility. In particular, data on feeding rates and mortality rates seem really important. I would expect that feeding rates are reduced in the presence of Pseudomonas. Reduced feeding rates would translate to lower consumed doses, and so even though the same concentration of virus is on a plate, it doesn't mean that the same quantity of virus is consumed. Likewise, if Pseudomonas is causing mortality of virus-infected hosts, it could give the impression of lower infection rates. Perhaps mortality rates are too small in the experimental setup to explain this pattern, but that isn't clear in the current version of the manuscript. Is mortality greatly impacted by knocking out quorum-sensing genes? Also, the authors explored susceptibility to infection, but completely ignored variation in virus shedding.

      I was also curious why the authors did not further explore the mechanism behind the quorum-sensing effect. Not sure whether this is possible, but would it be possible to add spent media to the infection plates where the spent media was from Pseudomonas that produce the quorum sensing compound but the plates contain OOP50, Pseudomonas, or the quorum sensing knockout of Pseudomonas? That would reveal whether it is the compound itself vs. something that the compound does.

      In addition, I was surprised by how much focus there was on the attenuation of infection and how little there was on the enhancement of infection. To me, enhancement seems like the more obvious thing to find a mechanism for -- is the bacteria suppressing immunity, preventing entry to gut cells, etc?

      I was a bit concerned about the "arbitrary units", which were used without any effort to normalize them. David Wang and Hongbing Jiang have developed a method based on tissue culture infectious dose 50 (TCID50) that can be used to measure infectious doses in a somewhat repeatable way. Without some type of normalization, it is hard to imagine how this study could be repeated. The 24-hour time period between exposure and glowing suggests very high doses, but it is still unclear precisely how high. Also, it is clear that multiple batches of virus were used in this study, but it is entirely unclear how variable these batches were.

      The authors in several places discuss high variability or low variability in incidence as though it is a feature of the virus or a feature of the host. It isn't. For infection data (or any type of binomial data) results are highly variable in the middle (close to 50% infection) and lowly variable at the ends (close to 0% or 100% infection). This is a result that is derived from a binomial distribution and it should not be taken as evidence that the bacteria or the host affect randomness. If you were to conduct dose-response experiments, on any of your bacterial food source treatments, you would find that variability is lowest at the extremely high and extremely low doses and it is most variable in the middle when you are at doses where about 50% of hosts are infected.

    1. Joint Public Review:

      Summary:<br /> In this interesting work, the authors investigated an important topical question: when we see travelling waves in cortical activity, is this due to true wave-like spread, or due to sequentially activated sources? In simulations, it is shown that sequential brain module activation can show up as a travelling wave - even in improved methods such as phase delay maps - and a variety of parameters is investigated. Then, in ex-vivo turtle eye-brain preparations, the authors show that visual cortex waves observable in local field potentials are in fact often better explained as areas D1 and D2 being sequentially activated. This has implications for how we think about travelling wave methodology and relevant analytical tools.

      Strengths:<br /> I enjoyed reading the discussion. The authors are careful in their claims, and point out that some phenomena may still indeed be genuine travelling waves, but we should have a higher evidence bar to claim this for a particular process in light of this paper and Zhigalov & Jensen (2023) (ref 44). Given this careful discussion, the claims made are well-supported by the experimental results. The discussion also gives a nice overview of potential options in light of this and future directions.

      The illustration of different gaussian covariances leading to very different latency maps was interesting to see.

      Furthermore, the methods are detailed and clearly structured and the Supplementary Figures, particularly single trial results, are useful and convincing.

      Weaknesses:<br /> The details of the sequentially activated Gaussian simulations give some useful results, but the fundamental idea still appears to be "sequential activation is often indistinguishable from a travelling wave", an idea advanced e.g. by Zhigalov & Jensen (2023). It takes a while until the (in my opinion) more intriguing experimental results.

      One of the key claims is that the spikes are more consistent with two sequentially activated modules rather than a continuous wave (with Fig 3k and 3l key to support this). Whilst this is *more* consistent, it is worth mentioning that there seems to be stochasticity to this and between-trial variability, especially for spikes.

    1. Ashby's law of requisite variety may also be at play for overloading our system 1 heuristic abilities with respect to misinformation (particularly in high velocity social media settings). Switching context from system 1 to system 2 on a constant basis to fact check everything in our (new digital) immediate environment can be very mentally and emotionally taxing. This can result in both mental exhaustion as well as anxiety.

    1. Joint Public Review:

      Randomized clinical trials use experimental blinding and compare active and placebo conditions in their analyses. In this study, Fassi and colleagues explore how individual differences in subjective treatment (i.e., did the participant think they received the active or placebo treatment) influence symptoms and how this is related to objective treatment. Authors address this highly relevant and interesting question using a powerful method by (re-)analyzing data from four published neurostimulation studies and including subjective treatment in statistical models explaining treatment response. The major strengths include the innovative and important research question, the inclusion of four different studies with different techniques and populations to address this question, sound statistical analyses, and findings that are of high interest and relevance to the field.

      The paper will have significant impact on the field. It will promote further investigation of the effects of sham vs active treatment by the introduction of the terms subjective treatment vs objective treatment and subjective dosage that can be used consistently in the future. The suggestions to assess the expectation of sham vs active earlier on in clinical trials will advance the understanding of subjective treatment in future studies. Overall, I believe the data will substantially contribute to the design and interpretation of future clinical trials by underscoring the importance of subjective treatment.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Osnes et al., describe a large and impressive study of the population and transmission dynamics of Neisseria gonorrhoeae using a global dataset of 9,732 genomes. This included dense sampling from Norway and the state of Victoria, Australia. Understanding the transmission dynamics of this increasingly drug-resistant pathogen is crucial for designing optimal public health interventions. This study provides useful insights into the differing transmission dynamics between two well-sampled populations.

      The authors have also developed novel techniques to address the size and complexity of the dataset, including an approach to account for recombination when building large phylogenies. While the authors have made significant efforts to account for sampling biases in the data, it is not clear that this has been sufficient to address the problem. The use of non-standard analysis techniques also requires further validation.

      Strengths:<br /> The size of the dataset and the comparisons between densely sampled regions are major strengths of the manuscript. While sampling biases may limit the generalizability of the results, as acknowledged by the authors, the characterization of local and inter-country transmission, will help to inform future studies into N. gonorrhoeae.

      Weaknesses:<br /> Sampling bias:<br /> The authors have gone to considerable efforts to acknowledge and account for biases in the sampling between different locations. Despite this, comparisons are still frequently made in the manuscript between populations with very different sampling profiles, which are likely to dominate the import, export and local transmission signals.

      To determine the sensitivity of their results to sampling, the authors randomly took subsamples of each population at varying sizes. While this would address issues with the overall number of genomes being considered, it is not obvious that it would account for biases in sampling including the differing dates over which each population was sampled.<br /> Randomly subsampling tips of the tree is unlikely to change the overall population structure of each dataset much. For example, subsampling a single outbreak would result in highly similar genomes each time. Subsampling clades would provide a better indication of how sensitive the results are to particular clusters within each population. Simulations would also help to determine under what conditions the inferred asymptotes for import and export fractions are likely to hold.

      The text states that Europe and the USA have 'older' transmission lineages than Norway and Victoria. Norway is also found to export more lineages than Victoria, which is likely to be heavily influenced by biases in the distribution of the 'rest of world' samples. Although the impact of sampling bias is acknowledged by the authors in cases such as these, it would be better to avoid making direct comparisons in the first place.

      Recombination detection and filtering:<br /> The authors introduce a novel pipeline for masking recombination before building phylogenetic trees, based on randomly subsetting the dataset and running the Gubbins algorithm. While I appreciate it is challenging to account for recombination in a dataset of this size, further verification needs to be done to demonstrate the effectiveness of this approach.

      In particular, this approach resulted in ~ 10% of sites being filtered out from a diverse set of genomes. This is considerably less than a previous publication that considered ~400 diverse gonococcal genomes, where just under 50% of sites were removed using the Gubbins algorithm (Sánchez-Busó et al., 2019).

      One reason for this is that the new approach requires recombination events called by Gubbins to meet additional filtering requirements before they are masked from the alignment. This may exclude rarer recombination events, which could subsequently impact the length of branches in the final phylogeny.

      Transmission clustering:<br /> The use of LineageHomology and ancestral state reconstruction to determine transmission clusters may be susceptible to biases in sampling between locations. As noted by the authors, locations with sparse sampling, such as the USA, are likely to have older ancestral nodes that are exclusive to that location. Biases in the sampling of countries that transmit to and from each location will also heavily impact the size of the inferred clusters.

      This could potentially explain the occurrence of larger 'mixed outbreaks' in Victoria when compared to Norway, as these clusters may be older and driven by a lack of observed isolates in the 'rest of world' subset.

      While it would not solve the problem entirely, a SNP-based cut-off as used in the original study of Victorian isolates by Williamson et al., is less likely to be as heavily biased.

      Import and export estimates:<br /> Using LineageHomology to define import and export estimates may have a similar problem with sampling biases. This is acknowledged by the authors and nicely described in Supplementary Figure 6. The authors make a comparison with the analysis of SARS-CoV-2 genomes by du Plessis et. al. (2021). However, in the analysis of SARS-CoV-2, the sampling times were far more consistent than those observed in the gonococcal dataset. To address this, the authors could compare their results to an analysis restricted to samples observed in a similar time period. This could most easily be achieved by cutting the inferred phylogeny at a particular date and re-running the LineageHomology analyses.

    1. Reviewer #1 (Public Review):

      This study examined a universal fractal primate brain shape. However, the paper does not seem well structured and is not well written. It is not clear what the purpose of the paper is. And there is a lack of explanation for why the proposed analysis is necessary. As a result, it is challenging to clearly understand what novelty in the paper is and what the main findings are. Additionally, several terms are introduced without adequate explanation and contextualization, further complicating comprehension. Does the second section, "2. Coarse-graining procedure", serve as an introduction or a method? Moreover, the rationale behind the use of the coarse-graining procedure is not adequately elucidated. Overall, it is strongly recommended that the paper undergoes significant improvements in terms of its structure, explanatory depth, and overall clarity to enhance its comprehensibility.

    1. Reviewer #1 (Public Review):

      This work seeks to understand how behaviour-related information is represented in the neural activity of the primate motor cortex. To this end, a statistical model of neural activity is presented that enables a non-linear separation of behaviour-related from unrelated activity. As a generative model, it enables the separate analysis of these two activity modes, here primarily done by assessing the decoding performance of hand movements the monkeys perform in the experiments. Several lines of analysis are presented to show that while the neurons with significant tuning to movements strongly contribute to the behaviourally-relevant activity subspace, less or un-tuned neurons also carry decodable information. It is further shown that the discovered subspaces enable linear decoding, leading the authors to conclude that motor cortex read-out can be linear.


      In my opinion, using an expressive generative model to analyse neural state spaces is an interesting approach to understanding neural population coding. While potentially sacrificing interpretability, this approach allows capturing both redundancies and synergies in the code as done in this paper. The model presented here is a natural non-linear extension of a previous linear model (PSID) and


      First, the model in the paper is almost identical to an existing VAE model (TNDM) that makes use of weak supervision with behaviour in the same way [1]. This paper should at least be referenced. If the authors wish they could compare their model to TNDM, which combines a state space model with smoothing similar to LFADS. Given that TNDM achieves very good behaviour reconstructions, it may be on par with this model without the need for a Kalman filter (and hence may achieve better separation of behaviour-related and unrelated dynamics).

      Second, in my opinion, the claims regarding identifiability are overstated - this matters as the results depend on this to some extent. Recent work shows that VAEs generally suffer from identifiability problems due to the Gaussian latent space [2]. This paper also hints that weak supervision may help to resolve such issues, so this model as well as TNDM and CEBRA may indeed benefit from this. In addition however, it appears that the relative weight of the KL Divergence in the VAE objective is chosen very small compared to the likelihood (0.1%), so the influence of the prior is weak and the model may essentially learn the average neural trajectories while underestimating the noise in the latent variables. This, in turn, could mean that the model will not autoencode neural activity as well as it should, note that an average R2 in this case will still be high (I could not see how this is actually computed). At the s