Reviewer #2 (Public Review):

Agrawal et al. propose an interesting model in which the autophagy pathway in adult mouse skeletal muscle fibers is orchestrated by two independent mechanisms: a) the activity of the NADPH oxidase (Nox) 2 enzyme necessary for autophagosome biogenesis and maturation and b) the level of acetylation of the microtubule (MT) network more selectively responsible for the fusion of the autophagosomes to the lysosomes. Using the well-known mdx mouse, a model for Duchenne muscular dystrophy, the authors perform a quite impressive (but rather traditional) biochemical characterization of the autophagy pathway and found that biogenesis and maturation of the autophagosomes are impaired in mdx mice muscle fibers by means of altered expression of components of the class III phosphatidylinositol 3-kinase complex (PI3K) such as Beclin, VPS15 (both upregulated in mdx mice), ATG14L and VPS34 (both downregulated), and by the reduced expression of JNK and JIP-1, required for the formation of the heterodimer between Beclin and ATG14L-VPS34. In mdx mice, defective nucleation of the phagophore appears to be coupled to altered elongation and expansion as confirmed by decreased expression of WIPI-1, an early marker of autophagosome formation, required for the assembly of the ATG5-12 complex. Clearance of sequestered cytosolic components necessitates the fusion of the autophagosome with the lysosome, a process that the authors found impaired in mdx mice due to altered formation of the SNARE tertiary complex (STX17-SNAP29-VAMP8), as a result of the marked reduction of STX17 expression.

In a previous work (Pal et al., Nat Commun 2014), the same group described the generation of an mdx-based mouse model where Nox2 activity was abolished by genetic ablation of the p47phox component. These mice presented with a better outcome in terms of dystrophic pathophysiology by means of reduced oxidative stress and improved autophagy. Further characterization of these mice in the present study reveals that in p47-/-/mdx mice abolishment of Nox2 activity restores autophagosome nucleation and maturation thanks to the increased expression of p-JNK, JIP-1 and improved stability of the Beclin-ATG14L complex, but no amelioration is observed on the formation of the SNARE tertiary complex indicating that the biogenesis of autophagosomes is dependent on Nox2 activity but not the fusion between autophagosomes and lysosomes. Given the existing body of evidence in non-muscle cells pointing at alpha-tubulin acetylation as a regulator of MT activity facilitating the fusion of autophagosomes to lysosomes, the authors thought to investigate the level of MT acetylation in mdx mice muscle fibers and found that acetylation is reduced but can be restored by inhibiting the HDAC6 enzyme via the FDA-approved, highly selective pharmacological inhibitor Tubastatin A (Tub A). Treatment of mdx mice at 3 weeks of age (before the onset of pathological manifestations) with Tub A not only restored the normal level of alpha-tubulin acetylation (without altering the organization and density of the MT network) but also curbed the intracellular redox status and improved the autophagic flux by stabilizing the SNARE tertiary complex. Interestingly, treatment of dystrophic mice with Tub A results in substantial improvement of the dystrophic phenotype as confirmed by a reduced level of apoptosis, diminished tissue inflammation, improved sarcolemma integrity, and superior force generation capacity in ex vivo experiments using the diaphragm and Extensor Digitorum Longus (EDL) muscle fibers of Tub A-treated mdx mice compared to untreated mdx and healthy counterparts.

The in-depth characterization of the steps orchestrating the autophagy pathway in the mdx mouse model on the one hand, and the comprehensive evaluation of the phenotype of the mdx mice treated with the HDAC6 inhibitor Tubastatin A on the other, support the conclusions proposed by the authors. Nonetheless, some aspects deserve consideration.

1) The effect of increased alpha-tubulin acetylation by means of genetic and pharmacological strategies (i.e., in vivo overexpression of alpha-tubulin acetyltransferase-aTAT1 and treatment with Tubacin or Tubastatin A, respectively) has been previously explored in isolated cardiomyocytes and skeletal muscle fibers and revealed that augmented MT acetylation, due to selective inhibition of HDAC6, increases cytoskeletal stiffness and favors Nox2 activation (Coleman et al., J Gen Physiol 2021).

2) Altered organization and density of the MT network in mdx FDB muscle fibers with loss of vertical directionality is not a novelty as well and it has been reported by others (see Randazzo et al., Hum Mol Genet 2019), who also observed that overexpression of a single beta-tubulin (tubb6) in normal Flexor Digitorum Brevis (FDB) muscle fibers mimic the disruption to the MT network of mdx FDB fibers, increases the level of detyrosinated tubulin and increases Nox2 activity (through elevated expression of gp91phox). Conversely, downregulation of the same beta-tubulin restores normal MT organization in mdx FDB. Previous work from the authors (Loehr et al., eLife 2018) reported that in p47-/-/mdx mice MT organization in diaphragm muscle fibers is normalized and autophagy improved. Accordingly, it is puzzling that increased alpha-tubulin acetylation determines such a wide range of ameliorations in terms of physiological and morphological aspects in dystrophic skeletal muscle fibers treated with Tubastatin A whereas no improvement in the overall MT organization is observed, as reported by Agrawal and colleagues.

3) Given that p47-/-/mdx mice present with levels of acetylated alpha-tubulin and HDAC6 expression comparable to mdx while showing significant improvement of the dystrophic phenotype despite partial rescue of the autophagic flux (as reported in Loehr et al., eLife 2018), it would have been of great interest to investigate the effect of HDAC6 inhibition in p47-/-/mdx mice as well.

Reviewer #2 (Public Review):

The study had an especially relevant aim for aging research and utilized various data types in an especially interesting human population. Multi-omics perspective adds great value to the work. The researchers aimed to evaluate how different indicators of biological age (BA) behave in children during their developmental stage. In the analysis, relationships between indicators of BA, health risk factors, and developmental factors were assessed in cross-sectional data comprising children aged 5-12 years. The manuscript is well-written and easy to follow. The methodology is good. The authors succeeded to reach the aim in most parts.

In the study, previously known and unknown biological age indicators were used. Known indicators included telomere length and Horvath's epigenetic age. Unknown (novel) indicators, transcriptomic and immunometabolic clocks, were developed in the present study and they showed a strong correlation with calendar age in this population, also in the validation data set. Although the transcriptomic and immunometabolic clocks have the potential of being true indicators of biological age, they are still lacking scientific evidence of being such indicators in adults. That is, their associations with age-related diseases and mortality are yet to be shown. Thus, the major remark of the study relates to the phrasing: these novel transcriptomic and immunometabolic clocks should be presented as BA indicator candidates waiting for the needed evidence.

Reviewer #2 (Public Review):

Wei et al. analysed the composition of immune cells, mostly macrophages, and neutrophils, in the context of zebrafish cardiac injury while utilizing clodronate liposomes (CL) to inhibit regeneration via alteration of the immune response. This work is a direct continuation of Shih-Lei et al. which compared the regenerative outcomes of zebrafish vs the non-cardiac regenerative medaka. In that work, the authors used CL to pre-deplete macrophages and showed significant effects on neutrophil clearance, revascularization, and cardiomyocyte proliferation. In this work, the authors used the same pre-depletion method to study the dynamics, composition, and transcriptomic state of macrophages and neutrophils, to overall assess the effect on cardiac regeneration. Using bulk RNA-seq at CL vs PBS treated hearts 7 and 21 days post cryo injury (dpci) a delayed\altered immune response was evident. Single-cell analysis at 1,3 and 7 dpci showed a wide range of immune populations in which most diverse are the macrophage populations. Pre-depletion using CL, altered the composition of immune cells resulting in the complete removal of a single resident macrophage population (M2) or dramatically reducing the overall numbers of other resident populations, while other populations were retained. Looking at the injury time course and distribution of macrophage populations, the authors identified several macrophage populations and neutrophil population 1 as pro-regenerative as their presence compared to CL-treated hearts correlates with regeneration. CL-treated hearts also show a marked sustained neutrophil retention suggesting that interaction with depleted macrophage populations is required for neutrophil clearance. As the marked reduction in populations 2 and 3 occurs after CL treatment, the authors tested whether early CL treatment (8 days or 1 month prior to injury) could reduce the non-recoverable populations and affect regenerative outcomes and indeed they observed a reduction in key genes characterizing M2 and M3 which caused marked reduction in revascularization, CM proliferation, neutrophil retention, and overall higher scaring of the heart.

The findings of this paper could be broadly separated into the characterization of myeloid cells after injury and in non-regenerating animals and assessing the effects of early pre-depletion of macrophages on various cardiac functions involved in regeneration. Both parts draw conclusions that are supported by the facts however several questions remain to be clarified.

1. In figures 2 and 3 the main claim is that the main resident macrophage populations, M2 and M3 are depleted and are largely unable to replenish after injury, similar to resident macrophages in mice 1. However, as the identification of this population is made solely using scRNA-seq, an alternative explanation would be that these cell populations do replenish but are sufficiently changed due to CL treatment (directly or indirectly) and thus would be a part of another cluster. To address this, we suggest:<br /> A. Run trajectory analysis to ascertain whether the different cell clusters are due to differentiating states of the cells<br /> B. Create a reporter line for M2 and M3 macrophages and assess whether they are indeed depleted or changing.

2. One of the major findings of this paper is that some macrophage populations can persist throughout injury and promote the regenerative response. Considering that macrophages have a half-life of less than a day in tissue 2 (although could be different in zebrafish and in this population), we estimate that the resident populations should be proliferative. As there is only a single proliferating macrophage population (M5) we speculate that it is a combination of several populations which are clustered together due to the high expression of cell cycle genes. To verify whether the resident populations are proliferating we suggest:<br /> A. Perform cell-cycle scoring and regression (found in Seurat package) and assess whether after regressing out cell cycle genes there are contributions of M5 to other clusters.<br /> B. Perform EDU labelling experiments with cell cycle identifiers (staining for hbaa1, Timp4.3) and assess their proliferative dynamics.

3. In connection to the previous point if indeed these resident macrophage populations are proliferative, even a smaller portion of remaining cells should be sufficient to partly replenish given sufficient time after CL 1. However as seen in Fig. 3B, the M2 population has a similar proportion of cells on days 1 and 3 after CL treatment and by day 7 it declines in numbers. Given that CL should not be present anymore, we expect this population to increase in numbers over time.

4. In Figure 6 the authors show a reduction in mpeg+ population however a persistent, large population ({plus minus}70% of the original mpeg+) is retained. The authors suggest that this population is comprised of other, non-macrophage, cell types however as this method is the very core of the paper and the persistence of macrophages could alter our understanding of the results, it must be verified.

Dick, S. A. et al. Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction. Nature Immunology 20, 29-39, doi:10.1038/s41590-018-0272-2 (2019).<br /> 2 Leuschner, F. et al. Rapid monocyte kinetics in acute myocardial infarction are sustained by extramedullary monocytopoiesis. J Exp Med 209, 123-137, doi:10.1084/jem.20111009 (2012).

Reviewer #2 (Public Review):

Here the effect of overall transcription blockade, and then specifically depletion of YAP/TAZ transcription factors was tested on cytoskeletal responses, starting from a previous paper showing YAP/TAZ-mediated effects on the cytoskeleton and cell behaviors. Here, primary endothelial cells were assessed on substrates of different stiffness and parameters such as migration, cell spreading, and focal adhesion number/length were tested upon transcriptional manipulation. Zebrafish subjected to similar manipulations were also assessed during the phase of intersegmental vessel elongation. The conclusion was that there is a feedback loop of 4 hours that is important for the effects of mechanical changes to be translated into transcriptional changes that then permanently affect the cytoskeleton.

The idea is intriguing, but it is not clear how the feedback actually works, so it is difficult to determine if the events needed could occur within 4 hrs. Specifically, it is not clear what gene changes initiated by YAP/TAZ translocation eventually lead to changes in Rho signaling and contractility. Much of the evidence to support the model is preliminary. Some of the data is consistent with the model, but alternative explanations of the data are not excluded. The fish washout data is quite interesting and does support the model. It is unclear how some of the in vitro data supports the model and excludes alternatives.

Major strengths: The combination of in vitro and in vivo assessment provides evidence for timing in physiologically relevant contexts, and rigorous quantification of outputs is provided. The idea of defining temporal aspects of the system is quite interesting.

Major weaknesses: The evidence for a "loop" is not strong; rather, most of the data can also be interpreted as a linear increase in effect with time once a threshold is reached. Washout experiments are key to setting up a time window, yet these experiments are presented only for the fish model. A major technical challenge is that siRNA experiments take time to achieve depletion status, making precise timing of events on short time scales problematic. Also, Actinomycin D blocks most transcription so exposure for hours likely leads to secondary and tertiary effects and perhaps effects on viability. No RNA profiling is presented to validate proposed transcriptional changes.

Reviewer #2 (Public Review):

The study by Liu et al. reports on the establishment and characterization of telencephalon eye structures that spontaneously form from human pluripotent stem cells. The reported structures are generated from embryonic cysts that self-form concentric zones (centroids) of telencephalic-like cells surrounded by ocular cell types. Interestingly, the cells in the outer zone of these concentric structures give rise to retinal ganglion cells (RGCs) based on the expression of several markers, and their neuronal morphology and electrophysiological activity. Single-cell analysis of these brain-eye centroids provides detailed transcriptomic information on the different cell types within them. The single-cell analysis led to the identification of a unique cell-surface marker (CNTN2) for the human ganglion cells. Use of this marker allowed the team to isolate the stem cell-derived RGCs.

Overall, the manuscript describes a method for generating self-forming structures of brain-eye lineages that mimic some of the early patterning events, possibly including the guidance cues that direct axonal growth of the RGCs. There are previous reports on brain-eye organoids with optic nerve-like connectivity; thus, the novel aspect of this study is the self-formation capacity of the centroids, including neurons with some RGC features. Notably, the manuscript further reports on cell-surface markers and an approach to generating and isolating human RGCs.

Reviewer #2 (Public Review):

This study proposed the AG fibroblast-neutrophil-ILC3 axis as a mechanism contributing to pathological inflammation in periodontitis. However, the immune response in the vivo is very complex. It is difficult to determine which is the cause and which is the result. This study explores the relevant issue from one dimension, which is of great significance for a deeper understanding of the pathogenesis of periodontitis. It should be fully discussed.

1) Many host cells participate in immune responses, such as gingival epithelial cells. AG fibroblast is not the only cell involved in the immune response, and the weight of its role needs to be clarified. So the expression in the conclusion should be appropriate.

2) This study cannot directly answer the issue of the relationship between periodontitis and systemic diseases.

Reviewer #2 (Public Review):

In this manuscript, Funabiki and colleagues investigated the co-evolution of DNA methylation and nucleosome remolding in eukaryotes. This study is motivated by several observations: (1) despite being ancestrally derived, many eukaryotes lost DNA methylation and/or DNA methyltransferases; (2) over many genomic loci, the establishment and maintenance of DNA methylation relies on a conserved nucleosome remodeling complex composed of CDCA7 and HELLS; (3) it remains unknown if/how this functional link influenced the evolution of DNA methylation. The authors hypothesize that if CDCA7-HELLS function was required for DNA methylation in the last eukaryote common ancestor, this should be accompanied by signatures of co-evolution during eukaryote radiation.

To test this hypothesis, they first set out to investigate the presence/absence of putative functional orthologs of CDCA7, HELLS and DNMTs across major eukaryotic clades. They succeed in identifying homologs of these genes in all clades spanning 180 species. To annotate putative functional orthologs, they use similarity over key functional domains and residues such as ICF related mutations for CDCA7 and SNF2 domains for HELLS. Using established eukaryote phylogenies, the authors conclude that the CDCA7-HELLS-DNMT axis arose in the last common ancestor to all eukaryotes. Importantly, they found recurrent loss events of CDCA7-HELLS-DNMT in at least 40 eukaryotic species, most of them lacking DNA methylation.

Having identified these factors, they successfully identify signatures of co-evolution between DNMTs, CDCA7 and HELLS using CoPAP analysis - a probabilistic model inferring the likelihood of interactions between genes given a set of presence/absence patterns. As a control, such interactions are not detected with other remodelers or chromatin modifying pathways also found across eukaryotes. Expanding on this analysis, the authors found that CDCA7 was more likely to be lost in species without DNA methylation.

In conclusion, the authors suggest that the CDCA7-HELLS-DNMT axis is ancestral in eukaryotes and raise the hypothesis that CDCA7 becomes quickly dispensable upon the loss of DNA methylation and/or that CDCA7 might be the first step toward the switch from DNA methylation-based genome regulation to other modes.

The data and analyses reported are significant and solid. However, using more refined phylogenetic approaches could have strengthened the orthologous relationships presented. Overall, this work is a conceptual advance in our understanding of the evolutionary coupling between nucleosome remolding and DNA methylation. It also provides a useful resource to study the early origins of DNA methylation related molecular process. Finally, it brings forward the interesting hypothesis that since eukaryotes are faced with the challenge of performing DNA methylation in the context of nucleosome packed DNA, loosing factors such as CDCA7-HELLS likely led to recurrent innovations in chromatin-based genome regulation.

Strengths:

- The hypothesis linking nucleosome remodeling and the evolution of DNA methylation.<br /> - Deep mapping of DNA methylation related process in eukaryotes.<br /> - Identification and evolutionary trajectories of novel homologs/orthologs of CDCA7.<br /> - Identification of CDCA7-HELLS-DNMT co-evolution across eukaryotes.

Weaknesses:

- Orthology assignment based on protein similarity.<br /> - No statistical support for the topologies of gene/proteins trees (figure S1, S3, S4, S6) which could have strengthened the hypothesis of shared ancestry.

Reviewer #2 (Public Review):

This manuscript by Walker et al describes an elegant study that synergizes our knowledge of virulence gene regulation of Vibrio cholerae. The work brings a new element of regulation for CRP, notably that CRP and the high density regulator HapR co-occupy the same site on the DNA but modeling predicts they occupy different faces of the DNA. The DNA binding and structural modeling work is nicely conducted and data of co-occupation are convincing. The work seeks to integrate the findings into our current state of knowledge of HapR and CRP regulated genes at the transition from the environment and infection. The strength of the paper is the nice ChIP-seq analysis and the structural modeling and the integration of their work with other studies. The weakness is that it is not clear how representative these data are of multiple hapR/CRP binding sites or how the work integrates as a whole with the entire transcriptome that would include genes discovered by others. Overall this is a solid work that provides an understanding of integrated gene regulation in response to multiple environmental cues.

Reviewer #2 (Public Review):

The paper by Maiti et al. reporting a highly interesting, previously un-noticed, phenomenon of cell size increase as part of the response to chronic proteotoxic stresses, such as heat shock, which the authors term "rewiring stress response". Furthermore, they establish that it is mediated via HSF1, and, strikingly, necessitates a certain threshold levels of HSP90. Dwelling deeper into the underlying mechanisms, they find that HSP90 help scale protein synthesis with the increased cell sizes, and when diminished, this scaling is impaired, and also cell viability in chronic stress is also compromised. These findings correspond with a previous study by this group on the lethality of HSP90 deficient mice, and moreover, have implications to our understanding of cellular adaptation to stress, and generate interesting hypotheses about the possible links of this mechanism to the impairments of the ability to cope with stress during aging and senescence.

This is an excellent study, with highly novel and important findings, which illuminate a new phenomenon related to cellular adaptation to chronic stress. I have only one major concern, about some technical aspects, specifically over-crowding effects, which could confound the results, which should be answered by the authors. Other than that, further details which I think are pertinent to the study most likely already exist in the experiments performed, and most could be answered with additional simple experiments and by further analyses of the proteomics data which has already been performed, but which results are not sufficiently shown in detail.

Reviewer #2 (Public Review):

Work of Rong Li´s lab, published in Nature 2017 (Ruan et al, 2017), led the authors to suggest that the mitochondrial protein import machinery removes misfolded/aggregated proteins from the cytosol and transports them to the mitochondrial matrix, where they are degraded by Pim1, the yeast Lon protease. The process was named mitochondria as guardian in cytosol (MAGIC).

The mechanism by which MAGIC selects proteins lacking mitochondrial targeting information, and the mechanism which allows misfolded proteins to cross the mitochondrial membranes remained, however, enigmatic. Up to my knowledge, additional support of MAGIC has not been published. Due to that, MAGIC is briefly mentioned in relevant reviews (it is a very interesting possibility!), however, the process is mentioned as a "proposal" (Andreasson et al, 2019) or is referred to require "further investigation to define its relevance for cellular protein homeostasis (proteostasis)" (Pfanner et al, 2019).

Rong Li´s lab now presents a follow-up story. As in the original Nature paper, the major findings are based on in vivo localization studies in yeast. The authors employ an aggregation prone, artificial luciferase construct (FlucSM), in a classical split-GFP assay: GFP1-10 is targeted to the matrix of mitochondria by fusion with the mitochondrial protein Grx5, while GFP11 is fused to FlucSM, lacking mitochondrial targeting information. In addition the authors perform a genetic screen, based on a similar assay, however, using the cytosolic misfolding-prone protein Lsg1 as a read-out.

My major concern about the manuscript is that it does not provide additional information which helps to understand how specifically aggregated cytosolic proteins, lacking a mitochondrial targeting signal could be imported into mitochondria. As it stands, I am not convinced that the observed FlucSM-/Lsg1-GFP signals presented in this study originate from FlucSM-/Lsg1-GFP localized inside of the mitochondrial matrix. The conclusions drawn by the authors in the current manuscript, however, rely on this single approach.

In the 2017 paper the authors state: "... we speculate that protein aggregates engaged with mitochondria via interaction with import receptors such as Tom70, leading to import of aggregate proteins followed by degradation by mitochondrial proteases such as Pim1." Based on the new data shown in this manuscript the authors now conclude "that MP (misfolded protein) import does not use Tom70/Tom71 as obligatory receptors." The new data presented do not provide a conclusive alternative. More experiments are required to draw a conclusion.<br /> In my view: to confirm that MAGIC does indeed result in import of aggregated cytosolic proteins into the mitochondrial matrix, a second, independent approach is needed. My suggestion is to isolate mitochondria from a strain expressing FlucSM-GFP and perform protease protection assays, which are well established to demonstrate matrix localization of mitochondrial proteins. In case the authors are not equipped to do these experiments I feel that a collaboration with one of the excellent mitochondrial labs in the US might help the MAGIC pathway to become established.

Reviewer #2 (Public Review):

This study follows up on a previous study by the group (Sibille et al Nature Communications 2022) in which high density Neuropixel probes were inserted tangentially through the superficial layers of the superior colliculus (SC) to record the activity of retinocollicular axons and postsynaptic collicular neurons in anesthetized mice. By correlating spike patterns, connected pairs could be identified which allowed the authors to demonstrate that functionally similar retinal axon-SC neuron pairs were strongly connected.

In the current study, the authors use similar techniques in vGAT-ChR2 mice and add a fiber optic to identify light-activated GABAergic and non-light-activated nonGABAergic neurons. Using their previously verified techniques to identify connected pairs, within regions of optogenetic activation they identified 214 connected pairs of retinal axons and nonGABAergic neurons and 91 pairs of connected retinal axons and GABAergic neurons. The main conclusion is that retinal activity contributed more to the activity of postsynaptic nonGABAergic SC neurons than to the activity of postsynaptic GABAergic SC neurons.

The study is very well done. The figures are well laid out and clearly establish the conclusions. My main comments are related to the comparison to other circuits and further questions that might be addressed in the SC.

It is stated several times that the superior colliculus and the visual cortex are the two major brain areas for visual processing and these areas are compared throughout the manuscript. However, since both the dorsal lateral geniculate nucleus (dLGN) and SC include similar synaptic motifs, including triadic arrangements of retinal boutons with GABAergic and nonGABAergic neurons, it might be more relevant to compare and contrast retinal convergence and other features in these structures.

The GABAergic and nonGABAergic neurons showed a wide range of firing rates. It might be interesting to sort the cells by firing rates to see if they exhibit different properties. For example, since the SC contains both GABAergic interneurons and projection neurons it would be interesting to examine whether GABAergic neurons with higher firing rates exhibit narrower spikes, similar to cortical fast spiking interneurons. Similarly, it might be of interest to sort the neurons by their receptive field sizes since this is associated with different SC neuron types.

The recording techniques allowed for the identification of the distance between connected retinocollicular fibers and postsynaptic neurons. It might also be interesting to compare the properties of connected pairs recorded at dorsal versus ventral locations since neurons with different genetic identities and response properties are located in different dorsal/ventral locations (e.g. Liu et al. Neuron 2023). Also, regarding the strength of connections, previous electron microscopy studies have shown that the retinocollicular terminals differ in density and size in the dorsal/ventral dimension (e.g Carter et al JCN 1991).

Was optogenetic activation of GABAergic neurons ever paired with visual activation? It would be interesting to examine the receptive fields of the nonGABAergic neurons before and after activation of the GABAergic neurons (as in Gale and Murphy J Neurosci 2016).

Reviewer #2 (Public Review):

Segas et al motivate their work by indicating that none of the existing myoelectric solution for people with trans-humeral limb difference offer four active degrees of freedom, namely forearm flexion/extension, forearm supination/pronation, wrist flexion/extension, and wrist radial/ulnar deviation. These degrees of freedom are essential for positioning the prosthesis in the correct plan in the space before a grasp can be selected. They offer a controller based on the movement of the stump.

The proposed solution is elegant for what it is trying to achieve in a laboratory setting. Using a simple neural network to estimate the arm position is an interesting approach, despite the limitations/challenges that the approach suffers from, namely, the availability of prosthetic hardware that offers such functionality, information about the target and the noise in estimation if computer vision methods are used. Segas et al indicate these challenges in the manuscript, although they could also briefly discuss how they foresee the method could be expanded to enable a grasp command beyond the proximity between the end-point and the target. Indeed, it would be interesting to see how these methods can be generalise to more than one grasp.

One bit of the results that is missing in the paper is the results during the familiarisation block. If the methods in "intuitive" I would have thought no familiarisation would be needed. Do participants show any sign of motor adaptation during the familiarisation block?

In Supplementary Videos 3 and 4, how would the authors explain the jerky movement of the virtual arm while the stump is stationary? How would be possible to distinguish the relative importance of the target information versus body posture in the estimation of the arm position? This does not seem to be easy/clear to address beyond looking at the weights in the neural network.

I am intrigued by how the Generic ANN model has been trained, i.e. with the use of the forward kinematics to remap the measurement. I would have taught an easier approach would have been to create an Own model with the native arm of the person with the limb loss, as all your participants are unilateral (as per Table 1). Alternatively, one would have assumed that your common model from all participants would just need to be 'recalibrated' to a few examples of the data from people with limb difference, i.e. few shot calibration methods.

Reviewer #2 (Public Review):

The study "A rapid microglial metabolic response controls metabolism and improves memory" by Drougard et al. provides evidence that short-term HFD has a beneficial effect on spatial and learning memory through microglial metabolic reprogramming. The manuscript is well-written and the statistics were properly performed with all the data. However, there are concerns regarding the interpretation of the data, particularly the gap between the in vivo observations and the in vitro mechanistic studies.

In the PLX-5622 microglial depletion study, it is unclear what happened to the body weight, food intake, and day-night behavior of these mice compared to the vehicle control mice. It is important to address the innate immunity-dependent physiology affected by a long period of microglial depletion in the brain (also macrophages in the periphery). Furthermore, it would be beneficial to validate the images presented in Fig.1F by providing iba1 staining in chow diet-fed mice with or without PLX-5622 for 7-10 days. Additionally, high-quality images, with equal DAPI staining and comparable anatomical level, should be provided in both chow diet-fed mice and HFD-fed mice with or without PLX-5622 in the same region of hypothalamus or hippocampus. These are critical evidences for this project, and it is suggested that the authors provide more data on the general physiology of these mice, at least regarding body weight and food intake.

It is also unclear whether the microglia shown in Fig.3A were isolated from mice 4 weeks after Tamoxifen injection. It is suggested that the authors provide more evidence, such as additional images or primary microglia culture, to demonstrate that the mitochondria had more fusion upon drp1 KO. It is recommended to use mito-tracker green/red to stain live microglia and provide good resolution images.

Regarding the data presented in Fig.5A, it is suggested that the authors profile the metabolomics of the microglial conditioned media (and provide the methods on how this conditioned media was collected) to determine whether there was already abundant lactate in the media. Any glucose-derived metabolites, e.g. lactate, are probably more preferred by neurons as energy substrates than glucose, especially in embryonic neurons (which are ready to use lactate in newborn brain).<br /> Finally, it is important to address whether PLX-5622 affects learning and spatial memory in chow diet-fed animals. Following the findings shown in Fig 5J and 5K, the authors should confirm these by any morphological studies on synapse, e.g. by synaptophysin staining or ultrastructure EM study in the area shown in Fig 5I.

Reviewer #2 (Public Review):

Breast cancer is the most common malignant tumor in women. One of subtypes in breast cancer is so called triple-negative breast cancer (TNBC), which represents the most difficult subtype to treat and cure in the clinic. Chemotherapy drugs including epirubicin and cisplatin are widely used for TNBC treatment. However, drug resistance remains as a challenge in the clinic. The authors uncovered a molecular pathway involved in chemotherapy drug resistance, and molecular players in this pathway represent as potential drug targets to overcome drug resistance. The experiments are well designed and the conclusions drawn mostly were supported by the data. The findings have potential to be translated into the clinic.

Reviewer #2 (Public Review):

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

Reviewer #2 (Public Review):

The authors embarked on a study to identify SNPs in clinical isolates of S. aureus that influence sensitivity to serum killing. Through a phenotypic screen of 300 previously sequenced S. aureus bacteremia (SAB) isolates, they identified ~40 SNPs causing altered serum survival. The remainder of the study focuses of tcaA, a gene with unknown function. They show that when tcaA is disrupted, it results in increased resistance to glycopeptides and antimicrobial components of human serum.

They perform an elegant series of experiments demonstrating how a tcaA knockout is more resistant to killing by whole serum. arachadonic acid, LL-37 and HNP-1. They provide compelling evidence that in the absence of tcaA resistance to arachidonic acid is mediated through release of wall teichoic acids from the cell wall, which acts as a decoy and sequesters the fatty acid.

Similarly, they suggest that resistance to cationic antimicrobial peptides is through alteration of the net charge of the cell wall due to loss of negatively charged WTAs based on reduced cytochrome C binding.

They continue to show that tcaA is induced in the presence of human serum, which causes increased resistance to the glycopeptide teichplanin.

They propose that tcaA disruption causes altered cell wall structure based on morphologic changes on TEM and increased sensitivity to lysostaphin and increased autolysis via triton x-100 assay.

Finally, they propose that tcaA influences mortality in SAB based on raw differences in 30-day morality. Interestingly they do decreased fitness during murine bacteremia model compared to wild-type.

The strengths of this manuscript are that it is well written and the identification of SNPs leading to altered serum killing is convincing and valuable data. The mechanism for tcaA-mediated resistance to arachadonic acid and AMPs is compelling and novel. The murine infection data demonstrating that tcaA mutants exhibit reduced virulence is important data.

The weakness of this manuscript mainly concerns the proposed mechanism that tcaA mutants show reduced peptidoglycan crosslinking. This conclusion is based on qualitative TEM images and increased sensitivity to lysostaphyin/autolysis. While these data are suggestive. it is difficult to draw such a conclusion without analysis of the cell wall by LC-MS.

Overall, I think this is a good submission and the majority of their conclusions are supported by the data. The mechanism behind the clinically relevant tcaA mutation is important, given its known role in glycopeptide resistance and therefore likely clinical outcomes. This manuscript would benefit from the inclusion of some additional experiments to help support their finding.

Reviewer #2 (Public Review):

Inorganic carbon (Ci) uptake by autotrophic organisms is often the rate-limiting process in overall photosynthetic productivity. Aquatic autotrophs including the cyanobacteria have evolved elaborate and metabolically expensive, yet very efficient CO2 concentrating mechanisms (CCMs) to over-come this limitation. The work examines the regulation of SbtA, which is a high affinity sodium dependent symporter. Current evidence suggests that this SbtA is highly regulated both at the transcriptional and post-transcriptional levels. For example, the sbtA gene is transcriptionally upregulated under conditions of inorganic carbon limitation and the transport activity of the expressed SbtA protein is apparently regulated allosterically by multiple factors, including those exerted by the binding of the small trimeric protein, SbtB. SbtB is a PII-type regulator that conditionally binds to the cytoplasmic face of the trimeric SbtA to form a hetero-complex apparently inactivating SbtA to which it is bound. The factors affecting this interaction remains to be clarified, but it is already clear that there is considerable complexity that needs to be unraveled since as with other PII proteins, multiple effector molecules act as ligands.

Using a novel protein-protein interaction assay combined with physiological analysis of various mutants, the authors present new information on the regulation of SbtA from Cyanobium sp. PCC7001 and Synechococcus elongatus PCC7942. Because of their novelty, additional validation may be important to establish their validity, yet they do appear to be robust overall..The work builds on earlier studies indicating negative regulation of SbtA and helps clarify other work, including detailed analysis of the orthologous, albeit somewhat more complex protein from Synechocystis PCC6803. The key significance of the present findings is that the energy charge of the adenylate system, a ubiquitous metabolic control mechanism in the biological world, is the prime and perhaps overriding regulatory parameter governing of SbtA activity. Based on this a model for the diurnal control transporter activity was proposed based on energy charge.

Reviewer #2 (Public Review):

N6-methyladenosine (m6A), the most abundant mRNA modification, is deposited by the m6A methyltransferase complexes (MTC). While MTC in mammals/flies/plants consists of at least six subunits, yeast MTC was known to contain only three proteins. Ensinck, Maman, et al. revisited this question using a proteomic approach and uncovered three new yeast MTC components, Kar4/Ygl036w/Dyn2. By applying sequence and structure comparisons, they identified Kar4, Ygl036w, and Slz1 as homologs of the mammalian METTL14, VIRMA. ZC3H13, respectively. While these proteins are essential for m6A deposition, the dynein light chain protein, Dyn2, is not involved in mRNA methylation. Interestingly, while mammalian and fly MTCs are configured as MAC (METTL3 and METTL14), and MACOM (other subunits) complexes, yeast MTC subunits appear to have different configurations. Finally, Kar4 has a different role as a transcription regulator in mating, which is not mediated by other MTC members. These data establish an important framework for the yeast MTC and also provide novel insights for those studying m6A deposition.

Reviewer #2 (Public Review):

The manuscript by Mastwal and colleagues explores how transient adolescent stimulation of ventral midbrain neurons that project to the frontal cortex may help to improve performance on certain memory tasks. The manuscript provides an interesting set of observations that DREADD-based activation over only 3 days during adolescence provides a fast-acting and long-lasting improvement in performance on Y-maze spontaneous alternation as well as aspects of neuronal function as assessed using in vivo imaging methods. While interesting, there are several weaknesses. First and foremost, it is not clear that the effects the authors are observing are mediated by dopamine. It has been clearly documented that the DAT-Cre line provides a better representation of midbrain dopamine cells in the mouse, particularly near the midline of the ventral midbrain (Lammel et al., Neuron 2015). This is precisely where the cells that project to the frontal cortex are located. Therefore, the selection of TH-Cre is problematic. It is very likely that the authors are labeling a substantial number of non-dopaminergic cells.

Reviewer #2 (Public Review):

The authors attempt to show distinct contributions of selective attention and neuromodulators (both cholinergic and catecholaminergic) during a spatial attention task. To do this, they had participants perform a Posner cueing task using random dot motion stimuli, with a typical 80/20 split of valid to invalidly cued trials. In addition, they designed a within-subjects paradigm wherein participants took placebo (PLA), Donepezil (DNP), or Atomoxetine (ATX). Both behaviour and EEG measures were taken in order to investigate the interaction or lack thereof of Drug and Cue factors with respect to these measures, and relative timing of EEG differences to derive potential neuromechanistic similarities/differences. In this context, an interaction of Drug and Cue factors (e.g. faster valid vs invalid RTs in ATX vs PLA) might indicate a role of that neuromodulator in the mechanisms of spatial attention. This is in fact not what they found, rather most findings pointed towards a lack of interaction of Drug and Cue, hence the central thesis of the paper of distinct contributions of neuromodulator and selective attention.

Strengths:<br /> - The experimental design is well done, especially the blinding of the drug taken in each session. However, it is an important caveat to any results that participants were obviously aware they had taken an active drug in ATX condition (Supp Info).<br /> - The analyses are in general quite solidly performed, with most analysis choices relating to behaviour and EEG making sense, albeit with exceptions below.<br /> - The research question and how it relates to the experiment is very interesting, and the question worthy of consideration.

Weaknesses:<br /> - The main weakness of the paper lies in the strength of evidence provided, and how the results tally with each other. To begin with, there are a lot of significance tests performed here, increasing the chances of false positives. Multiple comparison testing is only performed across time in the EEG results, and not across post-hoc comparisons throughout the paper. In and of itself, it does not invalidate any result per se, but it does colour the interpretation of any results of weak significance, of which there are quite a few. For example, the effect of Drug on d' and subsequent post-hoc comparisons, also effect of ATX on CPP amplitude and others.<br /> - The lack of an overall RT effect of Drug leaves any DDM result a little underwhelming. How do these results tally? One potential avenue for lack of RT effect in ATX condition is increased drift rate but also increased non-decision time, working against each other. However, it may be difficult to validate these results theoretically.<br /> - There is an interaction between ATX and Cue in terms of drift rate, this goes against the main thesis of the paper of distinct and non-interacting contributions of neuromodulators and attention. This finding is then ignored. There is also a greater EDAN later for ATX compared to PLA later in the results, which would also indicate interaction of neuromodulators and attention but this is also somewhat ignored.<br /> - The CPP results are somewhat unclear. Although there is an effect of ATX on drift rate algorithmically, there is no effect of ATX on CPP slope. On the other hand, even though there is no effect of DNP on drift rate, there is an effect of DNP on CPP slope. Perhaps one may say that the effect of DNP on drift rate trended towards significance, but overall the combination of effects here is a little unconvincing. In addition, there is an effect of ATX on CPP amplitude, but how does this tally with behaviour? Would you expect greater CPP amplitude to lead to faster or slower RTs? The authors do recognise this discrepancy in the Discussion, but discount it by saying the relationship between algorithmic and CPP parameters in terms of DDM is unclear, which undermines the reasoning behind the CPP analyses (and especially the one correlating CPP slope with DDM drift rate).<br /> - The posterior component effects are problematic. The main issue is the lack of clarification of and justification for the choice of posterior component. The analysis is introduced in the context of the target selection signal the N2pc/N2c, but the component which follows is defined relative to Cue, albeit post-target. Thus this analysis tells us the effect of Cue on early posterior (possibly) visual ERP components, but it is not related to target selection as it is pooled across target/distractor. Even if we ignore this, the results themselves wrt Drug lack context. There is a trending lower amplitude for ATX at later latencies at temporo-parietal electrodes, and more positive for DNP, relative to PLA. Is this what one would expect given behaviour? This is where the issue of correct component identification becomes critical in order to inform any priors on expected ERP results given behaviour.

Given the issues above; mainly a) weak statistical evidence, b) contradictory behavioural and EEG evidence, and c) lack of theoretical background to inform priors on what to expect from the EEG results in order to develop a coherent narrative, I would say that what remains is moderate/incomplete evidence towards the thesis of the paper. This work is however a very fruitful effort at approaching the research question as to whether there is an interaction of neuromodulators and spatial attention. I commend the authors on a transparent and rigorous analysis of the current data.

Reviewer #2 (Public Review):

In this study, the authors explore the structure/function of the DCLK kinases, most specifically DCLK1 as it is the most studied to date. Recently, the C-terminal domain has garnered attention as it was found to regulate the kinase domain, however, the different isoforms retain additional amino acid sequences with as-yet-undefined functions. The authors provide an evolutionary and biochemical characterization of these regions and provide evidence for some functionality for these additional C-terminal sequences. While these experiments are informative they do require that the protein is soluble and not membrane-bound as has been suggested to be important for functionality in other studies. Still, this is a major contribution to understanding the structure/function of these proteins that will be important in future experimental designs.

Reviewer #2 (Public Review):

Ruby et al. investigated whether demographic aging was absent in the naked-mole rat (Heterocephalus glaber); an exceptionally long-lived small mammal that appears to challenge Gompertzian patterns of increased mortality hazard with age. In particular, this study replicates a previous one in which the authors show that the mortality hazard does not increase with age as it is expected for mammals, especially small ones. The main motivation of this replication is to address the current controversy surrounding the "perpetual neoteny" reported by the authors. The study also extends to the exploration of the role of social factors on the observed patterns in mortality hazard across age and to a meta-analysis comparing mortality hazards across species of mole-rats which highlights the unique pattern of demographic aging (or the absence of) in naked mole-rats. This study is of broad interest to readers in the field of demography, aging, and life history evolution. The key claims of the manuscript state that naked-mole rats avoid an increase in mortality hazard as they age. Although this work raises new evolutionary questions concerning the unexpected gradual (or fully absent) increase versus Gompertzian increase in hazard among mammals, I also identified weaknesses that I discuss below.

Strengths:<br /> Sample sizes - The sample sizes across analyses are vast and the data curation described demonstrates careful thought during the data analysis processes.

Social factors - The analysis testing associations between body mass (as proxy for dominance) and colony size (as proxy for social competition) are novel and provide insights into potential evolutionary drivers for the observed lack of increase in mortality hazard.

Across species comparison - The analysis using Fukomys mole-rats offered a novel phylogenetic comparison of the mortality hazard across age and raises new evolutionary questions concerning the unexpected gradual versus Gompertzian increase in hazard. This study encourages new ones exploring alternative life histories among mammals.

Weaknesses:<br /> Censored data - A significant number of individuals remained alive (~50%) at the end of the study, and thus I wonder how much can the authors say about increased hazard if the individuals have not reach old ages. Maybe the individuals do live long and show increased hazard are very old ages.

Independence between studies - The study provides the replication of a prior study using the same captive population, but I understand that many observations are not independent across studies given repeated measurements. Although this provides reliability, I wonder how independent the conclusions are. This represents a weakness to me because we still do not know whether this is a unique evolutionary trait of this particular captive population. If this is the case, I agree this makes the population a great model for aging studies but do the authors findings have further implications across populations or species? I wonder if populations raised under different conditions would present similar patterns of mortality hazard across age.

Analysis - Another weakness concerns the analysis used. Authors make the claims that social hierarchy may affect mortality hazards and decide to explore associations between body mass and hazard. I wonder if a Cox regression model is more appropriate for the available continuous data, relative to a Kaplan-Meir method. A Cox regression will allow the authors to control for several continuous variables simultaneously, without the limitation of categorical assumptions. A Cox model could also be extended to time-varying covariates allowing for the hazard to change over time (if that is the case). If the authors understand that their approach is equivalent, I suggest a discussion on it. This also applies to the analysis on colony size.

In summary, I see value in this study. There is vast evidence for the penalty of becoming old among mammals. Thus, studies like this one reporting novel patterns are of high impact. I agree that such findings must be replicated and validated. I also see a lot of potential for the use of the available data for more extensive meta-analyses comparing life histories across social mammals or across species with similar use of habitat (underground). Such analyses may allow the authors to move beyond descriptions and discuss why such life history traits may have evolved. Yet, I am not sure how much novelty this study brings, relative to prior studies. It seems the authors may need more than 5 years to allow their individuals to reach older ages.

Reviewer #2 (Public Review):

This paper is an attempt to extend or augment muscle synergy and motor primitive ideas with task measures. The authors idea is to use information metrics (mutual information, co-information) in 'synergy' creation including task information directly. My reading of the paper is that the framework proposed radically moves from attempts to be analytic in terms of physiology and compositionality with physiological bases, instead into more descriptive ML frameworks that may not support physiological work easily.

This approach is very different from the notions of physiological compositional elements as muscle synergies and motor primitives, and to me seems to really be striving to identify task relevant coordinative couplings. This is a meta problem for more classical analyses. Classical analyses seek compositional elements stable across tasks. These elements may then be explored in causal experiments and generative simulations of coupling and control strategies. The present work does not convince me that the joint 'meta' analysis proposed with task information added is not unmoored from physiology and causal modeling in some important ways. It also neglects publications and methods that might be inconvenient to the new framework.

Information based separation has been used in muscle synergy analyses using infomax ICA, which is information not variance based at core. Though linear mixing of sources is assumed, minimized mutual information is the basis.

Physiological causal testing of synergy ideas is neglected in the literature reviews in the paper. Although these are in animal work, the clear connection of muscle synergy choices and analyses to physiology is important, and needs to be managed in the new methods proposed. Is any correspondence assumed? Possible?

Questions and concerns with the framework as an overall tool:

First, muscle based motor information sources have influences on different time scales in the task mechanics. Analyses of synergies in the methods proposed will be very much dependent on the number and quality of task variables included and how these are managed. Standardizing and comparing among labs, tasks sets and instrumentation differences is not well enough considered as a problem in this new proposed method toolset, at least in my reading. Will replication, and testing across groups ever be truly feasible in this framework? Muscle based motor information sources have influences on different time scales in the task mechanics. Kinematic analyses, dynamic analyses and force plate analyses of the same task may provide task variables that alter the results in the proposed framework it seems.

Second, there is a sampling problem in all synergy analyses. We cannot record all muscles or all task parameters. Examining synergies across multiple tasks seeks 'stationary' compositionality. Including task specific elements may or may not reinforce or give increased coordinative precision to the stationary compositionality.<br /> To me the new methods proposed seem partly orthogonal to the ideas of stable compositionality. The 'synergies' obtained will likely differ, and are more likely to be coordinative control groupings of recurrent task and muscle motifs (based on instrumentation) which may or may not relate to core compositionality in physiology. Is there any expectation that the framework should relate to core compositionality and physiology. This is not clear in the paper as written.

It would be useful to explore the approach with a range of neuromechanical models and controllers and simulated data to explore the issues I am raising and convince readers that this analysis framework adds clarity rather than dissolving the generalizability and interpretability of analyses in terms of underlying causal mechanisms.

The authors need to better frame their work in relation to causal analyses if they are claiming links to muscle synergies analyses and claim extension/refinement. Alternatively, these may not be linked, and instead parallel approaches exploring different hypotheses and goals using different organizational data descriptors.<br /> To me this appears a data science tool that may not help any reductionist efforts and leads into less interpretable descriptions of motor control. Not invalid, but sufficiently different that common term use muddies the water.

Reviewer #2 (Public Review):

The authors present a computational tool for high-throughput generation of bacterial strain-specific metabolic models. The study seems interesting. However, I have the following concerns.

1. In the results section "description of Bactabolize", the authors present technical details on how to generate a metabolic model. For the input and output, please provide concrete examples to show the functionality of Bactabolize.

2. KpSC pan-metabolic reference model is provided. Are they required as input for Bactabolize? Are the gene, metabolite information open accessible by users?

3. To generate metabolic models, the authors present comparison results with other methods. However, the authors only present the numbers in genes, metabolites and substrates. Since the interactions between gene, metabolite, and substrate are also critical, if possible, please provide the coverage details about these interactions. Venn diagram is recommended to compare these coverage differences.

4. Are quality control and gap-filling needed to be processed when constructing a new metabolic model?

5. Are there any visualization results to check the status of the generated draft model?

Reviewer #2 (Public Review):

This manuscript describes colony-growth phenotypes to measure the fitness of deletion mutants for 3509 non-essential S. pombe genes in 131 conditions. 3492 mutants, including 124 mutants of 'priority unstudied' proteins conserved in humans, providing varied functional clues.

Phenotype-correlation networks provide evidence for the roles of poorly characterized proteins through guilt by association with known proteins. Gene Ontology (GO) terms were predicted using machine learning methods that take advantage of protein-network and protein-homology data.

Integrated analyses produced 1,675 novel GO predictions for 783 genes, including 47 predictions for 23 priority unstudied proteins. Experimental validation for genes involved in cellular ageing were obtained.

A method called NET-FF, which combines network embeddings and protein homology data to predict GO annotations, was developed. The authors demonstrate NET-FF predicts GO terms better than random and compare the information content of the predicted terms with the PomBase GO annotations. The phenotypic data was used to filter the GO annotation predictions made by NET-FF and then explore specific biological examples supported by both datasets

This is a very impressive and rich resource of phenotypic data and it will be particularly useful for the S. pombe research community and generally useful for the functional characterization of highly conserved eukaryotic genes. Overall, the analysis is powerful and sound.

Reviewer #2 (Public Review):

The purpose of this study is to develop a tool that serves as a starting point for investigating and uncovering genes and pathways associated with aging. The tool utilizes information from the GTEx public database, which contains post-mortem human data. It focuses on identifying age-related gene expression changes across different age range, biological sexes, and medical histories, with a focus on specific tissues.

Additionally, the authors envision the platform as continuously evolving, with ongoing development and expansion to include new data and features, ensuring it remains a cutting-edge resource for researchers studying aging.

# Strengths<br /> voyAGEr presents a tool for exploring gene expression changes across multiple tissues in the context of aging. One of the main strengths of the tool is its intuitive and user-friendly interface, which allows for easy navigation and exploration of gene expression patterns for biologists. Users can explore changes in gene expression of single genes across multiple tissues, enabling them to identify genes of interest that can be further investigated.

A particularly noteworthy strength of the tool is its ability to show tissue-specific gene expression patterns. This feature is essential for elucidating the paradigm of tissue-specific asynchronous aging and provides a unique and valuable resource for the aging community.

Overall, the tool offers an entry point for further investigation of genes involved in aging, and its ability to show tissue-specific gene expression patterns provides a unique and valuable resource for the scientific community.

Lastly, the tool is accompanied by a clear and thorough tutorial that explains each of its functionalities and provides examples. The authors also acknowledge the limitations of the statistical inference tests used in the tool, which adds to its overall transparency.

# Weaknesses

## Underlying data analysis<br /> In this tool/resource paper, it is crucial that the data used is up-to-date to provide the most comprehensive and relevant information to users. However, the authors utilized GTEx v7, which is an outdated (2016) version of the dataset. It is worth noting that GTEx v8 includes over 940 individuals, representing a 35% increase in individuals, and a 50% increase in the total number of samples. The authors should check the newer versions of GTEx and update the data.

The authors did not address any correction for batch effects or RNA integrity numbers, which are known to affect transcriptome profiles. For instance, our analysis of GTEx v8 Cortex tissue revealed that after filtering out lowly expressed genes, in the same way authors did, PC1 (which accounts for 24% of the variation) had a Spearman's correlation value of 0.48 (p<6.1e-16) with RNA integrity number.

The data analyzed in the GTEx dataset is not filtered or corrected for the cause of death, which can range from violent and sudden deaths to slow deaths or cases requiring a ventilator. As a result, the data may not accurately represent healthy aging profiles but rather reflect changes in the transcriptome specific to certain diseases due to the age-related increase in disease risk. While the authors do acknowledge this limitation in the discussion, stating that it is not a healthy cohort and disease-specific analysis is not feasible due to the limited number of samples, it would be useful for users to have the option to analyze only cases of fast death, excluding ventilator cases and deaths due to disease. This is typically how GTEx data is utilized in aging studies. Alternatively, the authors should consider including the "cause of death" variable in the model.

The age distribution varies across tissues which may impact the results of the study. The authors' claim that age distribution does not affect the outcomes is inconclusive. Since the study aims to provide cross-tissue analysis, it is important to note that differing age distributions across tissues can influence the overall results. To address this, the authors should conduct downsampling to different age distributions across tissues and evaluate the level of tissue-specific or common changes that remain after the distributions are made similar.

The GTEx resource is extremely valuable, however, it comes with challenges. GTEx contains tissue samples from the same individuals across different tissues, resulting in varying degrees of overlap in sample origin across tissues as not all tissues are collected for all individuals. This could affect the similar/different patterns observed across tissues. As this tool is meant for broader use by the community, it is crucial for the authors to either rule out this possibility by conducting a cross-tissue comparison using a non-parametric model that accounts for the dependency between samples from the same individual, or to provide information on the degree of similarity between samples so that the users can keep this possibility in mind when using the tool for hypothesis generation.

## Visualisation and analysis platform<br /> The authors aimed to create an open-source and ever-evolving resource that could be adapted and improved with new functionality. However, this goal was only partially achieved. Although the code for the web app is open source, crucial components such as the statistical tests or the linear model are not included in the repository, limiting the tool's customizability and adaptability.

Furthermore, the authors' choice of visualization platform (R shiny) may not be the best fit for extensibility and open-source collaboration, as it lacks modularity. A more suitable alternative could be production-oriented platforms such as Flask or FastAPI.

To facilitate collaboration and improve the tool's adaptability, data resulting from the pre-processing pipeline should be made publicly available. This would make it easier for others to contribute and extend the tool's functionality, ultimately enhancing its value for the scientific community.

Reviewer #2 (Public Review):

In this manuscript, Birkbak and colleagues use a novel approach to transform multi-omics datasets in images and apply Deep Learning methods for image analysis. Interestingly they find that the spatial representation of genes on chromosomes and the order of chromosomes based on 3D contacts leads to best performance. This supports that both 1D proximity and 3D proximity could be important for predicting different phenotypes. I appreciate that the code is made available as a github repository. The authors use their method to investigate different cancers and identify novel genes potentially involved in these cancers. Overall, I found this study important for the field.

The major points of this manuscript could be grouped in three parts:

1. While the authors have provided validation for their model, it is not always clear that best approaches have been used.<br /> a. In the methods there is no mention of a validation dataset. I would like to see the authors training on a cancer from one cohort and predict on the same cancer from a different cohort. This will convince the reader that their model can generalise. They do something along those lines for the bladder cancer, but no performance is reported. At the very least they should withhold a percentage of the data for validation. Maybe train on 100 and validate on the remaining 300 samples. They might have already done something along these lines, but it was not clear from the methods.<br /> b. It was not clear how they used "randomised cancer types as the negative control". Why not use normal tissue data or matched controls?<br /> c. If Figure 2B, the authors claim they have used cross validation. Maybe I missed it, but what sort of cross validation did they use?<br /> 2. Potential improvement to the method<br /> a. It is very encouraging the use of HiC data, but the authors used a very coarse approach to integrate it (by computing the chromosome order based on interaction score). We know that genes that are located far away on the same chromosome can interact more in 3D space than genes that are relatively close in 1D space. Did the authors consider this aspect? Why not group genes based on them being located in the same TAD?<br /> b. Authors claim that "given that methylation negatively correlates with gene expression, these were considered together". This is clearly not always the case. See for example https://genomebiology.biomedcentral.com/articles/10.1186/s13059-022-02728-5. What would happen if they were not considered together?<br /> 3. Interesting results that were not explained.<br /> a. In Figure 3A methylation seems to be the most important omics data, but in 3B, mutations and expression are dominating. The authors need to explain why this is the case.

Reviewer #2 (Public Review):

Murata et al have characterized a new transcription activator termed PFG, which regulates gene expression in female gametocytes. The authors show solid evidence that PFG is a partner of the previously described transcription factor AP2-FG and describe three sets of genes: genes activated by PFG or AP2-FG alone and genes activated by the complex. The authors also show differential binding to the target DNA sequences by AP2-FG to either a 10bp, if in a complex with PFG or a 5bp motif if alone. In all, this is a useful study which further elucidates the underlying regulatory network that drives development of sexual stages and ultimately transmission to mosquitoes. The data presented are clear and solid and the conclusions drawn are mostly supported by the results shown. However, in the absence of evidence of physical interaction, it remains unclear if AP2-FG and PFG actually interact directly or as part of the same complex.

Reviewer #2 (Public Review):

This manuscript focused on why aging leads to decreased beiging of white adipose tissue. The authors used an inducible lineage tracing system and provided in vivo evidence that de novo beige adipogenesis from Pdgfra+ adipocyte progenitor cells is blocked during early aging in subcutaneous fat. Single-cell RNA sequencing of adipocyte progenitor cells and in vitro assays showed that these cells have similar beige adipogenic capacities in vitro. Single-cell nucleus RNA sequencing of mature adipocytes indicated that aged mice have more Npr3 high-expressing adipocytes in the subcutaneous fat from aged mice. Meanwhile, adipocytes from aged mice have significantly lower expression of genes involved in de novo lipogenesis, which may contribute to the declined beige adipogenesis.

The mechanism that leads to age-related impairment of white adipose tissue beiging is not very clear. The finding that Pdgfra+ adipocyte progenitor cells contribute to beige adipogenesis is novel and interesting. It is more intriguing that the aging process represses Pdgfra+ adipocyte progenitor cells from differentiating into beige adipocytes during cold stimulation. Mature adipocytes that have high de novo lipogenesis activity may support beige adipogenesis is also novel and worth further pursuing. The study was carried out with a nice experimental design, and the authors provided sufficient data to support the major conclusions. I only have a few comments that could potentially improve the manuscript.

1. It is interesting that after three days of cold exposure, aged mice also have much fewer beige adipocytes. Is de novo adipogenesis involved at this early stage? Or does the previous beige adipocyte that acquired white morphology have a better "reactivation" in young mice? It would be nice if the author could discuss the possibilities.<br /> 2. Is the absolute number of Pdgfra+ cells decreased in aged mice? It would be nice to include quantifications of the percentage of tomato+ beige adipocytes in total tomato+ cells to reflect the adipogenic rate.

Reviewer #2 (Public Review):

This is a very interesting paper about the coupling of Slack and Nav1.6 and the insight this brings to the effects of quinidine to treat some epilepsy syndromes.

Slack is a sodium-activated potassium channel that is important to hyperpolarization of neurons after an action potential. Slack is encoded by KNCT1 which has mutations in some epilepsy syndromes. These types of epilepsy are treated with quinidine but this is an atypical antiseizure drug, not used for other types of epilepsy. For sufficient sodium to activate Slack, Slack needs to be close to a channel that allows robust sodium entry, like Nav channels or AMPA receptors. but more mechanistic information is not available. Of particular interest to the authors is what allows quinidine to be effective in reducing Slack.

In the manuscript, the authors show that Nav, not AMPA receptors, are responsible for Slack's sensitization to quinidine blockade, at least in cultured neurons (HeK293, primary cortical neurons). Most of the paper focuses on the evidence that Nav1.6 promotes Slack sensitivity to quinidine.

The paper is very well written although there are reservations about the use of non-neuronal cells or cultured primary neurons rather than a more intact system. I also have questions about the figures. Finally, riluzole is not a selective drug, so the limitations of this drug should be discussed. On a minor point, the authors use the term in vivo but there are no in vivo experiments.

Reviewer #2 (Public Review):

This paper introduces a new model that aims to explain the generators of temporal decoding matrices (TGMs) in terms of underlying signal properties. This is important because TGMs are regularly used to investigate neural mechanisms underlying cognitive processes, but their interpretation in terms of underlying signals often remains unclear. Furthermore, neural signals are often variant over different instances of stimulation despite behaviour being relatively stable. The author aims to tackle these concerns by developing a generative model of electrophysiological data and then showing how different parameterizations can explain different features of TGMs. The developed technique is able to capture empirical observations in terms of fundamental signal properties. Specifically, the model shows that complexity is necessary in terms of spatial configuration, frequencies and latencies to obtain a TGM that is comparable to empirical data.

The major strength of the paper is that the novel technique has the potential to further our understanding of the generators of electrophysiological signals which are an important way to understand brain function. Furthermore, the used techniques are state-of-the-art and the developed model is publicly shared in open source code.

On the other hand, the results of comparisons between simulations and real data are not always clear for an inexperienced reader. For example, the comparisons are qualitative rather than quantitative, making it hard to draw firm conclusions. Relatedly, it is unclear whether the chosen parameterizations are the only/best ones to generate the observed patterns or whether others are possible. In the case of the latter, it is unclear what we can actually conclude about underlying signal generators. It would have been different if the model was directly fitted to empirical data, maybe of different cognitive conditions. Finally, the neurobiological interpretation of different signal properties is not discussed. Therefore, taken together, in its currently presented form, it is unclear how this method could be used exactly to further our understanding of the brain.

Reviewer #2 (Public Review):

The article presents 'Mesotrode,' a technique that integrates chronic widefield calcium imaging and electrophysiology recordings using tetrodes in head-fixed mice. This approach allows recording the activity of a few single neurons in multiple cortical/subcortical structures, in which the tetrodes are implanted, in combination with widefield imaging of dorsal cortex activity on the mesoscale level, albeit without cellular resolution. The authors claim that Mesotrode can be used to sample different combinations of cortico-subcortical networks over prolonged periods of time, up to 60 days post-implantation. The results demonstrate that the activity of neurons recorded from distinct cortical and subcortical structures are coupled to diverse but segregated cortical functional maps, suggesting that neurons of different origins participate in distinct cortico-subcortical pathways. The study also extends the capability of Mesotrode by conducting electrophysiological recordings from the facial motor nerve. It demonstrates that facial nerve spiking is functionally associated with several cortical areas( PTA, RSP, and M2), and optogenetic inhibition of the PTA area significantly reduced the facial movement of the mice.

Studying the relationship between widefield cortical activity patterns and the activity of individual neurons in cortical and subcortical areas is very important, and Murphy's lab has been a pioneer in the field. However, the choice of low-yield recording methods (tetrode) instead of more high-yield recording techniques, such as silicon probes, makes the approach presented in this study somewhat less appealing. Also, the authors claim that a tetrode-based approach can allow chronic recordings of single neural activity over days - a topic that is very controversial. In terms of results, I was under the impression that most of the conclusions presented in the bulk of the paper ( Figures 1-5) are very similar to what previous work from Murphy's lab and other labs has shown using acute preparation. In this respect, the paper can benefit from a more in-depth analysis of the heterogeneity of single-neuron functional coupling. The last part of the facial nerve recording is interesting (Figure 6), but I think it can be integrated better into the rest of the paper.

Reviewer #2 (Public Review):

In this manuscript, Hoops et al., using two different model systems, identified key developmental changes in Netrin-1 and UNC5C signaling that correspond to behavioral changes and are sensitive to environmental factors that affect the timing of development. They found that Netrin-1 expression is highest in regions of the striatum and cortex where TH+ axons are travelling, and that knocking down Netrin-1 reduces TH+ varicosities in mPFC and reduces impulsive behaviors in a Go-No-Go test. Further, they show that the onset of Unc5 expression is sexually dimorphic in mice, and that in Siberian hamsters, environmental effects on development are also sexually dimorophic. This study addresses an important question using approaches that link molecular, circuit and behavioral changes. Understanding developmental trajectories of adolescence, and how they can be impacted by environmental factors, is an understudied area of neuroscience that is highly relevant to understanding the onset of mental health disorders. I appreciated the inclusion of replication cohorts within the study.

Reviewer #2 (Public Review):

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

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

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

The task seems to have lower approach bias than some other AAC tasks in the literature. Although this was inferred by looking at Fig 2 (it doesn't seem to drop below 46%) and Fig 3d seems to show quite a strong approach bias when using a reward/punishment sensitivity index. It would be good to confirm some overall stats on % of trials approached/avoided overall.

Weaknesses:<br /> The negative reliability of punishment learning rate is concerning as this is an important outcome.

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

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

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

Reviewer #2 (Public Review):

The authors combine genetic tools, dye fills and connectome analysis techniques to generate a "first-of-its-kind", near complete, synaptic resolution map of the head bristle neurons of Drosophila. While some of the BMN anatomy was already known based on previous work by the authors and other researchers, this is the first time a near complete map has been created for the head BMNs at electron microscopy resolution.

Strengths:<br /> 1. The authors cleverly use techniques that allow moving back and forth between periphery (head bristle location) and brain, as well as moving between light microscopy and electron microscopy data. This allows them to first characterize the pathways taken by different head BMNs to project to the brain and also characterize anatomical differences among individual neurons at the level of morphology and connectivity.<br /> 2. The work is very comprehensive and results in a near complete map of all head BMNs.<br /> 3. Authors also complement this anatomical characterization with a first-level functional analysis using optogenetic activation of BMNs that results in expected directed grooming behavior.

Weaknesses:<br /> 1. The clustering analysis is compelling but cluster numbers seem to be arbitrarily chosen instead of by using some informed metrics.<br /> 2. It could help provide context if authors revealed some of the important downstream pathways that could explain optogenetics behavioral phenotypes and previously shown hierarchical organization of grooming sequences.<br /> 3. In contrast to the rigorous quantitative analysis of the anatomical data, the behavioral data is analyzed using much more subjective methods. While I do not think it is necessary to perform a rigorous analysis of behaviors in this anatomy focused manuscript, the conclusions based on behavioral analysis should be treated as speculative in the current form e.g. calling "nodding + backward walking" as an avoidance response is not justified as it currently stands. Strong optogenetic activation could lead to sudden postural changes that due to purely biomechanical constraints could lead to a couple of backward steps as seen in the example videos. Moreover since the quantification is manual, it is not clear what the analyst interprets as backward walking or nodding. Interpretation is also concerning because controls show backward walking (although in fewer instances based on subjective quantification).

Summary:<br /> The authors end up generating a near-complete map of head BMNs that will serve as a long-standing resource to the Drosophila research community. This will directly shape future experiments aimed at modeling or functionally analyzing the head grooming circuit to understand how somatotopy guides behaviors.

Reviewer #2 (Public Review):

The manuscript provides new insight into a family of human enzymes. It demonstrates that STEAP2 can reduce iron and STEAP1 can be promiscuous regarding the source of electron donors that it can use. The quality of the kinetics experiment and the structural analysis is excellent. I am less enthusiastic about the interpretation of data and the take-home message that the manuscript intends to deliver. Above all, the work combines data on STEAP2 and STEAP1 and it remains unclear which questions are ultimately addressed. A second critical point is about the interpretation of the experiment demonstrating that STEAP1 can be reduced by cytochrome b5 reductase. The abstract states that "We show that STEAP1 can form an electron transfer chain with cytochrome b5 reductase" whereas the main text discusses the data by suggesting that "we speculate that FAD on b5R may partially dissociate to straddle between the two proteins". The two statements seem to be partly contradictory. Cytochrome b5 reductases do not easily release FAD but rather directly donate electrons to heme-protein acceptors (PMID: 36441026). According to the methods section, no FAD was added to the reaction mix used for the cytochrome b5 reductase experiment. Overall, the data seem to indicate that the reductase might reduce the heme of STEAP1 directly. Would it be possible to check whether FAD addition affects the kinetics of the process? And to perform a control experiment to check that NAD(P)H does not directly reduce the heme of STEAP1 (though unlikely)? A final point concerns the "slow Fe3+-NTA reduction by STEAP2". The reaction is not that slow as the initial phase is 2 per second. The reaction does not show dependence on the substrate concentration suggesting "poor substrate binding". I am not convinced by this interpretation. Poor substrate binding would give rise to substrate dependency as saturation would not be achieved. A possible interpretation could be that substrate binding is instead tight and the enzyme is saturated by the substrate. Can it be that the reaction is limited by non-productive substrate-binding and/or by interconversions between active and non-active conformations?

Roosevelt does not seem to have much faith in the words of the charter itself, but seems to call for example and action throughout her defense and explanation of the charter. She believed that only living the character would guide the actions and behavior of others. This hope that Roosevelt have would become real, as the U.N's declaration of human rights has become a point of behavioral guidance for humanity, as can be seen in the 50th anniversary of the U.N's declaration of human rights.

Roosevelt highlights this point which is very interesting, because the United Nations does not enforce the Declaration of human rights. Despite Roosevelt's assertive comments about human rights and the push for the U.N's declaration of human rights to be completed, the declaration of human rights has only served as moral guidance for the world.

Roosevelt here appeals to pathos to encourage motivation about the attempt of creating effort toward freedom and individual rights for everyone, where everyone has individual freedom and rights that are not controlled but belong to the individual, and are respected. The U.N has accomplished Roosevelt's vision of what the U.N's declaration of human rights should be to people and the world as is seen in the below documentation of the U.N's declaration of human rights' 50th anniversary.

Eleanor agrees to the inclusion of economic rights at the request of Russia. Russia argued that a declaration of human rights should include social and economic rights, not just political rights. The U.N's declaration of human rights originally included political rights, but not economic or social rights. Despite this, Russia still did not assent to the U.N's declaration of human rights, Roosevelts move here was to appease the Russians to draw them towards assenting to the U.N's declaration of human rights through persuasion by being agreeable to Russia's appeal to logos. This however did not work.

Despite what Roosevelt states here, she did not have the same approach to Russia when drafting the United Nations Declaration of human rights. She was often frustrated with their push to redefine human rights, and their push to include economic and social rights into the declaration of human rights. Despite her including economic rights in the declaration of human rights. Russia still did not want to agree with the content in the declaration of human rights.

The focal point of Roosevelt's essay is her frustration with communist countries. The attack on the U.N's declaration of human rights is primarily definitional in substance (though ideological in dispute). Although The U.N's declaration of human rights is presumptive about the terms democracy and human freedom, there is not universal agreement on what those terms mean.

Reviewer #2 (Public Review):

Weaver et al. used video analysis of flies that were feeding in their previously developed FLIC assay to begin to dissect the mechanisms of feeding. FLIC or Fly Liquid Interaction Counter records electrical signals that are generated when a fly touches a liquid food substrate with its legs or proboscis or both. Using video data of the liquid food interactions in the FLIC assay allowed the authors to precisely identify what a fly is doing in the feeding chamber and what the relationship is between the flies' behavior and the electrical signal recorded in the assay. This analysis produced the first detailed behavioral profile of feeding flies and allowed the authors to categorize different types of feeding in the FLIC assay, from tasting food (using their legs) to fast and long feeding bouts (using their proboscis).

After establishing what FLIC signals correspond to the different types of feeding, they used these signals to examine the food choices of starved and sated flies when presented with a sugar-rich (2% sucrose) or protein-rich (2% yeast + 1% sucrose) liquid food source. To represent hedonic feeding, they also presented flies with a choice between super sweet (20% sucrose) food or protein-rich (2% yeast + 1% sucrose) liquid food. Although fully fed flies show no difference in the number of times they visit either food choice, the flies spend more time feeding during their visits on 20% sucrose food than they do on regular sugar and on the yeast food source, suggesting that 20% sucrose is a more pleasurable food source. To make sure this was not due to the higher caloric content of 20% sucrose, they also offered flies food with the same sweetness as 20% sucrose (2% sucrose + 18% arabinose) but without caloric content and food with the same caloric content but the sweetness of 2% sucrose (2% sucrose + 18% sorbitol). This experiment showed that sweetness was the driver for the longer feeding bouts, confirming that sweeter food is apparently perceived as more pleasurable. They also looked at the effect of starving flies on the hedonic drive and found that starvation increases the time spent feeding on pleasurable food, consistent with findings in mammals that homeostatic feeding affects the hedonic drive.

To begin dissecting circuits underlying hedonic drive, the authors used CaMPARI expression in all neurons. CaMPARI is a green fluorescent reporter that turns red in the presence of Ca2+ (a measure of neuronal activity) and UV exposure. Fully fed flies in the super sweet food choice condition showed more red fluorescence in the mushroom bodies. Inhibiting a subset of these neurons acutely shows that horizontal lobes are required for the increased duration of feeding bouts on super sweet food. These lobes are innervated by a cluster of DA neurons and inhibiting them also blocks the increased super sweet feeding times.

The data in the paper largely support the conclusions. The application of this tool to distinguish between homeostatic and hedonic feeding is innovative and very compelling. As proof of the principle of the strength of their paradigm, the authors identify a distinct brain circuit involved in hedonic feeding. The methods established in the paper make a deeper understanding of feeding mechanisms possible at both a genetic and brain circuit level.

Reviewer #2 (Public Review):

Carla de la Fuente et al., utilize a diversity of approaches to understand which plant traits contribute to the stress resilience of pearl millet in the Sahelian desert environment. By comparing data resulting from crop modeling of pearl millet growth and meteorological data from a span of 20 years, the authors clearly determined that early season drought resilience is contributed by accelerated growth of the seedling primary root, which confirms a hypothesis generated in a previous study, Passot et al., 2016. To determine the genetic basis for this trait, they performed a combination of GWAS, QTL analysis, and RNA sequencing and identified a previously unannotated coding sequence of a glutaredoxin C9-like protein, PgGRXC9, as the strongest candidate. Phenotypic analysis using a mutant of the closest Arabidopsis homolog AtROXY19 suggests the broad conservation of this pathway. Comparisons between the transcript of PgGRXC9 by in situ hybridization (this work) and AtROXY19 pattern expression (Belin et al., 2014) support the hypothesis that this pathway acts in the elongation zone of the root. Additional analysis of cell production and elongation rates in root apex in both pearl millet and A. thaliana suggests that PgGRXC9 specifically regulates primary root through the promotion of cell elongation. While several studies have established the connection between redox status of cells and root growth, the current study represents an important contribution to the field because of the agricultural importance of the plant studied, and the connection made between this developmental trait and stress resilience in a specific and stressful environmental context of the Sahelian desert.

While the study presents a compelling narrative that is based on a diverse range of approaches, some aspects require further refinement to be fully convincing.

First, while it is appreciated that working with pearl millet presents certain technical challenges regarding genetic characterization, and the authors have done outstanding work by combing the power of GWAS and QTL mapping to reproducibly identify genetic loci associated with root growth, the related work in Arabidopsis is not fully substantiated. In particular, only one mutant allele was utilized to test the function of this gene in root growth. The lack of a second characterized allele or evidence of genetic complementation makes it difficult to definitively contribute the root developmental defects to the characterized mutation in ROXY19.

The role of redox status in contributing to root growth differences between accessions was not directly tested here. The manuscript is not able to mechanistically link the molecular function of ROXY19 to the change in root growth rate, however, this limitation of the study was not clearly described in the text.

The authors state the use of cell elongation rate (Morris and Silk, 1992) as a parameter to estimate the difference in root growth between contrasted pearl millet lines and A. thaliana roxy19 mutant versus wild type; however, there are inconsistencies in what data are presented. First, in Figure 2E, regarding the comparison between different genotypes of pearl millet lines, they use the parameter of maximum cell length but when authors compare cell elongation between A. thaliana genotypes, in Figure 4D, they use the elongation rate parameter. Second, while the cell elongation rate is based exclusively on the cell length data of the "elongation only" zone (Morris and Silk, 1992), the authors profile the cell length in the whole root apex, from the quiescent center to the beginning of the differentiation zone and it is not clear how they discriminate between each developmental zone and what data was used to estimate elongation rate.

Reviewer #2 (Public Review):

In this manuscript the authors present and characterize LOVtag, a modified version of the blue-light sensitive AsLOV2 protein, which functions as a light-inducible degron in Escherichia coli. Light has been shown to be a powerful inducer in biological systems as it is often orthogonal and can be controlled in both space and time. Many optogenetic systems target regulation of transcription, however in this manuscript the authors target protein degradation to control protein levels in bacteria. This is an important advance in bacteria, as inducible protein degradation systems in bacteria have lagged behind eukaryotic systems due to protein targeting in bacteria being primarily dependent on primary amino acid sequence and thus more difficult to engineer. In this manuscript, the authors exploit the fact that the J-alpha helix of AsLOV2, which unwinds into a disordered domain in response to blue light, contains an E-A-A amino acid sequence which is very similar to the C-terminal L-A-A sequence in the SsrA tag which is targeted by the unfoldases ClpA and ClpX. They truncate AsLOV2 to create AsLOV2(543) and combine this truncation with a mutation that stabilizes the dark state to generate AsLOV2*(543) which, when fused to the C-terminus of mCherry, confers light-induced degradation. The authors do not verify the mechanism of degradation due to LOVtag, but evidence from deletion mutants contained in the supplemental material hints that there is a ClpA dominated mechanism. They demonstrate modularity of this LOVtag by using it to degrade the LacI repressor, CRISPRa activation through degradation of MCP-SoxS, and the AcrB protein which is part of the AcrAB-TolC multidrug efflux pump. In all cases, measurement of the effect of the LOVtag is indirect as the authors measure reduction in LacI repression, reduction in CRISPRa activation, and drug resistance rather than directly measuring protein levels. Nevertheless the evidence is convincing, although seemingly less effective than in the case of mCherry degradation, although it is hard to compare due to the different endpoints being measured. The authors further modify LOVtag to contain a known photocycle mutation that slows its reversion time in the dark, so that LOVtag is more sensitive to short pulses of light which could be useful in low light conditions or for very light sensitive organisms. They also demonstrate that combining LOVtag with a blue-light transcriptional repression system (EL222) can decrease protein levels an additional 269-fold (relative to 15-fold with LOVtag alone). Finally, the authors apply LOVtag to a metabolic engineering task, namely reducing expression of octanoic acid by regulating the enzyme CpFatB1, an acyl-ACP thioesterase. The authors show that tagging CpFatB1 with LOVtag allows light induced reduction in octanoic acid titer over a 24 hour fermentation. In particular, by comparing control of CpFatB1 with EL222 transcriptional repression alone, LOVtag, or both the authors show that light-induced protein degradation is more effective than light-induced transcriptional repression. The authors suggest that this is because transcriptional repression is not effective when cells are at stationary phase (and thus there is no protein dilution due to cell division), however it is not clear from the available data that the cells were in stationary phase during light exposure. Overall, the authors have generated a modular, light-activated degron tag for use in Escherichia coli that is likely to be a useful tool in the synthetic biology and metabolic engineering toolkit.

Reviewer #2 (Public Review):

This is a well-written paper using gene expression in tree sparrow as model traits to distinguish between genetic effects that either reinforce or reverse initial plastic response to environmental changes. Tree sparrow tissues (cardiac and flight muscle) collected in lowland populations subject to hypoxia treatment were profiled for gene expression and compared with previously collected data in 1) highland birds; 2) lowland birds under normal condition to test for differences in directions of changes between initial plastic response and subsequent colonized response.

The question is an important and interesting one but I have several major concerns on experimental design and interpretations.

1) The datasets consist of two sources of data. The hypoxia treated birds collected from the current study and highland and lowland birds in their respective native environment from a previous study. This creates a complete confounding between the hypoxia treatment and experimental batches that it is impossible to draw any conclusions. The sample size is relatively small. Basically correlation among tens of thousands of genes was computed based on merely 12 or 9 samples.

2) Genes are classified into two classes (reversion and reinforcement) based on arbitrarily chosen thresholds. More "reversion" genes are found and this was taken as evidence reversal is more prominent. However, a trivial explanation is that genes must be expressed within a certain range and those plastic changes simply have more space to reverse direction rather than having any biological reason to do so.

3) The correlation between plastic change and evolved divergence is an artifact due to the definitions of adaptive versus maladaptive changes. For example, the definition of adaptive changes requires that plastic change and evolved divergence are in the same direction (Figure 3a), so the positive correlation was a result of this selection (Figure 3d).

Reviewer #2 (Public Review):

Balmas et al., continue the previous work from multiple groups that suggested the implication of uterine ILC2s and signals that activate them, i.e., IL-33/ST2 axis, in healthy and complicated pregnancies and move forward to understand further their role. The authors leverage available and appropriate tools to address more specifically the role of ILC2s during pregnancy and endotoxin-induced abortion, namely mouse models of selective ILC2 deficiency (Roraflox/floxIl7raCre/wt) and transcriptomic analysis of the immune response.

The authors demonstrate, and therewith confirm findings by Bartemes et al. (2018), that ILC2 reside in the mouse uterus, depend on IL-33 and expand during pregnancy. Moreover, they show the Il33 expression by CD45- cells of the uterine stroma. What remains unclear is the kinetics of Il33 expression and ILC2 expansion upon gestation and whether the local ILC2 population expands or arrives from the periphery.

Lack of maternal ILC2, in a mouse genetic model, resulted, as expected, in the absence of uILC2 but also in lighter fetuses at term, similar to the phenotype observed in the absence of maternal IL-33. It would be interesting to understand whether the effect of the IL-33 signaling is a direct ILC2 mediated effect, as for example by using the ST2flox/flox mice. Do the fetuses catch up in weight with their WT controls during weaning time? Do they have any long-term cognitive/behavioral impairment?<br /> The authors showcase the impairment in the remodeling of uterine wall vessels in dams lacking ILC2. It remains to be verified whether this is dependent on IL-33 and whether it is a direct effect of ILC2 or ILC2-dependent infiltration of eosinophils. Further, the absence of ILC2 is accompanied by an increase in Il1b in the uterine tissue suggesting that uILC2 contribute to the uterine microenvironment.

The authors perform RNA sequencing analysis on the bulk samples of uterine ILC2, where uILC2 cluster separately from corresponding lung and LN cells and are featured with higher expression of typical ILC2 markers. Somewhat odd, the authors report on the Foxp3/FoxP3 expression among uILC2, however the staining is not very bright and a Treg control as well as biological negative control should be provided. Moreover, FoxP3 is also not expressed among intestinal ILC2 with regulatory function (Wang et al. 2017). I suggest this data panel to be re-evaluated. A scRNA-Seq analysis would probably be more comprehensive in this case, but might be beyond the scope of this publication.

Absence of uILC2 results in the increased numbers of DCs, macrophages and neutrophils in the uterus, an impact which is not visible in the spleen, which is why the authors argue that this is a uterus-restricted phenomenon, although perturbances in the large intestine and lungs could be expected. Moreover, it remains to be investigated whether these effects are restricted to mid-term pregnancy or preserved until term.

Upon establishing the role of uILC2 in maintaining healthy pregnancy, the authors demonstrate a role for uILC2 in endotoxin-mimicked bacterial infection and abortion. An impressive set of data demonstrate that dams that lack uILC2 have a significantly higher fetus resorption rate than WT dams upon LPS challenge. It remains to be understood whether this phenotype is also dependent on IL-33. Finally, mechanistically, using a somewhat reductionist in vitro model, the authors suggest a protective feedback mechanism between type 2-secreting uILC2 and IL-1b-expressing DCs. This is an interesting concept that still needs a formal confirmation in vivo. Are uILC2 also subjected to plasticity upon IL-1b treatment (Ohne et al. 2016)?

In conclusion, the authors provide a well-conceived study that will be useful for reproductive and tissue immunologists. The data are collected using validated models and methodology and analyzed in a solid manner and can be used as a starting point for further mechanistic studies, assessing the protective potential of uILC2 in pregnancy during infections.

Reviewer #2 (Public Review):

Clary et al. utilized 2-photon intravital imaging techniques to investigate the dynamic behavior of Merkel cells and their innervation during homeostasis and hair regeneration. The authors demonstrated that both Merkel cells (Atoh1-GFP) and the branched axons (TrkC) innervating them undergo significant plasticity and remodeling during homeostasis. Merkel cells were added, removed, and relocated, while axons showed growth and regression. By taking advantage of live imaging, the authors identified two different ways in which Merkel cells interact with axons: creating the stable kylikes and the previously undescribed dynamic Bouton structure. Using live imaging and extensive quantification tools, the authors thoroughly characterized Merkel cell and axon plasticity. They found that Merkel cell plasticity is associated with the hair cycle, while axon plasticity is not. Moreover, newly generated Merkel cells have a short lifespan. By comparing the survival of afferents associated with Merkel cells to empty ones and analyzing Atoh1 cKO, the authors concluded that Merkel cells have a stabilizing effect on axon terminals.

Strengths:

The authors developed an intravital imaging system that enables the simultaneous tracking of both Merkel cells and axon branches. Live imaging, combined with numerous quantification tools, enabled an in-depth characterization of the different behaviors and dynamic nature of Merkel cells, axon branches, and their interaction. The authors' approach has the particular strength of allowing for the comparison of the dynamic behavior of axons associated with Merkel cells to those not innervating Merkel cells within the same touch dome, as well as describing a Bouton structure as a novel morphology mediating Merkel cell and nerve interaction.

Weaknesses:

Although the authors provide an in-depth analysis of Merkel cell dynamics and its association with hair growth, these concepts have been previously reported by the authors and others. Therefore, the extent of novel concepts and scientific advances should be better explained.

The authors suggest that Merkel cell association is a stabilizing factor on innervated axon branches by comparing branch plasticity between branches connected to Merkel cells and empty ones and using Atoh cKO. While the first set of experiments are compelling and provide interesting observations, additional experimental models, such as Merkel cell ablation in adults, may better strengthen the authors' claims. The authors currently use K14-Cre;Atoh1 cKO to support their observations. However, the absence of Merkel cell development in this model, might also lead to developmental defects in nerve patterning (absence of target organ) leading to the phenotype observed by the authors.

Finally, the authors use intravital imaging to describe the Bouton structure and dynamic. Though very interesting there is not enough data to support authors claim for interaction between axon and Merkel cells through the Bouton structure. The paper can benefit from additional functional analysis of this structure.

Reviewer #2 (Public Review):

In this study, Isoe and team produced an atlas of the telencephalon of the adult medaka fish with which they better defined pallial and subpallial regions, characterized the expression of neurotransmitters, and performed clonal analysis to address their organization and maintenance during the continuous neurogenesis. They show that pallial anatomical regions are formed by independent clonal units. Furthermore, the authors demonstrate that pallial compartments exhibit region-specific chromatin landscapes, suggesting that gene expression is differentially regulated. Specifically, synaptic genes have a distinct chromatin landscape and expression in one of the regions of the dorsal pallium, the Dd2. Using the region-specific RNA expression and chromatin accessibility data they have generated; the authors propose several transcription factors as candidate regulators of Dd2 specification. Lastly, the authors use the enrichment of transcription factor binding motifs to establish homology between medaka and human telencephalon, aiming to describe an evolutionary origin for the Dd2 region.

Overall, the study carefully describes diverse aspects of neurogenesis in the telencephalon of the adult medaka fish. As such, the manuscript has the potential to contribute insights to the understanding of circuits and neurogenesis in teleosts and the medaka fish, as well as the evolution of cellular heterogeneity and organization of the telencephalon. Furthermore, the atlas, if easily accessible to the broader community, could be a substantial resource to researchers interested in medaka and teleosts neuroscience. However, there are some conceptual and technical concerns that should be addressed to strengthen this work.

Improving the atlas: The different interpretations of the imaging data generated remain isolated or fragmented and could be better integrated to describe anatomical, connectivity, and ontogeny differences through pallial and subpallial regions.<br /> Molecular differences across regions and species: Differential gene expression and chromatin accessibility throughout regions should be better and more deeply characterized and presented, exhibiting more region-specific features, and leading to a better description of candidate transcription factors that could differentially regulate regional gene expression. The comparison between medaka fish and human telencephalon regions would benefit from a more extensive molecular analysis. Comparison of gene expression and accessible regions could expand the analysis together with TF-binding motif enrichment.<br /> Lineage tracing: The authors claim that the functional compartmentalization of the pallium relies on different cell lineages, which also mostly share connectivity patterns and, at least to some extent, expression patterns. It would be interesting to see how homogenous these lineages are at the molecular level and whether their compartmentalization is retained when neurons reach maturity.

Reviewer #2 (Public Review):

This study provides the proteomic and phosphoproteomics data for our understanding of the molecular alterations in adipose tissue and skeletal muscle from women with PCOS. This work is useful for understanding of the characteristics of PCOS, as it may provide potential targets and strategies for the future treatment of PCOS. While the manuscript presents interesting findings on omics and phenotypic research, the lack of in-depth mechanistic exploration limits its potential impact.

The study primarily presents findings from omics and phenotypic research, but fails to provide a thorough investigation into the underlying mechanisms driving the observed results. Without a thorough elucidation of the mechanistic underpinnings, the significance and novelty of the study are compromised.

Reviewer #2 (Public Review):

Human bactericidal/permeability-increasing protein (huBPI) is known to have in vitro antibacterial activity against Pa, but in vivo, its antibacterial activity is significantly lowered due to binding by autoantibodies called BPI-ANCA. The authors of this study hypothesized that non-human BPIs would escape neutralization by intrinsic BPI-ANCA and retain full antibacterial activity against Pa. Through bioinformatic analysis, the authors anticipated that scorpion BPI (scoBPI) has enough similarity with huBPI to retain antimicrobial activity while escaping recognition by BPI-ANCA. This hypothesis is supported by the following observations: 1) scoBPI fails to capture any BPI-ANCA, 2) scoBPI prevents E. coli- and Pa-LPS dependent inflammatory responses like huBPI and 3) scoBPI exhibits remarkable antimicrobial activity against MDR-Pa in the nanomolar range. Antimicrobial activity of scoBPI was also demonstrated against E. coli suggesting a conserved mechanism of activity against Gram-negative bacteria. The authors use immobilization methods to demonstrate that scoBPI does not bind BPI-ANCA, but a drawback of this method is that some molecular interactions may be disrupted due to immobilization. Moreover, any inhibitory effects of BPI-ANCA on scoBPI activity in the bactericidal assays were not explored. Regardless, the results of this study clearly support their original hypothesis. These findings have broad implications in identifying novel chemotherapies to treat drug-resistant Gram-negative bacterial infections.

Reviewer #2 (Public Review):

The paper by Arribas et al. examines the coding properties of adult-born granule cells in the hippocampus at both single cell and network level. To address this question, the authors combine electrophysiology and modeling. The main findings are:<br /> - Noisy stimulus patterns produce unreliable spiking in adult-born granule cells, but more reliable responses in mature granule cells.<br /> - Analysis of spike patterns with a spike response model (SRM) demonstrates that adult-born and mature GCs show different coding properties.<br /> - Whereas mature GCs are better decoders on the single cell level, heterogeneous networks comprised of both mature and adult-born cells are better encoders at the network level.

Based on these results, the authors conclude that granule cell heterogeneity confers enhanced encoding capabilities to the dentate gyrus network.

Although the manuscript contains interesting ideas and initial data, several major points need to be addressed.

Major points:<br /> 1. The authors use and noisy stimulation paradigm to activate granule cells at a relatively high frequency. However, in the intact network in vivo, granule cells fire much more sparsely. Furthermore, granule cells often fire in bursts. How these properties affect the coding properties of granule cells proposed in the present paper remains unclear. At the very least, this point needs to be better discussed.

2. The authors induce spiking in granule cells by injection of current waveforms. However, in the intact network, neurons are activated by synaptic conductances. As current and conductance have been shown to affect spike output differently, controls with conductance stimuli need to be provided. Dynamic clamp is not a miracle anymore these days.

3. The greedy procedure is a good idea, but there are several issues with its implementation. First, it is unclear how the results depend on the starting value. What we end up with the same mixed network if we would start with adult-born cells? Second, the size of the greedy network is very small. It is unclear whether the main conclusion holds in larger networks, up to the level of biological network size (1 million). Finally, the fraction of adult-born granule cells in the optimal network comes out very large. This is different from the biological network, where clearly four or five-week-old granule cells cannot represent the majority. Much more work is needed to address these issues.

4. Likewise, the idea of dynamic pattern separation seems quite nice. However, the authors focus on the differences between mixed and pure networks, which are extremely small. Furthermore, the correlation coefficients of "low", "medium", and "high" correlation groups are chosen completely arbitrarily. A correlation coefficient of 0.99, considered low here, would seem extremely high in other contexts. Whether dynamic pattern separation is possible over a wider range of input correlation coefficients is unclear (see O'Reilly and McClelland, 1995, Hippocampus, for a possible relationship). Finally, aren't code expansion and lateral inhibition the key mechanisms underlying pattern separation? None of these potential mechanisms are incorporated here.

5. A main conclusion of the paper is that while mature GCs are better decoders on the single cell level, heterogeneity in mixtures improves coding in neuronal networks. However, this seems to be true only for r^2 as a readout criterion (Fig. 4F). For information, the result is less clear (Fig. 4G). The results must be discussed in a more objective way. Furthermore, intuitive explanations for this paradoxical observation are not provided. Saying that "this is an interesting open question for future work" is not enough.

6. The authors ignore possible differences in the output of mature and adult-born granule cells in their thinking. If mature and adult-born granule cells had different outputs, this could affect their contributions to the code (either positively or negatively). At the very least, this possibility should be discussed.

Reviewer #2 (Public Review):

In this manuscript Toshima et al document the use of sophisticated microscopy - with powerful spatial and time resolution - to image markers of the yeast endosomal system.

The initial work documented in this paper does a good job of defining the compartment endocytic cargoes internalise to. This is convincingly shown to be a compartment that is not marked by Sec7 but is instead a distinct (sub)compartment marked by the SNARE protein Tlg2. This agrees with many previous studies, (including biochemical experiments and microscopy of cargoes in a series of membrane trafficking mutants) but has different conclusions to another study (Day et al 2018 - Developmental Cell). Although the microscopy techniques used in the two studies are different, the yeast system and many of the reporters (FP tagged Tlg1, Sec7, Vps21 and fluorescently labelled mating factor) are the same. The Day et al study is suitably referenced throughout the manuscript but as to why the authors have come to fundamentally different answers about endocytic cargoes internalising to a Sec7+ compartment, is not discussed.

The work goes on to show endocytic carriers (marked by Abp1) and endocytic cargoes like fluorescently labelled mating factor internalise to the Tlg2+ compartment. The forward trafficking of these molecules is then observed to transit to a later endosome compartment labelled by Vps21. The super-resolution and time lapse imaging, sometimes even using 3 colours - is of very high quality and fully support the model presented at the end of the paper for this trafficking itinerary. Trafficking mutants are also used (such as a defective allele of arp3 and deletion of VPS21 / YPT52 GTPases) to interrupt trafficking routes and define the pathways followed by endocytosed mating factor.

The endocytic trafficking from Tlg2+ to Vps21 compartments is shown to be defective in mutants lacking GGA adaptors (gga1∆ gga2∆), with cargoes accumulating in the Tlg2+ compartment and other clathrin adaptor mutants not causing this defect. This research avenue also reveals that the GGA proteins are required to maintain the distinct Tlg2 sub compartment.

The final section of the paper uses the same tools to analyse the localisation of the recycling v-SNARE protein Snc1. This is arguably the most important set of experiments in the paper, not only is Snc1 a putative v-SNARE that functionally interacts with Tlg2, but this cargo, unlike pheromone, allows the investigation of recycling back to the PM from TGN/endosomes. However, the authors do not comment on the fact that Snc1 does not localise to the plasma membrane in either experiments using different microscopy techniques (Figure 5A + 5B), calling into question whether the recycling pathway is operating properly or that the FP-tagged machinery has disrupted processing? The steady state localisation of Snc1 in WT cells only looks normal in Supplemental figure, this discrepancy should be discussed or addressed.

Reviewer #2 (Public Review):

This study describes the emergence of virulent strains of the rice bacterial blight pathogen Xanthomonas oryze pv. oryzae in the Morogoro rice-growing region in Tanzania. The aims of the study were to describe the virulence features of the emerging population, as compared to previous bacterial blight outbreaks in Africa, and generate an elite rice variety that is resistant to both pathogen populations. To achieve these aims, the authors characterized the genetic basis of the virulence of these new strains by sequencing the genomes of three representative strains and phenotyping using excellent genetic resources for identifying the susceptibility gene targets of this pathogen in rice. They then used two rounds of hybrid CRISPR-Cas9/Cpf1 to successfully edit six targets of the pathogen in an East African rice variety, which conferred resistance to all strains tested.

The strengths of this paper are the systematic analysis of the virulence of emerging pathogen strains relative to strains from previous outbreaks and the successful creation of edited lines that will form the basis for continued efforts to gain regulatory approval for the introduction of resistant rice in East Africa. The creation of the edited line is a substantial and important contribution, indeed, the authors include strains collected in 2021 and include disease severity data from 2022 in the supplementary data, illustrating the urgent need for solutions.

The weaknesses of the study are largely related to the quick turnaround between data collection and manuscript submission.<br /> (1) Different strains are used for different experimental work and sequence analysis, making relationships between different parts of the work unclear and also more challenging for the reader to follow because of changing strain designations. CIX4457, CIX4458, and CIX4462 were virulent on rice near-isogenic-lines, CIX4457 and CIX4505 were used for identifying SWEET targets and phenotyping edited lines, while whole genome sequencing was conducted with CIX4462, CIX4506, CIX4509.<br /> (2) Disease survey results from 2022 are listed in Supplementary Table 2, but it is challenging for the reader to summarize across many lines of data, which appear to represent individual samples.<br /> (3) The focus of the editing is Komboka but bacterial blight in 2022 was mostly on other varieties. It would be helpful to have more context on this variety and what has prevented adoption by the growers in the Morogoro region to date.

Reviewer #2 (Public Review):

The study "Postinspiratory complex acts as a gating mechanism regulating swallow-breathing coordination and other laryngeal behaviors" by Huff et al., provides additional insight into the role of the recently discovered postinspiratory complex during swallow-breathing coordination. The authors used optogenetics in mice to show that activation of the PiCo during inspiration or at the start of post-inspiration can evoke swallowing. At later stages of expiration, PiCo activation activates undefined laryngeal activities. The analysis of respiratory phase reset leads to the conclusion that the PiCo is important for central gating of swallow. In conclusion, the authors claim that swallow-breathing coordination depends on a defined microcircuit compromising the PiCo and the pre-Botzinger complex.

Reviewer #2 (Public Review):

The manuscript "Phosphorylation of tyrosine 90 in SH3 domain is a new regulatory switch controlling Src kinase" describes efforts to understand how phosphorylation of tyrosine (Y90) in the SH3 domain of Src affects the activity and function of this multi-domain kinase. The authors find that an Src variant containing a phospho-mimetic mutation (Glu) at position 90 demonstrates elevated activation levels in lysates and cells (Figure 1) and adopts a less compact autoinhibited conformation within the context of a SrcFRET biosensor in lysates (Figures 3A, 3B). A series of pulldown experiments with an isolated SH3 domain (Figure 2A, 2B) or full-length Src (Figure 2C, 2D) that contain the phospho-mimetic Y90E mutation demonstrates that phosphorylation of Tyr90 would likely disrupt the interaction of Src's SH3 domain with intermolecular binding partners and the linker that couples SH2 domain/C-tail binding to autoinhibition, which provides a mechanistic basis for the observed elevated kinase activity of Src Y90E. By performing a series of imaging experiments with a SrcFRET biosensor, the authors show that the Y90E mutation does not show enhanced localization at focal adhesions like a hyperactivated Src mutant (Y527F) that contains a non-phosphorylatable C-tail (Figure 4A). However, using ImFCS combined with TIRF microscopy (Figure 4B), the authors demonstrate that Src Y90E shows similarly reduced mobility (relative to the WT SrcFRET biosensor) at the plasma membrane (especially at focal adhesions) as Src Y527F. Consistent with the elevated kinase activity of Src Y90E, the authors go on to demonstrate that the Src Y90E variant shows an ability to transform fibroblasts-at levels that are intermediate between wild-type Src and the hyperactive Src mutant Y527F (Figure 5). Similarly, Src Y90E confers an intermediate level (between wild-type Src and Src Y527F) of invasiveness and ability to form spheroids. Together, these comprehensive experiments with a Y90 phospho-mimetic strongly support a model where phosphorylation of Src's SH3 domain at Tyr90 would lead to a more intramolecularly disengaged SH3 regulatory domain and enhanced kinase activity in cells.

Most of the conclusions in this paper are well supported by solid data, but confidence in several assays would be higher if additional technical detail or controls were provided and the biological significance of these findings would be higher if the role that Y90 phosphorylation plays in Src regulation and function were better delineated.

1) The kinase activity assays in Figures 1C,1D, and 7A need to be scaled to the Src variant levels present in the lysate (quantification of relative Src levels is not provided).

2) More details are required for the experiments quantifying Y90 phosphorylation levels in Figure 3C. The experimental states that equal amounts of IP'd proteins were used for these analyses but there are no details on how this was confirmed. In addition, the experimental states that normalized intensities were used for your quantifying the Y90 phospho-peptide but no details are provided on how normalization was performed (the legend states that a base peptide was used but it is unclear what this means).

3) A key question is whether Y90 phosphorylation serves a regulatory role in Src's cellular activity and, if so, what is the regulatory network that mediates this phospho-event. Using a mass spectrometry readout with three Src variants (wild type vs. Y527F vs. E381G) that possess differing kinase activities, the authors demonstrate that Y90 phosphorylation levels correlate to Src's kinase activity (Figure 3C), which they suggest is an indication that this residue is an autophosphorylation site (or phosphorylated by another Src family kinase). However, as Src's kinase activity correlates with SH3 domain disengagement (which leads to a more accessible Y90), it is also entirely possible that another tyrosine kinase is responsible for this phosphorylation event. More importantly, it is unclear under which signaling regime Y90 phosphorylation would play a significant regulatory role. This phospho-event was observed in a previous phospho-proteomic study but it is unclear whether the phosphorylation levels of this site occur high enough stoichiometry to modulate the intracellular function of Src and whether there is a regulatory signaling network that influences Y90 phosphorylation levels.

Reviewer #2 (Public Review):

In their manuscript, Markicevic et al. report that manipulation of D1 spiny neurons in the right dorsomedial striatum results in a behavioral effect observed in motor movement. This behavioral effect is accompanied by changes in BOLD fMRI changes as estimated by a classification approach and pairwise regional correlation. These brain-wide analyses reveal a number of important outcomes. First, alterations in signal dynamics are observed in the striatum most dominantly in the injection site when contrasting excitation to inhibition. Second, thalamic regions that have reciprocal anatomical connections with the injection site show greater classification accuracy. Third, evaluation of cortical regions demonstrates increased classification accuracy for unimodal regions including primary motor, visual, primary somatosensory, and posterior parietal association regions. Lastly, using pairwise correlations, a decrease is observed when comparing excitation to either inhibition or no modulation of activity in the primary motor cortex, anterior cingulate, and retrosplenial cortices.

This report effectively demonstrates that excitation or inhibition of a large population of D1 spiny neurons results in disruption of basic motor behavior. The greatest strength of the work is derived from identifying that features in the time-series of regions in the thalamus that project and receive projections to the injected site are impacted as well unimodal cortical regions. Moreover, a differential effect is observed for excitatory drive relative to both no drive and inhibition. The use of the approach by Fulcher and Jones (2017) provides an important addition to the more commonly used pairwise correlation approach as it relies on the dynamics of the fMRI signal.

While the methods adopted by the authors to acquire the data and evaluate the experimental manipulations are robust and the obtained results are compelling, the current analysis comes short of relating whether variation that can be estimated across the animals has an impact on these results. Specifically, the authors do not leverage the individual animal viral expression or impact on behavior to constrain and estimate the observed responses reported subsequently. Several reports in humans have used individual variability to estimate the relation between behavior and changes in the BOLD fMRI responses at rest, and a basic demonstration of this type of result has been achieved in mice. Applying a similar approach here would further strengthen the result reported here by identifying which regions are linked to the behavioral deficit (e.g., whether the primary motor cortex is linked to contraversive/ipsiversive rotations at the individual level).

Complementing linking the behavior of individual animals to changes in the fMRI signal, an estimation of structure-function that is driven by each individual animal's expression map may enhance the current analysis approach by leveraging potential subtle expression variations to reveal whether the observed changes can be explained by the extent to which expression is different across animals. In addition, a quantification of the difference between the excitatory and inhibitory cohorts will rule out that differences in the impact on the fMRI signal were a result of unintentional group differences in expression extent.

A significant weakness in the current version of the manuscript is the lack of quantification of the viral expression. Currently, the authors do not provide enough information on the extent of coverage of viral expression on average or at the individual level. In particular, while the authors are careful to use the Allen Mouse Brain Connectivity atlas to constrain the fMRI results, they do not relate the specific expression extent, to clearly communicate to the readers, which regions within the striatum are likely to have better representation given the actual expression levels. Moreover, the authors do not use their own nor the Allen Institute data to carry out a formal structure-function analysis (following Stafford et al., 2014 PNAS, for example). This is critical since the authors wish to infer on the impact of their manipulation on both cortical and thalamic regions while the precise region in the striatum that they affect is never quantified.

Reviewer #2 (Public Review):

This is a very dense and thorough analysis of the role of Uso1 in Aspergillus using genetics, pulldown assays, and modelling.<br /> Uso1 has been established as an essential tethering factor that acts in conjunction with Rab1 to deliver ER-derived vesicles to the Golgi. The current picture is that Uso1 is a Rab1 effector, but the authors challenge this interpretation using a combination of genetics experiments, biochemical analysis of protein-protein interactions, and alphafold2 prediction.

While Rab1 is essential, they identify strains of Aspergillus that bypass the need for Rab1, which carry two mutations in Uso1. They go on to show that Uso1 binds directly to the Bos1 and Bet1 components of the SNARE complex and that the rescue mutations cause tighter binding of the Uso1 globular head domain to Bos1 and (hypothetically) to the membrane. They support their genetics and biochemical analysis by doing structure predictions with alphafold2 and suggesting how these mutants might act. They also show that an overexpressed mutated monomeric globular domain of Uso1 (without the coiled-coil 'tether' that causes dimerisation) rescues growth defects of delta Uso1, suggesting the essential activity of Uso1 is not the tethering but its being part of the SNARE complex.

The data is solid, and the interpretation is convincing, showing Uso1 is not 'merely' a tethering factor. It has multiple roles, and this study opens up new questions regarding what exactly is Uso1's function as part of the SNARE bundle, and also in which way the Rab1-mediated tethering and the SNARE complex aspects of Uso1 are linked and/or regulated.

However, there are some aspects of this work that need to be strengthened/clarified including some of the modelling and the interpretation of the role of Uso1 dimerisation. Also, given the availability of models for all homologues, it would be interesting to test whether analogous Uso1 mutant in S.cerevisiae can also rescue rab1- lethality. This would suggest the new proposed role of Uso1 is a general feature, at least for fungi, rather than a particularity of Aspergillus.

Reviewer #2 (Public Review):

The authors of this study describe a goal of elucidating the signaling pathways that are upregulated in tendinopathy in order to target these pathways for effective treatments. Their goal is honorable, as tendinopathy is a common debilitating condition with limited treatments. The authors find that IL-6 signaling is upregulated in human tendinopathy samples with transcriptomic and GSEA analyses. The evidence of their initial findings are strong, providing a clinically-relevant phenotype that can be further studied using animal models.

Along these lines, the authors continue with an advanced in vitro system using the mouse tail tendon as the core with progenitors isolated from the Achilles tendon as the external sheath embedded in a hydrogel matrix. One question that comes to mind is whether the fibroblast progenitors in the extrinsic sheath of Achilles tendon is similar to those surrounding the tail tendon. The similarity of progenitors between different tendons is assumed with this model. I would consider this to be a minor issue, and would consider the in vitro system to be an additional strength of this study.

In order to address the IL-6 signaling pathway, the authors use core tendons from IL-6 knockout mice and progenitors from wild-type mice. The reasoning behind this approach was a little confusing... is IL-6 expressed solely in the tendon core compared to the extrinsic sheath? Furthermore, is a co-culture system for 7 days appropriate to model tendinopathy without the supplementation of exogenous inflammatory compounds? The transcriptomic differences in Figure 3 seem to be subtle, and may perhaps suggest that it could be a model that more closely resembles steady state compared to tendinopathy. If so, is IL-6 still relevant during steady state?

Nevertheless, the results presented in Figures 4 and 5 are impressive, demonstrating a link between IL-6 and fibroblast progenitor numbers and migration. Their experimental design in these figures show strong evidence, using Tocilizumab and recombinant IL-6 to rescue shown phenotypes. I would reduce the claims on proliferation, however, unless a proliferation-specific marker (e.g., Ki67, BrdU, EdU) is included in confocal analyses of Scx+ progenitors. The Achilles tendon injury model provides a nice in vivo confirmation of Scx-progenitor migration to the neotendon.

Given their goal to elucidate signaling pathways that could be targeted in the clinic, I think it would significantly strengthen the study if they could measure tendon healing in IL-6 knockouts or in wild-type mice treated with IL-6 inhibitors, since conventional ablation of IL-6 may lead to the elevation of compensatory IL-6 superfamily ligands that could activate STAT signaling. The authors claim that reducing IL-6 signaling decreases transcriptomic signatures of tendinopathy, but IL-6 may be necessary to promote normal healing of the tendon following injury. It is supposed that a lack of Scx+ progenitor migration would delay tendon healing.

Overall, the authors of this study elucidated IL-6 signaling in tendinopathy and provided a strong level of evidence to support their conclusions at the transcriptomic level. However, functional studies are needed to confirm these phenotypes and fully support their aims and conclusions. With these additional studies, this work has the potential to significantly influence treatments for those suffering from tendinopathy.

Reviewer #2 (Public Review):

The authors aimed to examine the role of a group of neurons expressing Foxb1 in behaviors through projections to the dlPAG. Standard chemogenetic activation or inhibition and optogentic terminal activation or inhibition at local PAG were used and results suggested that, while activation led to reduced locomotion and breathing, inhibition led to a small degree of increased locomotion.

The observed effects on breathing are evident and dramatic. However, this study needs significant improvements in terms of data analysis and presentation and some of studies seem incomplete; and therefore the data may not yet support the conclusion.

1) Fig.1 has no experimental data and needs to be replaced with detailed pictures from the viral injected mice showing the projections diagrammed.

2) Fig. 3 needs control pictures and statistical comparison with different conditions in c-Fos. Also expression in other nearby regions needs to be presented to demonstrate the specificity of the expression.

3) Fig. 5, a great effort has been made to illustrate the point that CCK and Foxb1 are differentially expressed. Why not just perform a double in situ experiment to directly illustrate the point?

4) Fig. 7 data on optogenetic stimulation on immobility and breathing, since not all mice showed the same phenotype, what is the criterion for allocating these mice to hit or no hit groups? Given the dramatically reduced breathing and locomotion, what is the temperature response? More data needs to be gathered to support that this is a defense behavior.

5) The authors claim to target dlPAG. However, in the picture shown in Fig. 8C, almost all PAG contains ChR2 fibers and it is likely all the fibers will be activated by light. Thus, as presented, the data does not support the claim of the specificity on dlPAG. Also c-Fos data needs to be presented on the degree of activation of downstream PAG neurons after light exposure.

6) Fig. 9 only showed one case. A statistical comparison needs to be presented.

7) Optogentic terminal activation in the PAG will likely elicit back-propagation and subsequent activation of additional downstream brain sites of Foxb1 neurons. More experiments need to be done to assess this and as presented, the data does not support the role of PAG necessarily.

8) The authors claim negative data from PVH-Cre mice. More data need to be presented to make this case.

The conclusion, even as presented, adds to the known evidence of the PAG in the defense behavior.

Reviewer #2 (Public Review):

This manuscript investigates the role of Perk (Protein kinase RNA-like endoplasmic reticulum kinase) and Atf4 (Activating Transcription Factor-4) in neurodegenerative and regenerative responses following optic nerve injury. The authors employed conditional knockout mice to examine the impact of the Perk/Atf4 pathway on transcriptional responses, with a particular focus on canonical Atf4 target genes and the involvement of C/ebp homologous protein (Chop).

The study demonstrates that Perk primarily operates through Atf4 to stimulate both pro-apoptotic and pro-regenerative responses after optic nerve injury. This Perk/Atf4-dependent response encompasses canonical Atf4 target genes and limited contributions from Chop, exhibiting overlap with c-Jun-dependent transcription. Consequently, the Perk/Atf4 pathway appears crucial for coordinating neurodegenerative and regenerative responses to central nervous system (CNS) axon injury. Additionally, the authors observed that neuronal knockout of Atf4 mimics the neuroprotection resulting from Perk deficiency. Moreover, Perk or Atf4 knockout hinders optic axon regeneration facilitated by the deletion of the tumor suppressor Pten.

These findings contrast with the transcriptional and functional outcomes reported for CRISPR targeting of Atf4 or Chop, revealing a vital role for the Perk/Atf4 pathway in orchestrating neurodegenerative and regenerative responses to CNS axon injury.

However, the main concern is the overall data quality, which appears to be suboptimal. The transfection efficiency of AAV2-hSyn1-mTagBFP2-ires-Cre used in this study does not seem highly effective, as evidenced by the data presented in Supplementary Figure 1. The manuscript also contains several inconsistencies and a mix of methods in data collection, analysis, and interpretation, such as the labeling and quantification of RGCs and the combination of bulk and single-cell sequencing results.

Despite these limitations, the study offers valuable insights into the role of the Perk/Atf4 pathway in determining neuronal fate after axon injury, emphasizing the significance of understanding the molecular mechanisms that govern neuronal survival and regeneration. This knowledge could potentially inform the development of targeted therapies to promote neuroprotection and CNS repair following injury.

Reviewer #2 (Public Review):

This is an exceptional study that provides conclusive evidence for the existence of a descending pathway from the brain that inhibits nociceptive behavioral outputs in larvae of Drosophila melanogaster. The authors identify both molecular and neuronal/cellular components of this pathway. Converging lines of evidence and conclusive genetic experiments indicate that the neuropeptide, drosulfakinin (DSK), and its receptors (CCK1 and CCK2) function to inhibit nociception behaviors. Interestingly, the authors show that the relevant DSK neurons have cell bodies that are in the larval brain and that these neurons send projections into the thoracic ganglion and ventral nerve cord. Several lines of evidence support the hypothesis that fourth-order nociceptive neurons called Goro, are one relevant target for these outputs. RNAi knockdown of the CCK1 receptor in these cells sensitizes behavioral and physiological responses to noxious heat. Second, the axons of DSK neurons form physical contact with processes of Goro neurons as revealed by GRASP analysis. However, the authors' careful experiments indicate that the contacts between axons and Goro neurites might not be indicative of direct synapses and instead might operate through the bulk transmission of the peptidergic signals. The study raises many interesting questions for future study such as what behavioral contexts might depend on this pathway. Using the CAMPARI approach, the authors do not find that the DSK neurons are activated in response to nociceptive input but instead suggest that these cells may be tonically active in gating nociception. Future studies may find contexts in which the output of the DSK neurons is inhibited to facilitate nociception or contexts in which the cells are more active to inhibit nociception.

Reviewer #2 (Public Review):

Lauterbur et al. present a description of recent additions to the stdpopsim simulation software for generating whole-genome sequences under population genetic models, as well as detailed general guidelines and best practices for implementing realistic simulations within stdpopsim and other simulation software. Such realistic simulations are critical for understanding patterns in genetic variation expected under diverse processes for study organisms, training simulation-intensive models (e.g., machine learning and approximate Bayesian computation) to make predictions about factors shaping observed genetic variation, and for generating null distributions for testing hypotheses about evolutionary phenomena. However, realistic population genomic simulations can be challenging for those who have never implemented such models, particularly when different evolutionary parameters are taken from a variety of literature sources. Importantly, the goal of the authors is to expand the inclusivity of the field of population genomic simulation, by empowering investigators, regardless of model or non-model study system, to ultimately be able to effectively test hypotheses, make predictions, and learn about processes from simulated genomic variation. Continued expansion of the stdpopsim software is likely to have a significant impact on the evolutionary genomics community.

Strengths:

This work details an expansion from 6 to 21 species to gain a greater breadth of simulation capacity across the tree of life. Due to the nature of some of the species added, the authors implemented finite-site substitution models allowing for more than two allelic states at loci, permitting proper simulations of organisms with fast mutation rates, small genomes, or large effect sizes. Moreover, related to some of the newly added species, the authors incorporated a mechanism for simulating non-crossover recombination, such as gene conversion and horizontal gene transfer between individuals. The authors also added the ability to annotate and model coding genomic regions.

In addition to these added software features, the authors detail guidelines and best practices for implementing realistic population genetic simulations at the genome-scale, including encouraging and discussing the importance of code review, as well as highlighting the sufficient parameters for simulation: chromosome level assembly, mean mutation rate, mean recombination rate or recombination map if available, effective size or more realistic demographic model if available, and mean generation time. Much of these best practices are commonly followed by population genetic modelers, but new researchers in the field seeking to simulate data under population genetic models may be unfamiliar with these practices, making their clear enumeration (as done in this work) highly valuable for a broad audience. Moreover, the mechanisms for dealing with issues of missing parameters discussed in this work are particularly useful, as more often than not, estimates of certain model parameters may not be readily available from the literature for a given study system.

Weaknesses:

An important update to the stdpopsim software is the capacity for researchers to annotate coding regions of the genome, permitting distributions of fitness effects and linked selection to be modeled. However, though this novel feature expands the breadth of processes that can be evaluated as well as is applicable to all species within the stdpopsim framework, the authors do not provide significant detail regarding this feature, stating that they will provide more details about it in a forthcoming publication. Compared to this feature, the additions of extra species, finite-site substitution models, and non-crossover recombination are more specialized updates to the software.

Reviewer #2 (Public Review):

In this manuscript, Dominici et al. aim to determine whether the reversible inhibition of the type I protein arginine methyltransferases (PRMT) would maintain the stemness of muscle stem cells in culture and enable subsequent regenerative capacities. They demonstrate that the type I PRMT inhibitor MS023 enhances self-renewal and in vitro expansion of muscle stem cells isolated from mice. Using a very rigorous single-cell RNA-sequencing approach, they further demonstrate that distinct sub-populations of cells emerge under type I PRMT inhibition and that these cells entered the differentiation program more efficiently. Moreover, they revealed a shift in metabolism in these cells, which they confirmed in vitro. Finally, they demonstrate that MS023 enhances muscle stem cell engraftment in vivo and that the direct injection of MS023 increases muscle strength in a mice model of Duchenne muscular dystrophy.

This study will have a great impact on the field of stem cells and offer potential therapeutic avenues for diseases such as Duchenne muscular dystrophy.

Reviewer #2 (Public Review):

The study describes and names a new marine reptile taxon on the basis of an incomplete postcranial skeleton from the early Triassic of China. The morphologial description and comparison is well concucted/informative and very detailed. The paper and results (phylo. analyseis and hypothesis on ancestral body shape) of Wang et al. 2022 should be discussed in more detail.

Reviewer #2 (Public Review):

The existence of PAG-USV-producing neurons has been recently established, alongside two independent pathways, POA->PAG, and AMG->PAG, that promote and inhibit the production of ultrasound vocalizations in female and male mice, respectively. Because vocalizations can be modulated in a variety of contexts, such as in the presence of a predator, the authors first show that the AMG->PAG pathway is activated in situations where mice stop vocalizing, such as in the presence of a predator or aggressive conspecifics, and can inhibit natural vocalizations in contexts where females vocalize (extending to their previous findings in male mice). Interestingly, AMG->PAG neurons also receive input from POA neurons that are known to promote vocalizations via their connection to PAG interneurons that inhibit PAG-USV-producing neurons. This POA->AMG and PAG pathway is inhibitory and therefore its capacity to promote vocalizations via these two parallel pathways might be achieved by its inhibition of AMG and PAG neurons that inhibit the PAG-USV producing neurons. While these results hint at possible mechanisms that could underlie the hierarchical control of vocalization, and how different external signals impinge on existing pathways to produce behavior flexibility, the study is missing important elements to draw such conclusions. Overall, the study is also missing important information on how experiments were performed.

Reviewer #2 (Public Review):

Gaucher disease is a rare genetic disorder that is commonly treated by either administration of a functional enzyme or reduction of the substrate. Some patients receiving enzyme replacement therapy develop avascular osteonecrosis (AVN), but the risk factors were not known. In this study, a cohort of 155 patients was followed longitudinally for two decades, and their risk of developing AVN was analyzed. The data convincingly shows that patients with heterozygous N409S mutation, a past history of AVN, receiving velaglucerase therapy, or with higher serum glucosylsphingosine levels have a higher risk of AVN. These findings will provide a means to identify Gaucher disease patients at higher risk of AVN and to provide them with an optimal treatment. In addition, the study establishes that it is prudent to achieve a low glucoylspingosine level as a therapeutic goal in Gaucher patients with risk of AVN.

Reviewer #2 (Public Review):

In their manuscript, Van Creveld et al. set out to demonstrate divergent functions for two clades of flavodoxin in diatoms. To achieve their goals, the authors combined metatranscriptomic results originating from three separate research cruises in the North Pacific Ocean with laboratory experiments with a clade I flavodoxin knock-out mutant in the diatom Thalassiosira pseudonana. Overall, their field study confirmed that Clade II flavodoxin is mostly up-regulated under iron limitation in most diatoms that were represented in their metatranscriptomic data (Figure 5 A-F). Their field study also demonstrated that clade I flavodoxin is expressed at levels that are several orders of magnitude lower than clade II flavodoxin (figure 5H). The lower expression of clade I flavodoxin was also observed in laboratory culture experiments (Figure 2). The laboratory experiments also demonstrated that the clade I flavodoxins were responsive to iron limitation in some of the species studied (Their Figure 2C), such that the assignment of function based solely on the clade I and clade II flavodoxin classification may not always be straight forward, and that exceptions will likely be found as more diatom species are studied.

In their quest to determine whether Clade I flavodoxin plays a role in adaptation to oxidative stress, the authors created several knock-out mutants where the clade I flavodoxin is not functional. These mutant strains responded to iron limitation in the same way as the WT strains. However, the mutant strains defective in the clade I flavodoxin were more slightly more sensitive to oxidative stress (created by exposure to lethal doses of hydrogen peroxide) than the wild-type strains. The results of the oxidative stress challenges would have been stronger if a broader concentration range of hydrogen peroxide had been used in the experiments leading to a dose-response curve for both the mutant and wild-type strains.

The supplemental information provided in the main manuscript holds a lot of important information. Take for example Figure S4 showing the placement of reads for Clade I and Clade II in a Maximum-likelihood tree for flavodoxin in the North Pacific Ocean. The results show that clade II flavodoxin is much more commonly found in the transcripts than clade I flavodoxin. Perhaps different results would have been obtained by conducting a similar sampling of metatranscriptome in the Atlantic Ocean that is less subject to iron limitation.

Overall, the authors have provided results that support a role for Clade I flavodoxin in alleviating oxidative stress in Thalassiosira pseudonana, however, whether or not this role is universal for clade I flavodoxin in other diatom species will require further studies.

Reviewer #2 (Public Review):

This paper introduces a method to quantify how genetic ancestry drives non-random mating in admixed populations. Admixed American populations are structured by racial, gender, and class hierarchies. This has the potential to cause both ancestry-related assortative mating, in which the ancestry of mates tends to be correlated, and ancestry-related sex bias, in which individuals have a preference for mates with a particular ancestry composition. By applying their method to several African American and Latin American populations, Sandoval et al. further our understanding of ancestry-based population structure in this region more broadly.

Strengths<br /> As many others have recently done, Sandoval et al. leverage the ability of a neural network to predict demographic parameters from high-dimensional population genomic data. Sandoval et al. first develop a clever probabilistic model of mating by defining the probability of a male and female mating as a function of the difference in ancestry between the individuals. They use this model to simulate population genomic data under various demographic scenarios, and then train a neural network on these simulated data. Finally, they apply the neural network to empirical data and learn the parameters of the underlying probability distribution, which can be related back to assortative mating and sex bias.

One clear strength of this paper is their ability to jointly assess assortative mating and sex bias, as well as their ability to apply their model to multiple contemporary admixed populations.

Importantly, the authors couch their results in an intersectional understanding of populations and consistently refer to research from historians and other social scientists throughout their paper, which reflects a very thoughtful awareness of the interdisciplinary nature of this research.

Weaknesses<br /> The definition of assortative mating is conceptually confusing - in the text, assortative mating is introduced as genetic similarity between mates, i.e. positive assortative mating. However, based on the definition of assortative mating in their model, a population can have high assortative mating for a particular ancestry component even when there is non-zero sex bias for that component (e.g. males with low Native American ancestry are more likely to mate with females with high Native American ancestry). Fundamentally, this scenario cannot reflect positive assortative mating; rather, it reflects negative assortative mating (i.e. there is structured genetic dissimilarity between mates). However, the authors do not discuss the fact that the interpretation of the assortative mating parameter changes with the value of the sex bias parameter.

In addition, the results of the inference in ASW are difficult to interpret. They find that males of high African ancestry are more likely to mate with females of low African ancestry. This result seems counterintuitive given the body of literature that suggests sex-biased admixture in African Americans has greater male European and female African contributions. The authors do not suggest potential explanations for this observation.

Lastly, the authors have not done any simulations to assess how accurate parameter estimates are if the demographic model is misspecified, which weakens the interpretability of the results.

Reviewer #2 (Public Review):

The authors improved significantly a previously published luminescence-based assay for the detection of MVB-derived exosome secretion, by using a membrane-impermeable Nluc inhibitor to make sure only intact vesicles and not cellular debris are quantified. Using this improved assay they confirmed prior reports that exposure to the Ca2+ ionophore ionomycin triggers exosome release. They then build on this by showing that exosomes are also released when Ca2+ influx is caused by plasma membrane (PM) wounding, using pore-forming toxins or mechanical stress. Investigating possible molecular mechanisms involved in Ca2+-regulated MVB exocytosis/exosome release, the authors use proteomics to identify proteins recruited to purified MVBs in an ionomycin-dependent fashion. One of these proteins is ANX6, which interestingly was previously implicated in the repair of PM wounds in other cell types. The paper then explores the possible role of ANX6, showing that ionophore-dependent exosome secretion is inhibited in ANX6-depleted intact cells, or in permeabilized cells reconstituted with cytosol in the presence of anti-ANX6 antibodies. These results are convincing and very consistent with prior findings from other groups. The interesting advance is the demonstration that Ca2+ influx through PM lesions also triggers exocytosis of MVBs, and not only mature lysosomes as previously described. This reveals that PM injury, a frequent event in vivo, could play a role in the extensively documented detection of extracellular exosomes in biological fluids.

They also present some imaging data suggesting that ANX6 inhibition stalls MVBs at the cell surface and that ANX6 may promote MVB exocytosis and exosome release by tethering different intracellular membranes. These results are consistent with the author's interpretation but less compelling since they are based on limited confocal imaging without markers for specific compartments such as the PM and without quantification.

Another limitation of the study is that most experiments were performed using 30 min of cell exposure to micromolar concentrations of ionomycin, and the kinetics of exosome secretion after shorter times of ionophore exposure is not shown. The improved luminescence assay is described as sensitive and linear, but a linear time course over 24 h is only shown for constitutive exosome release, not for cells treated with ionomycin. Nocodazole experiments led to the conclusion that microtubules are required for 'sustained' exosome release, but this is somewhat misleading since ionophores markedly enhance exocytosis, raising questions as to whether the process is still linear after 30 min in the presence of ionomycin. The permeabilized-cell reconstitution assay apparently detected a requirement for ANX6 after just 2 min, which is reassuring but also raises the possibility that exosome release may not be sustained up to 30 min. PM resealing is a rapid process, completed in 1-2 min, so if one of the goals was to explore a connection between MVB exocytosis and PM repair, shorter time points would make more sense. This is particularly important since prolonged exposure to micromolar concentrations of ionomycin is known to cause extensive cytotoxicity, including actin cytoskeleton alterations, changes in ATP levels, and apoptosis (the authors perform only one limited control for apoptosis, a western that did not detect PARP cleavage).

Overall, this is an interesting study that brings together earlier observations but places them in a new context - that Ca2+-dependent exosome release from MVBs may occur in the context of PM wounding, and thus might play a role in PM resealing. Strong evidence was presented for the ANX6 requirement in ionophore-induced exosome release. However, since most previous studies implicating ANX6 in PM repair in other cell types involved a non-physiological form of laser wounding, it is still unclear if ANX6 is required for PM resealing after mechanical wounding, in the cells used in this study.

Reviewer #2 (Public Review):

This study characterized the mice deficient for PARL and concluded that mitochondrial defects lead to ferroptosis and spermatogenic cell death. In mammalian germ cells, the existence of ferroptosis is not known so far. Interestingly, a study using C. elegans recently established the occurrence of germ cell ferroptosis (Perez et al., Dev Cell 2020: PMID: 32652074). Thus, if the conclusion of this study is valid, this study can be a timely demonstration of germ cell ferroptosis in mammals. I understand the potential value of this study. However, in this study, although several indirect data were provided, I do not think the results firmly established the occurrence of germ cell ferroptosis. Further, some major technical barriers prevent the interpretation of these results. In general, perturbations in mitochondria dynamics could be expected to disrupt spermatogenesis. It would be necessary to establish germ-cell ferroptosis to explain the specific phenotype of the PARL mutants. Overall, I appreciate the potential impact; but I am not fully convinced by the main conclusion reported in this study.

Reviewer #2 (Public Review):

By elegantly designing experiments, MaBouDi et al. elucidated honeybee's behavioral strategy to quantitatively associate sensory cues with valences. The description is simple and concise enough to understand the logic. Particularly, the authors clearly demonstrated how sensory evidence and reward likelihood quantitatively affect the decision-making process and animals' response time. Their behavioral characterization approach and proposed model could also be helpful for studies using higher animal species. I have a few doubts regarding the definition of rejection behavior and the structure of the model that is critical to lead their main conclusions.

Reviewer #2 (Public Review):

This manuscript describes that CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance. Overall, the data are presented clearly. The distinct roles of CCR4 and CCR7 at different phases of thymocyte deletion (shown in Figure 6C) are novel and important. However, the conclusion that expression profiles of CCR4 and CCR7 are different during DP to SP thymocyte development was documented previously. More importantly, the data presented in this manuscript do not support the conclusion that CCR7 is uncoupled from medullary entry. Moreover, it is unclear how the short-term thymus slice culture experiments reflect thymocyte migration from the cortex to the medulla.

1. Differential profiles in the expression of chemokine receptors, including CCR4, CCR7, and CXCR4, during DP to SP thymocyte development were well documented. Previous papers reported an early and transient expression of CCR4, a subsequent and persistent expression of CCR7, and an inverse reduction of CXCR4 (Campbell, et al., 1999, Cowan, et al., 2014, and Kadakia, et al. 2019). The data shown in Figures 1, 2, and 3 are repetitive to previously published data.

2. The manuscript describes the lack of CCR7 at early stages during DP to SP thymocyte development (Figure 1-3). However, CCR7 expression is detected insensitively in this study. Unlike CCR4 detection with a wide fluorescence range between 0 and 2x10*4 on the horizontal axis, CCR7 detection has a narrow range between 0 and 2x10*3 on the vertical axis (Figure 1C, 1D, 4B, 4C, 6B, S2, S3), so that flow cytometric CCR7 detection in this study is 10-times less sensitive than CCR4 detection. It is therefore likely that the "CCR7-negative" cells described in this manuscript actually include "CCR7-low/intermediate" thymocytes described previously (for example, Figure S5A in Van Laethem, et al. Cell 2013 and Figure 6 in Kadakia, et al. J Exp Med 2019).

3. Low levels of CCR7 expression could be functionally evaluated by the chemotactic assay as shown in Figure 2. However, the data in Figure 2 are unequally interpreted for CCR4 and CCR7; CCR4 assays are sensitive where a migration index at less than 1.5 is described as positive (Figure 2A and 2B), whereas CCR7 assays are dismissal to such a small migration index and are only judged positive when the migration index exceeds 10 or 20 (Figure 2C and 2D). CCR7 chemotaxis assays should be carried out more sensitively, to equivalently evaluate the chemotactic function of CCR4 and CCR7 during thymocyte development.

4. Together, this manuscript suffers from the poor sensitivity for CCR7 detection both in flow cytometric analysis and chemotactic functional analysis. Conclusions that CCR7 is absent at early stages of DP to SP thymocyte development and that CCR7 is uncoupled from medullary entry are the overinterpretation of those results with the poor sensitivity for CCR7. The oversimplified scheme in Figure 3D is misleading.

5. The short-term thymus slice culture experiments should be described more carefully in terms of selection events during DP to SP thymocyte development, which takes at least 2 days for CD4 lineage T cells and approximately 4 days for CD8 lineage T cells (Saini, et al. Sci Signal 2010 and Kimura, et al. Nat Immunol 2016). The slice culture experiments in this manuscript examined cellular localization within 12 hours and chemokine receptor expression within 24 hours (Figures 4, 5) even for the development of CD8 lineage T cells (Figure S2), which are too short to examine entire events during DP to SP thymocyte development and are designed to only detect early phase events of thymocyte selection.

6. It is unclear what the medullary density alteration measured in the thymus slice culture experiments represents. Although the manuscript describes that the increase in the medullary density reflects the entry of cortical thymocytes to the medulla (Figure 4E and S2E), this medullary density can be affected by other mechanisms, including different survival of the cells seeded on the top of different thymus microenvironments. Thymocytes seeded on the medulla may be more resistant to cell death than thymocytes seeded in the cortex, for example, because of the rich supply of cytokines by the medullary cells. So, the detected alterations in the medullary density may be affected by the differential survival of thymocytes seeded in the cortex and the medulla. Also, the medullary density is measured only within a short period of up to 12 hours. The use of MHC-II-negative slices and CCR4- or CCR7-deficient thymocytes in the thymus slice cultures may verify whether the detected alteration in the medullary density is dependent on TCR-initiated and chemokine-dependent cortex-to-medulla migration.

Reviewer #2 (Public Review):

This study evaluated the effect of population-based HPV vaccination programs in India which is suffering from the disease burden of cervical cancer. The authors used model simulations for estimating the outcomes by adopting the latest available data in the literature. The findings provide evidence-based support for policymakers to devise efficient strategies to reduce the impacts of cervical cancer in the country.

Strengths.<br /> The study investigated the potential impact of cervical cancer elimination when HPV vaccination was disrupted (e.g., during the COVID-19 pandemic) and for meeting the WHO's initiatives. The authors considered several settings from the low to high effects of vaccination disruption when concluding the findings. The natural history was calibrated to local-specific epidemiological data which helps highlight the validity of the estimation.

Weaknesses.<br /> Despite the importance and strengths, the current study may likely be improved in several directions. First, the study considered the scenario of using a recently developed domestic HPV vaccine but assuming vaccine efficacy based on another foreign HPV vaccine that has been developed and used (overseas) for more than 10 years. More information should be provided to support this important setting.

Second, the authors are advised to discuss the vaccine acceptability and particularly the feasibility to achieve high coverage scenarios in relatively conservative countries where HPV vaccines aim to prevent sexually transmitted infection. Third, as the authors highlighted, the health economics of gender-neutral strategies, which is currently missing in the manuscript, would be a substantial consideration for policymakers to implement a national, population-based vaccination program.

Reviewer #2 (Public Review):

This study by Masser et al. analyzes global replication timing and gene expression in rif-1 null zebrafish. This work is an extension of their previous report on the normal replication timing pattern during wild-type zebrafish development. The major valuable finding here is that Rif1 is not essential for viability in zebrafish, and - counter to expectation from studies in cultured cells and other species - late replication does not strongly depend on Rif1. Instead, the data suggest that Rif1 subtly sharpens replication timing pattern during normal development rather than function generally to delay replication timing. In the absence of Rif1, the normal pattern establishment is somewhat delayed. The authors also document some changes in expression during development with more genes being repressed by Rif1 than activated at some early stages.

The study and analysis are generally rigorous, and the conclusions are supported by convincing data. The manuscript is well written, though there are aspects of the presentation that could be improved for a broader scientific audience. Given the strong link between replication timing and cell type/development, studying timing in a whole developing organism is important. The experimental approach is technically challenging, particularly the bioinformatic analysis. The scientific advance here is largely confined to documenting the timing of Rif1-affected transcription, the unanticipated effect of the rif1 deletion on replication timing and on sex determination, though the latter is not explored. The work is descriptive and feels like two relatively unconnected studies, transcription and replication plus a small bit of development, and the difference in timing of the transcription phenotypes and replication phenotypes suggests they may be very distinct Rif1 roles. There isn't a lot of new insight into the mechanism of how Rif1 affects either replication timing or gene expression. As such, the overall study is an useful set of findings and detailed data for future work, but it doesn't make a big step forward in understanding the role of Rif1 or the biological processes it affects.

Weaknesses worth addressing include the following:

1. Loss of Rif1 did not affect viability, but it did strongly influence sex determination, resulting in a lower population of females. This effect is the strongest organismal phenotype, but the study provides no explanation for the loss of females from the data gathered here.<br /> 2. The approach to distinguish nascent zygotically expressed mRNAs from maternal mRNAs is a strength. Are the differentially expressed genes related at all to regions of the genome whose replication timing is most affected? Are any of them related to the sex determination or developmental phenotypes?

Reviewer #2 (Public Review):

In their manuscript entitled "DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation", Mullen et al. describe an interesting mechanism of inducing antigen presentation. The manuscript includes a series of experiments that demonstrate that blockade of pyrimidine synthesis with DHODH inhibitors (i.e. brequinar (BQ)) stimulates the expression of genes involved in antigen presentation. The authors provide evidence that BQ mediated induction of MHC is independent of interferon signaling. A subsequent targeted chemical screen yielded evidence that CDK9 is the critical downstream mediator that induces RNA Pol II pause release on antigen presentation genes to increase expression. Finally, the authors demonstrate that BQ elicits strong anti-tumor activity in vivo in syngeneic models, and that combination of BQ with immune checkpoint blockade (ICB) results in significant lifespan extension in the B16-F10 melanoma model. Overall, the manuscript uncovers an interesting and unexpected mechanism that influences antigen presentation and provides an avenue for pharmacological manipulation of MHC genes, which is therapeutically relevant in many cancers. However, a few key experiments are needed to ensure that the proposed mechanism is indeed functional in vivo.

The combination of DHODH inhibition with ICB reflects more of an additive response instead of a synergistic combination. Moreover, the temporal separation of BQ and ICB raises the question of whether the induction of antigen presentation with BQ is persistent during the course of delayed ICB treatment. To confidently conclude that induction of antigen presentation is a fundamental component of the in vivo response to DHODH inhibition, the authors should examine whether depletion of immune cells can reduce the therapeutic efficacy of BQ in vivo. Moreover, they should examine whether BQ treatment induces antigen presentation in non-malignant cells and APCs to determine the cancer specificity. Finally, although the authors show that DHODH inhibition induces expression of both MHC-I and MHC-II genes at the RNA level, only MHC-I is validated by flow cytometry given the importance of MHC-II expression on epithelial cancers, including melanoma, MHC-II should be validated as well.

Overall, the paper is clearly written and presented. With the additional experiments described above, especially in vivo, this manuscript would provide a strong contribution to the field of antigen presentation in cancer. The distinct mechanisms by which DHODH inhibition induces antigen presentation will also set the stage for future exploration into alternative methods of antigen induction.

Reviewer #2 (Public Review):

The manuscript from Qi et. al. provides novel structures for connexin 43 (Cx43) gap junction channels (GJCs) and hemichannels, which they claim correspond to the closed conformations of these channels. This leads the authors to propose a mechanism of gating that implicates the existence of lipids in the pore, which could stabilize the N-terminal domain as the gate region within the pore. The authors performed a lipidomic assay in their structures and identified a dehydroepiandrosterone (DHEA), a sterol compound specifically enriched in their Cx43 purified samples. However, at the current structural resolution, they cannot conclude whether DHEA is the small lipid-like density found at the pore of closed channels. Further studies, including functional studies, are needed to determine whether DHEA is a gating intermediary. Interestingly, other recently published structures of large-pore channels support the notion that lipids are found inside the pore. However, this evidence is only supported by Cryo-EM structures and is an issue generating major controversy in the field, particularly when these molecules are implicated in the gating mechanisms. The finding of putative lipids-pore interactions is a very intriguing observation, but it should be interpreted carefully. A major concern is that channel reconstitution is performed in an excess of lipids and detergents that could lead to artifacts. Thus, these lipid-like densities observed in Cx43 (and other structures) after single particle analysis could not represent native lipid-protein interactions. Subsequently, all conclusions for the role of lipids in gating could rely on a potential protein purification-induced artifact. Also, it is hard to visualize how the lipids can move in/out of the pore during gating, particularly from this putative lipids-pore conformation to an open conformation.

Another important aspect of this work is that provided structures for both Cx43 GJCs and hemichannels. As expected, there are differences in extracellular loops rearrangements between these two structures. One issue, however, is that the resolution for Cx43 hemichannels is still low (3.98 Å), thus interpretations need to be taken with caution. In addition, the intracellular domains that are important for the gating and regulation of Cx43, including the intracellular loop and the carboxyl-terminal domain were not resolved in these structures. Nevertheless, this is a common issue for other connexin Cryo-EM structures reported in the literature.

Reviewer #2 (Public Review):

Harris et al. have described the cryo-EM structure of PI3K p110gamma in a complex with a nanobody that inhibits the enzyme. This provided the first structure of full-length of PI3Kgamma in the absence of a regulatory subunit. This nanobody is a potent allosteric inhibitor of the enzyme, and might provide a starting point for developing allosteric, isotype-specific inhibitors of the enzyme. One distinct effect of the nanobody is to greatly decrease the dynamics of the enzyme as shown by HDX-MS, which is consistent with a growing body of observations suggesting that for the whole PI3K superfamily, enzyme activators increase enzyme dynamics.

The most remarkable outcome of the study is that upon observing the site of nanobody binding, the authors searched the literature and found that there was a previous report of a PKCbeta phosphorylation of PI3Kgamma in the helical domain that is near the nanobody binding site. This led the authors to re-examine the consequence of the phosphorylation armed with better structural models and the tools to study the effects of this phosphorylation on enzyme dynamics. They found that the site of phosphorylation is buried in the helical domain, suggesting that a large conformational change would have to take place to enable the phosphorylation. HDX-MS showed that phosphorylation at three sites clustered in the helical domain generate a distinctly different conformation with rapid deuterium exchange. This suggests that the phosphorylation locks the enzyme in a more dynamic state. Their enzyme kinetics show that the phosphorylated, dynamic enzyme is activated.

While this phosphorylation was reported before, the authors have provided a mechanism for why this activates the enzyme, and they have shown why binders that stabilise the helical domain (such as binding to the p101 regulatory subunit and the nanobody) prevent the phosphorylation. It is this insight into the dynamics of the PI3Kgamma that will likely be the long-lasting influence of the work.

The paper is well written and the methods are clear.

Reviewer #2 (Public Review):

In this work the authors describe the shape and interconnectedness of intracellular structures of malaria blood stage parasites by taking advantage of expansion microscopy. Compared to previous microscopy work with these parasites, the strength of this paper lies in the increased resolution and the fact that the NHE ester highlights protein densities. Together with the BodipyC membrane staining, this results in data that is somewhere in between EM and standard fluorescence microscopy: it has higher resolution than standard fluorescence microscopy and provides some points of reference of different cellular structures due to the NHE ester/BodipyC.

This study makes many interesting and useful observations and although it is somewhat "old school descriptory" in its presentation, researchers working in many different areas will find something of interest here. This ranges from mitosis, to organisation and distribution of major cellular structures, endocytosis and invasion, overall providing a rich and interesting resource. The results section is long but by taking the space to explain everything in detail, it has the advantage that it clearly transpires how things were done and on how many cells a conclusion is based on. Further the authors often also included a brief interpretation of their findings with a very open assessment what it does and what it does not show, highlighting interesting questions left by the data.

Overall this is a very nice and useful paper that will be of interest to many, particularly those working on nuclear division, cytokinesis, endocytosis or invasion in malaria parasites. The spatiotemporal arrangement and interconnection of subcellular structures will also give a framework for specific functional studies.

Reviewer #2 (Public Review):

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

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

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

Reviewer #2 (Public Review):

In this manuscript by Popova et al., the authors report the pathological impact of Rubella virus (RV) infection on human brain development. In particular, they uncovered a selective tropism of Rubella virus for microglial cells in cultured slices of human developing brain and 2D mixed fetal brain cell culture. Their results suggest that RV infection of microglia relies on the presence of diffusible factors from other cell populations. Moreover, the authors showed that RV infection of human brain organoids supplemented or not with microglia leads to interferon response and dysregulation of gene involved in brain development. This set of data will help understanding the cellular specificity and pathological mechanisms occurring in the developing brain upon RV infection. The data provided are overall of high quality and shed new light on the cellular tropism and the pathomechanisms of RV infection.

Reviewer #2 (Public Review):

This study focuses on the association between weight at birth and area, volume and thickness of the cerebral cortex measured at timepoints throughout the lifespan. Overall, the study is well designed, and supported by evidence from a large sample drawn from three geographically distinct cohorts with robust analytical and statistical methods.

The authors test three hypotheses: (1) that higher birth weight is associated with greater cortical area in later life; (2) that associations are robust across samples and age; and (3) that associations are stable across the lifespan. Analyses are performed separately in three cohorts: ABCD, UKBB and LCBC and the pattern of associations compared by means of spatial correlations. They find that BW is positively associated with cortical area (and, as a consequence, cortical volume) across most of the cortex, with effect sizes being greatest in frontal and temporal regions. These associations remain largely unchanged when accounting for age, sex, length of gestation and (in one cohort) ethnicity. Variations due to MRI scanner and site are accounted for statistically. Measures are taken to determine within sample replicability through split-half analyses.

The authors conclude that BW, as a marker of early development, is consistently associated with brain characteristics throughout the lifespan, acting as an 'intercept' and promoting brain reserve, i.e.: the capacity of the brain to withstand aging effects. Indeed, the authors calculate that 600g lower BW results in reductions in cortical volume akin to 6-7 years of aging in middle to later life. This is perhaps a startling statistic but one that is not entirely supported by the data presented.

A key piece of information lacking from this study is the functional importance of the reported associations. That lower BW is associated with lower cortical volume and that cortical volume decreases with age is perhaps not surprising - the same could be said for height - one cannot conclude that the same processes underpin the two factors without examining the functional consequences of BW-related volume reductions in older age. The notion of 'brain reserve' indicates a protective effect. If this is the case, one might expect to see a mediating effect of BW on age-related cognitive effects. Without this data, it is difficult to reach the authors conclusions that decreased birthweight has the same effect as 7 years of aging in later life.

In addition, it is not clear to what degree the association between BW and cortical area/volume is simply reflecting overall somatic growth: brain mass scales with body height, and birth weight and length are associated with adult height. While the specificity of the associations between cortical area/volume and BW are not fully tested, the effects are significantly diminished when controlling for a related measure of somatic growth: intracranial volume (Fig S5). In this context, additional commentary on the specificity of the reported BW-brain associations (or lack thereof) would be helpful.

Finally, the authors use linear models to model brain area, thickness and volume as a function of age. The authors' previous studies have demonstrated nonlinear trajectories of cortical thickness in the LCBC cohort across most of the cortex. A stronger rationale (e.g.: theoretical or model selection) supporting the use of GLM in this study would be more compelling.

Reviewer #2 (Public Review):

The authors evaluate whether non time reversible models fit better data presenting strand-specific substitution biases than time reversible models. Specifically, the authors consider what they call NREV6 and NREV12 as candidate non time-reversible models. On the one hand, they show that AIC tends to select NREV12 more often than GTR on real virus data sets. On the other hand, they show using simulated data that NREV12 leads to inferred trees that are closer to the true generating tree when the data incorporates a certain degree of non time-reversibility. Based on these two experimental results, the authors conclude that "We show that non-reversible models such as NREV12 should be evaluated during the model selection phase of phylogenetic analyses involving viral genomic sequences". This is a valuable finding, and I agree that this is potentially good practice. However, I miss an experiment that links the two findings to support the conclusion: in particular, an experiment that solves the following question: does the best-fit model also lead to better tree topologies?

On simulated data, the significance of the difference between GTR and NREV12 inferences is evaluated using a paired t test. I miss a rationale or a reference to support that a paired t test is suitable to measure the significance of the differences of the wRF distance. Also, the results show that on average NREV12 performs better than GTR, but a pairwise comparison would be more informative: for how many sequence alignments does NREV12 perform better than GTR?

Reviewer #2 (Public Review):

The eleven paralogs of SLC26 proteins in humans exhibit a remarkable range of functional diversity, spanning from slow anion exchangers and fast anion transporters with channel-like properties, to motor proteins found in the cochlear outer hair cells. In this study, the authors investigate human SLC26A6, which functions as a bicarbonate (HCO3-)/chloride (Cl-) and oxalate (C2O42-)/Cl- exchanger, combining cryo-electron microscopy, electrophysiology, and in vitro transport assays. The authors provide compelling evidence to support the idea that SLC26A6's exchange anions at equimolar stoichiometry, leading to the electroneutral and electrogenic transport of HCO3-/ Cl- and C2O42-/Cl-, respectively. Furthermore, the structure of SLC26A6 reveals a close resemblance to the fast, uncoupled Cl- transporter SLC26A9, with the major structural differences observed within the anion binding site. By characterizing an amino acid substitution within the SLC26A6 anion binding site (R404V), the authors also show that the size and charge variance of the binding pocket between the two paralogs could, in part, contribute to the differences in their transport mechanisms.

This is a well-executed study, and the strength of this work lies in the reductionist, in vitro approach that the authors took to characterize the transport process of SLC26A6. The authors used and developed an array of functional experiments, including two electrogenic transport assays - a fast kinetic (electrophysiology) and a slow-kinetic (fluorescent-based ACMA) - and two electroneutral transport assays, probing for Cl- (lucigenin) and HCO3- (europium), which are well executed and characterized. The structural data is also of high quality and is the first structure of an SLC26 coupled anion exchanger, providing essential information for clarifying our understanding of the functional diversity between the SLC26 family of proteins.

Reviewer #2 (Public Review):

In the manuscript Watanuki et al. want to define the metabolic profile of HSCs in stress/proliferative (myelosuppression with 5-FU), and mitochondrial inhibition and homeostatic conditions. Their conclusions are that during proliferation HSCs rely more on glycolysis (as other cell types) while HSCs in homeostatic conditions are mostly dependent on mitochondrial metabolism. Mitochondrial inhibition is used to demonstrate that blocking mitochondrial metabolism results in similar features of proliferative conditions.

The authors used state-of-the-art technologies that allow metabolic readout in a limited number of cells like rare HSCs. These applications could be of help in the field since one of the major issues in studying HSCs metabolism is the limited sensitivity of the "standard" assays, which make them not suitable for HSC studies.

However, the observations do not fully support the claims. There are no direct evidence/experiments tackling cell cycle state and metabolism in HSCs. Often the observations for their claims are indirect, while key points on cell cycle state-metabolism, OCR analysis should be addressed directly.

Specifically, there are several major points that rise concerns about the claims:

1. The gating strategy to select HSCs with enlarged Sca1 gating is not convincing. I understand the rationale to have a sufficient number of cells to analyze, however this gating strategy should be applied also in the control group. From the FACS plot seems that there are more HSCs upon 5FU treatment (Figure S1b). How that is possible? Is it because of the 20% more of cycling cells at day 6? To prove that this gating strategy still represents a pure HSC population, authors should compare the blood reconstitution capability of this population with a "standard" gated population. If the starting population is highly heterogeneous then the metabolic readout could simply reflect cell heterogeneity.

2. S2 does not show major differences before and after sorting. However, a key metabolite like Lactate is decreased, which is also one of the most present. Wouldn't that mean that HSCs once they move out from the hypoxic niche, they decrease lactate production? Do they decrease anaerobic glycolysis? How can quiescent HSC mostly rely on OXPHOS being located in hypoxic niche?

3. The authors performed challenging experiments to track radiolabeled glucose, which are quite remarkable. However, the data do not fully support the conclusions. Mitochondrial metabolism in HSCs can be supported by fatty acid and glutamate, thus authors should track the fate of other energy sources to fully discriminate the glycolysis vs mito-metabolism dependency. From the data on S2 and Fig1 1C-F, the authors can conclude that upon 5FU treatment HSCs increase glycolytic rate.

4. In Figure S1, 5-FU leads to the induction of cycling HSCs and in figure 1, 5-FU results in higher activation of glycolysis. Would it be possible to correlate these two phenotypes together? For example, by sorting NBDG+ cells and checking the cell cycle status of these cells?

5. FIG.2B-C: Increase of Glycolysis upon oligomycin treatment is common in many different cell types. As explained before, other radiolabeled substrates should be used to understand the real effect on mitochondria metabolism.

6. Why are only ECAR measurements (and not OCR measurements) shown? In Fig.2G, why are HSCs compared with cKit+ myeloid progenitors, and not with MPP1? The ECAR increased observed in HSC upon oligomycin treatment is shared with many other types of cells. However, cKit+ cells have a weird behavior. Upon oligo treatment cKit+ cells decrease ECAR, which is quite unusual. The data of both HSCs and cKit+ cells could be clarified by adding OCR curves. Moreover, it is recommended to run glycolysis stress test profile to assess the dependency to glycolysis (Glucose, Oligomycin, 2DG).

7. Since HSCs in the niche are located in hypoxic regions of the bone marrow, would that not mimic OxPhos inhibition (oligomycin)? Would that not mean that HSCs in the niche are more glycolytic (anaerobic glycolysis)?

FIG.3 A-C. As mentioned previously, the flux analyses should be integrated with data using other energy sources. If cycling HSCs are less dependent to OXPHOS, what happen if you inhibit OXHPHOS in 5-FU condition? Since the authors are linking OXPHOS inhibition and upregulation of Glycolysis to increase proliferation, do HSCs proliferate more when treated with oligomycin?

8. FIG.4 shows that in vivo administration of radiolabeled glucose especially marks metabolites of TCA cycle and Glycolysis. The authors interpret enhanced anaerobic glycolysis, but I am not sure this is correct; if more glycolysis products go in the TCA cycle, it might mean that HSC start engaging mitochondrial metabolism. What do the authors think about that?

9. FIG.4: the experimental design is not clear. Are BMNNCs stained and then put in culture? Is it 6-day culture or BMNNCs are purified at day 6 post 5FU? FIG-4B-C The difference between PBS vs 5FU conditions are the most significant; however, the effect of oligomycin in both conditions is the most dramatic one. From this readout, it seems that HSCs are more dependent on mitochondria for energy production both upon 5FU treatment and in PBS conditions.

10. In Figure 5B, the orange line (Glucose+OXPHOS inhibition) remains stable, which means HSCs prefer to use glycolysis when OXPHOS is inhibited. Which metabolic pathway would HSCs use under hypoxic conditions? As HSCs resides in hypoxic niche, does it mean that these steady-state HSCs prefer to use glycolysis for ATP production? As mentioned before, mitochondrial inhibition can be comparable at the in vivo condition of the niche, where low pO2 will "inhibit" mitochondria metabolism.

11. FIG.6H should be extended with cell cycle analyses. There are no differences between 5FU and ctrl groups. If 5FU induces HSCs cycling and increases glycolysis I would expect higher 2-NBDG uptake in the 5FU group. How do the authors explain this?

12. In S7 the experimental design is not clear. What are quiescent vs proliferative conditions? What does it mean "cell number of HSC-derived colony"? Is it a CFU assay? Then you should show colony numbers. When HSCs proliferate, they need more energy thus inhibition of metabolism will impact proliferation. What happens if you inhibit mitochondrial metabolism with oligomycin?

13. In FIG 7 since homing of HSCs is influenced by the cell cycle state, should be important to show if in the genetic model for PFKFB3 in HSCs there's a difference in homing efficiency.

Reviewer #2 (Public Review):

The study by Ellis et al. documents the development of a CRISPR interference (CRISPRi) screen aiming at identifying virulence-critical genes of Legionella pneumophila, the facultative intracellular bacterium causing Legionnaires' disease. L. pneumophila employs the Dot/Icm type IV secretion system to translocate more than 300 different "effector proteins" into host cells. Many effector proteins appear to have redundant functions, and therefore, depleting several of them is required to observe a strong intracellular replication phenotype. In the current study, Ellis et al. develop a "multiplex, randomized CRISPRi sequencing" (MuRCiS) approach to silence several effector genes simultaneously and randomly, thereby possibly causing synthetic lethality for L. pneumophila upon infection of host cells.

The MuRCiS approach comprises the ligation of different CRISPR spacers flanked by repeats in presence of "dead end" oligonucleotide pairs capping a random array of building blocks to be inserted into a library vector. Thus, spacer arrays with an average of 3.3 spacers per array were obtained. As a proof-of-concept, spacer arrays targeting 44 transmembrane effector-encoding L. pneumophila genes were employed to screen for intracellular growth defects in macrophages and amoeba. Consequently, novel pairs of synergistically functioning effector genes were identified by comparative next-generation sequencing of the input and output pools of spacer arrays.

A major strength of this well-written and straightforward study is the construction and use of random and multiplexed CRISPRi arrays, allowing an unbiased and comprehensive screen for multiple genes affecting the intracellular growth of L. pneumophila. The ingenious approach established by Ellis et al. will be useful for further genetic analysis of L. pneumophila infection and might also be adopted for other pathogens employing a large set of (functionally redundant) virulence factors.

Reviewer #2 (Public Review):

In this manuscript, Smith et al. delineated novel mechanistic insights into the structure-function relationships of the C-terminal repeat domains within the mouse DUX protein. Specifically, they identified and characterised the transcriptionally active repeat domains, and narrowed down to a critical 6aa region that is required for interacting with key transcription and chromatin regulators. The authors further showed how the DUX active repeats collaborate with the C-terminal acidic tail to facilitate chromatin opening and transcriptional activation at DUX genomic targets.

Although this study attempts to provide mechanistic insights into how DUX4 works, the authors will need to perform a number of additional experiments and controls to bolster their claims, as well as provide detailed analyses and clarifications.

Reviewer #2 (Public Review):

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

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

Reviewer #2 (Public Review):

The manuscript by Thomen et al. FKBP secures ribosome homeostasis in Plasmodium falciparum and focuses on the importance of PfKBP35 protein, its interaction with the FK506 compound, and the role of PfKBP35 in ribosome biogenesis. The authors showed the interaction of the PfKBP54 with FK506, but the part of the FK506 and PfKBP54 in ribosome biogenesis based on the data is unclear.

The introduction is plotted with two parallel stories about PfKBP35 and FK506, with ribosome biogenesis as the central question at the end. In its current form, the manuscript suffers from two stories that are not entirely interconnected, unfinished, and somewhat confusing. Both stories need additional experiments to make the manuscript(s) more complete. The results from PfFBP35 need more evidence for the proposed ribosome biogenesis pathway control. On the other hand, the results from the drug FK506 point to different targets with lower EC50, and other follow-up experiments are needed to substantiate the authors' claims.

The strengths of the manuscript are the figures and experimental design. The combination of omics methods is informative and gives an opportunity for follow-up experiments.

Reviewer #2 (Public Review):

Wu et al. conducted longitudinal single-nucleus RNA sequencing in a Drosophila transgenic line expressing pathogenic tau (Arg406 ->Trp) and control to study presenile degenerative dementia with bitemporal atrophy. Their data is consistent with previous findings on Tau neurotoxicity, which significantly affects excitatory neurons in human brain samples and transgenic mice. Authors identify aging-like signatures, and an innate immune glial response, including the NFKB pathway, in the transgenic animals.

Strength: This is a great resource for the dissection of dynamic, age-dependent gene expression changes at cellular resolution for the fly community. The article's conclusions are largely supported by the data.

Weakness: No additional orthogonal validation is done on the identified pathways using immunohistochemistry. Also, the authors hypothesized that innate immune signatures might serve as predictors of neuronal subtype vulnerability in tauopathies. Although their data support stronger immune responses in the mutant lines, these findings are not validated. Moreover, the Authors need to use appropriate control animals to compare the mutant Tau animals.

Reviewer #2 (Public Review):

On the whole, I think this paper is a nice demonstration of how current and past aversive experiences shape an animal's behavior, and how this experience is shaped/encoded by neuromodulation. While most past work has focused on passive environmental cues such as chemical, physical, and electromagnetic perturbation, this work focuses on inter-species conflict, which is an important environmental factor that is understudied and would benefit from more research. The authors have created a nice paradigm to investigate this phenomenon further with an organism (C. elegans) that can be easily genetically modified to uncover genetic factors that influence this behavior.

The authors initially present evidence that animals avoid food patches, and egg laying on these patches, in response to predation from P. pacificus and P. uniformis. P. pacificus is quite aggressive, and the RS5194 strain kills all prey animals after 20 hours. Even prior to death, animals exposed to this species experience significant cuticle damage that can be detected by the expression of NLP-29, a known antimicrobial peptide. After 6 hours, animals have a strong aversion to laying eggs on a bacterial lawn that is shared with this species.

However, the authors choose to not use this species, and instead use P. uniformis males which do not lay eggs, and which do not appear to damage the cuticle (or at least sufficiently to induce nlp-29 expression). Nevertheless, their presence appears to cause a slight aversion to laying eggs on food. The authors then screen for neuromodulatory mutants that may alter this behavior, and identify dopamine signaling as an important contributor to this behavior. The authors do a nice job of rescuing the mutant effect with both cell-specific rescue, and general rescue with dopamine administration.

This work is an important contribution to our understanding of predator-induced stresses on prey, and how dopamine neuromodulation alters prey behavior.

My primary criticism of this work is how the data are quantified and explained. Worms perform random walks on and off food, the statistics of which are modified based on environmental cues and internal states. This drive to perform stochastic trajectories is a fundamental feature of these organisms (Klein et al, eLife, 2017). In all assays, the worms lay eggs throughout the arena (diameter ~ 6 mm), with a higher probability of laying eggs on food (diameter ~ 3mm). However, the data are presented as median egg distances from the edge of the food, with each data point representing an assay median from a distribution that spans the entire length of the arena. The recorded effect sizes for different conditions are a fraction of a millimeter for distributions that span the entire arena. These effect sizes are smaller than the length of a worm. Also, after 20 hours of worms crawling on food, the edge of the lawn is more diffuse, with a variance that exceeds the effect size.

The authors present this as evidence of an intentional avoidance of food, but a simpler hypothesis is that the statistics of the worm's random walk have been altered as a response to predation. A larger rate of diffusion would also explain why the variance of body position and egg laying increases upon predation, and would cause the (very small) shift in median distance from the edge of the food. This is also consistent with the proposed role of dopamine, which is known to promote egg-laying during roaming (Cermak et al, 2020). The authors propose that predation increases dopamine release, which in turn leads to food avoidance, but an increased rate of egg-laying during roaming would also produce this effect.

Given the high variance and very small effect sizes observed, a simpler hypothesis of changes to random walk statistics is more parsimonious with the data, and what is already known about C. elegans random walk behavior, and how environmental cues and internal state alter the statistics of this behavior.

Reviewer #2 (Public Review):

In this essential study for the field, McComas et al. use a combination of MD simulations and experiments to construct a unifying transport cycle for a single GLUT protein, GLUT5. The authors demonstrate that GLUT5 likely moves through a transient, intermediate-occluded state like that observed in PfHT1. They also demonstrate that substrate-binding, the specificity of which is regulated by allosteric coupling of the substrate binding site to the extracellular gate, lowers the energetic barriers for the transition from outward- to inward-facing states. The manuscript is clearly and logically written, the data is presented clearly, and the conclusions are sound.

Reviewer #2 (Public Review):

The authors here study the electromechanical coupling in HCN1 channels using molecular dynamics simulations and electrophysiological data. They proposed that the interfaces between S4, S5, S6, and lipids contribute to the coupling mechanism. Their simulations showed state-dependent interactions at the S4-S5 and S5-S6 interfaces, as well as at the interface between the S4-S5 linker and the C-linker. These later interactions were also shown with Cd2+ crosslinking experiments. Furthermore, lipids were also shown to have state-dependent interactions in their simulations and were proposed to be crucial for hyperpolarization-dependent gating. Finally, they propose a domino-like mechanism of activation of HCN channels.

This is a well-written manuscript on a hot topic. The study would attract many readers.

Reviewer #2 (Public Review):

Building on their previous studies, Parab et al used a larger collection of genetically modified zebrafish lines to map the precise expression domains of different VEGF isoforms in the brain and demonstrated that different combinations of VEGF isoforms differentially control the formation of fenestrated vessels at different locations in the 0brain.

The authors used three Wnt signaling mutants to convincingly show wnt signaling is essential for parenchymal angiogenesis, but not required for fenestrated vessel development, such as those in choroid plexus, suggesting fenestrated vessel and barrier vessel are differentially regulated. The previous work from this group has established that VEGF isoforms are critical for myelencephalic choroid plexus development. In this study, they carefully documented the developmental vessel patterning in the diencephalic choroid plexus/pineal gland interface. They also documented the local expression pattern of VEGF isoforms with a set of BAC transgenic fish, together with the phenotype of a series of VEGF mutant fish, the data well support that different combinations of VEGF isoforms regulate fenestrated vessel development at different brain locations.

Given a larger temporal and spatial domain, VEGFs are critical for all forms of vessel development, there are potential redundancy mechanisms to maintain hemostasis of VEGF signaling, in this study, no data is provided to address whether LOF of one form of VEGF affects the expression of other isoforms.

This work provided detailed evidence of different isoform combinations of VEGF regulate formation/patterning of the fenestrated vessel at CP, OVLT, and NH in zebrafish. It will be interesting to follow in the mammalian system, how well these findings are conserved, for example, which isoform of VEGF is critical for vascular patterning during the developmental stages of the pineal gland? How VEGF isoforms participate in choroid plexus development at different ventricle regions and subsequence secretory function maintenance. However, these tasks are challenging without a good genetic tool to locally manipulate VEGF isoform expression during mammalian brain vessel development.

Reviewer #2 (Public Review):

The authors convert the AHBA dataset into a dense cortical map and conduct an impressively large number of analyses demonstrating the value of having such data.

I only have comments on the methodology. First, the authors create dense maps by simply using nearest neighbour interpolation followed by smoothing. Since one of the main points of the paper is the use of a dense map, I find it quite light in assessing the validity of this dense map. The reproducibility values they calculate by taking subsets of subjects are hugely under-powered, given that there are only 6 brains, and they don't inform on local, vertex-wise uncertainties). I wonder if the authors would consider using Gaussian process interpolation. It is really tailored to this kind of problem and can give local estimates of uncertainty in the interpolated values. For hyperparameter tuning, they could use leave-one-brain-out for that.

I know it is a lot to ask to change the base method, as that means re-doing all the analyses. But I think it would strengthen the paper if the authors put as much effort in the dense mapping as they did in their downstream analyses of the data.

It is nice that the authors share some code and a notebook, but I think it is rather light. It would be good if the code was better documented, and if the user could have access to the non-smoothed data, in case they was to produce their own dense maps. I was only wondering why the authors didn't share the code that reproduces the many analyses/results in the paper.

Roosevelt does not seem to have much faith in the words of the charter itself, but seems to call for example and action throughout her defense and explanation of the charter. She believed that only living the character would guide the actions and behavior of others. This hope that Roosevelt have would become real, as the U.N's declaration of human rights has become a point of behavioral guidance for humanity, as can be seen in the 50th anniversary of the U.N's declaration of human rights.

Roosevelt here appeals to pathos to encourage motivation about the attempt of creating effort toward freedom and individual rights for everyone, where everyone has individual freedom and rights that are not controlled but belong to the individual, and are respected. The U.N has accomplished Roosevelt's vision of what the U.N's declaration of human rights should be to people and the world as is seen in the below documentation of the U.N's declaration of human rights' 50th anniversary.

Eleanor agrees to the inclusion of economic rights at the request of Russia. Russia argued that a declaration of human rights should include social and economic rights, not just political rights. The U.N's declaration of human rights originally included political rights, but not economic or social rights. Despite this, Russia still did not assent to the U.N's declaration of human rights, Roosevelts move here was to appease the Russians to draw them towards assenting to the U.N's declaration of human rights through persuasion by being agreeable to Russia's appeal to logos. This however did not work.

The focal point of Roosevelt's essay is her frustration with communist countries. The attack on the U.N's declaration of human rights is primarily definitional in substance (though ideological in dispute). Although The U.N's declaration of human rights is presumptive about the terms democracy and human freedom, there is not universal agreement on what those terms mean.

Despite what Roosevelt states here, she did not have the same approach to Russia when drafting the United Nations Declaration of human rights. She was often frustrated with their push to redefine human rights, and their push to include economic and social rights into the declaration of human rights. Despite her including economic rights in the declaration of human rights. Russia still did not want to agree with the content in the declaration of human rights.

Roosevelt highlights this point which is very interesting, because the United Nations does not enforce the Declaration of human rights. Despite Roosevelt's assertive comments about human rights and the push for the U.N's declaration of human rights to be completed, the declaration of human rights has only served as moral guidance for the world.

Reviewer #2 (Public Review):

The manuscript by Jahncke and colleagues is centered on the CCK+ synaptic defects that are a consequence of Dystroglycanopathy and/or impaired dystroglycan-related protein function. The authors use conditional mouse models for Dag1 and Pomt2 to ablate their function in mouse forebrain neurons and demonstrate significant impairment of CCK+/CB1R+ interneuron (IN) development in addition to being prone to seizures. Mice lacking the intracellular domain of Dystroglycan have milder defects, but impaired CCK+/CB1R+ IN axon targeting. The authors conclude that the milder dystroglycanopathy is due to the partially reduced glycosylation that occurs in the milder mouse models as opposed to the more severe Pomt2 models. Additionally, the authors postulate that inhibitory synaptic defects and elevated seizure susceptibility are hallmarks of severe dystroglycanopathy and are required for the organization of functional inhibitory synapse assembly.

The manuscript is overall, fairly well-written and the description of the phenotypic impact of disruption of Dystroglycan forebrain neurons (and similar glycosyltransferase pathway proteins) demonstrate impairment in axon targeting and organization. There are some questions with regards to interpretation of some of the results from these conditional mouse models. The study is mostly descriptive, and some validation of subunits of the dystroglycan-glycoprotein complex and laminin interactions would go towards defining the impact of disruption of dystroglycan's function in the brain. The statistics and basic analysis of the manuscript appear to be appropriate and within parameters for a study of this nature. Some clarification between the discrepancies between the Walker Warburg Syndrome (WWS) patient phenotypes and those observed in these conditional mouse models is warranted. This manuscript has the potential to be impactful in the Dystroglycanopathy and general neurobiology fields.

Reviewer #2 (Public Review):

In this work, the authors elaborate on an analytically tractable, continuous-attractor model to study an idealized neural network with realistic spiking phase precession/procession. The key ingredient of this analysis is the inclusion of a mechanism for slow firing-rate adaptation in addition to the otherwise fast continuous-attractor dynamics. The latter which continuous-attractor dynamics classically arises from a combination of translation invariance and nonlinear rate normalization.

For strong adaptation/weak external input, the network naturally exhibits an internally generated, travelling-wave dynamics along the attractor with some characteristic speed. For small adaptation/strong external stimulus, the network recovers the classical externally driven continuous-attractor dynamics. Crucially, when both adaptation and external input are moderate, there is a competition with the internally generated and externally generated mechanism leading to oscillatory tracking regime. In this tracking regime, the population firing profile oscillates around the neural field tracking the position of the stimulus. The authors demonstrate by a combination of analytical and computational arguments that oscillatory tracking corresponds to realistic phase precession/procession. In particular the authors can account for the emergence of a unimodal and bimodal cells, as well as some other experimental observations with respect the dependence of phase precession/procession on the animal's locomotion.

The strengths of this work are at least three-fold: 1) Given its simplicity, the proposed model has a surprisingly large explanatory power of the various experimental observations. 2) The mechanism responsible for the emergence of precession/procession can be understood as a simple yet rather illuminating competition between internally driven and externally driven dynamical trends. 3) Amazingly, and under some adequate simplifying assumptions, a great deal of analysis can be treated exactly, which allows for a detailed understanding of all parametric dependencies. This exact treatment culminates with a full characterization of the phase space of the network dynamics, as well as the computation of various quantities of interest, including characteristic speeds and oscillating frequencies.

As mentioned by the authors themselves, the main limitation of this work is that it deals with a very idealized model and it remains to see how the proposed dynamical behaviors would persist in more realistic models. For example, the model is based on a continuous attractor model that assumes perfect translation-invariance of the network connectivity pattern. Would the oscillating tracking behavior persist in the presence of connection heterogeneities? Can the oscillating tracking behavior be observed in purely spiking models as opposed to rate models as considered in this work? Another important limitation is that the system needs to be tuned to exhibit oscillation within the theta range and that this tuning involves a priori variable parameters such as the external input strength. Is the oscillating-tracking behavior overtly sensitive to input strength variations? The author mentioned that an external pacemaker can serve to drive oscillation within the desired theta band but there is no evidence presented supporting this. A final and perhaps secondary limitation has to do with the choice of parameter, namely the time constant of neural firing which is chosen around 3ms. This seems rather short given that the fast time scale of rate models (excluding synaptic processes) is usually given by the membrane time constant, which is typically about 15ms. I suspect this latter point can easily be addressed.

Despite these limitations, it is my opinion that the authors convincingly achieved their aims in this work.

Reviewer #2 (Public Review):

It is increasingly recognized that the cerebellum is involved in a wide range of cognitive and behavioral processes beyond motor coordination and motor learning. This work contributes to the recent body of work showing functional connections between the cerebellum and many other brain regions. This study uses a combination of in vivo electrophysiology, viral tracing, and optogenetics to identify pathways from the deep cerebellar nuclei (DCN) to the nucleus accumbens (NA) core and medial shell running through "nodes" in the ventral tegmental area (VTA) and centromedial and parfascicular nuclei of the thalamus. The significance of this work is in providing function data and anatomical pathways that may underlie the role of the cerebellum in reward behavior.

This work makes two significant contributions to the field. First, the authors show that electrical stimulation in the DCN (the output of the cerebellar circuit) elicits (primarily excitatory) responses in neurons of the NA core and medial shell. Previous studies have shown that stimulation in the cerebellum increases dopamine in the NA, but this study is the first to use in vivo electrophysiology to measure changes in neuronal firing rates. Responses in NA neurons are primarily excitatory, with a small number of neurons showing inhibitory or mixed excitatory/inhibitory responses. The data here are clear and support the conclusions. The only caveat, acknowledged by the authors, is the use of ketamine/xylazine to anesthetize the mice may alter the firing properties of NA neurons and the balance of excitation and inhibition in neuronal responses. The specific mechanisms (neurotransmitters, synapses, or circuits) resulting in excitation or inhibition of NA neurons are not investigated here, though this may be an interesting avenue of future work.

The second significant contribution of this work is identifying anatomical pathways that connect DCN to the NA. The identification of these pathways is well supported by the viral injection data. The data using cre-expressing AAV in the DCN and floxed td-tomato AAV in the VTA or thalamus is particularly convincing. However, the inclusion of additional controls would strengthen the conclusions (see below).

In general, the conclusions are well-supported by the data. However, in a few places inadequate controls or description of the experiments weakens the conclusions.

1. In Figure 4, the authors injected a retrograde tracer in the NA and an anterograde tracer in DCN to find potential "nodes" of overlap. From this experiment, the authors identify the VTA and regions of the thalamus as potential areas of tracer overlap, but it is unclear how many other brain regions were examined. Did the authors jump straight to likely locations of overlap based on previous findings, or were large swaths of the brain examined systematically? If other brain regions were examined, which regions and how was this done? A table listing which brain regions were examined and the presence/intensity of ctb-Alexa568 and GFP fluorescence would be helpful.<br /> 2. In Figure 5, the authors inject AAV1-Cre in DCN and AAV-FLEX-tdTomato in VTA or thalamus. This is an interesting experiment, but controls are missing. An important control is to inject AAV-FLEX-tdTomato in the VTA or thalamus in the absence of AAV1-Cre injection in DCN. Cre-independent expression of tdTomato should be assessed in the VTA/thalamus and the NA.

Reviewer #2 (Public Review):

Place cells fire sequentially during hippocampal theta oscillations, forming a spatial representation of behavioral experiences in a temporally-compressed manner. The firing sequences during theta cycles are widely considered as essential assemblies for learning, memory, and planning. Many theoretical studies have investigated the mechanism of hippocampal theta firing sequences; however, they are either entirely extrinsic or intrinsic. In other words, they attribute the theta sequences to external sensorimotor drives or focus exclusively on the inherent firing patterns facilitated by the recurrent network architectures. Both types of theories are inadequate for explaining the complexity of the phenomena, particularly considering the observations in a previous paper by the authors: theta sequences independent of animal movement trajectories may occur simultaneously with sensorimotor inputs (Yiu et al., 2022).

In this manuscript, the authors concentrate on the CA3 area of the hippocampus and develop a model that accounts for both mechanisms. Specifically, the model generates extrinsic sequences through the short-term facilitation of CA3 cell activities, and intrinsic sequences via recurrent projections from the dentate gyrus. The model demonstrates how the phase precession of place cells in theta sequences is modulated by running direction and the recurrent DG-CA3 network architecture. To evaluate the extent to which firing sequences are induced by sensorimotor inputs and recurrent network architecture, the authors use the Pearson correlation coefficient to measure the "intrinsicity" and "extrinsicity" of spike pairs in their simulations.

I find this research topic to be both important and interesting, and I appreciate the clarity of the paper. The idea of combining intrinsic and extrinsic mechanisms for theta sequences is novel, and the model effectively incorporates two crucial phenomena: phase precession and directionality of theta sequences. I particularly commend the authors' efforts to integrate previous theories into their model and conduct a systematic comparison. This is exactly what our community needs: not only the development of new models, but also understanding the critical relationships between different models.

Reviewer #2 (Public Review):

The authors had two aims in this study. First, to develop a tool that lets them quantify the synaptic strength and sign of upstream neurons in a large network of cultured neurons. Second, they aimed at disentangling the contributions of excitatory and inhibitory inputs to spike generation.

For the quantification of synaptic currents, their methods allows them to quantify excitatory and inhibitory currents simultaneously, as the sign of the current is determined by the neuron identity in the high-density extracellular recording. They further made sure that their method works for nonstationary firing rates, and they did a simulation to characterize what kind of connections their analysis does not capture. They did not include the possibility of (dendritic) nonlinearities or gap junctions or any kind of homeostatic processes. I see a clear weakness in the way that they quantify their goodness of fit, as they only report the explained variance, while their data are quite nonstationary. It could help to partition the explained variance into frequency bands, to at least separate the effects of a bias in baseline, the (around 100 Hz) band of synaptic frequencies and whatever high-frequency observation noise there may be. Another weak point is their explanation of unexplained variance by potential activation of extrasynaptic receptors without providing evidence. Given that these cultures are not a tissue and diffusion should be really high, this idea could easily be tested by adding a tiny amount of glutamate to the culture media.

For the contributions of excitation and inhibition to neuronal spiking, the authors found a clear reduction of inhibitory inputs and increase of excitation associated with spiking when averaging across many spikes. And interestingly, the inhibition shows a reversal right after a spike and the timescale is faster during higher network activity. While these findings are great and provide further support that their method is working, they stop at this exciting point where I would really have liked to see more detail. A concern, of course is that the network bursts in cultures are quite stereotypical, and that might cause averages across many bursts to show strange behaviour. So what I am missing here is a reference or baseline or null hypothesis. How does it look when using inputs from neurons that are not connected? And then, it looks like the E/(E+I) curve has lots of peaks of similar amplitude (that could be quantified...), so why does the neuron spike where it does? If I would compare to the peak (of similar amplitude) right before or right after (as a reference) are there some systematic changes? Is maybe the inhibition merely defining some general scaffold where spikes can happen and the excitation causes the spike as spiking is more irregular?<br /> The averaged trace reveals a different timescale for high and low activity states. But does that reflect a superposition of EPSCs in a single trial or rather a different jittering of a single EPSC across trials? For answering this question, it would be good to know the variance (and whether/ how much it changes over time). Maybe not all spikes are preceded by a decrease in inhibition. Could you quantitify (correlate, scatterplot?) how exactly excitation and inhibition contributions relate for single postsynaptic spikes (or single postsynaptic non-spikes)? After all, this would be the kind of detail that requires the large amount of data that this study provides.

For the first part, the authors achieved their goal in developing a tool to study synaptic inputs driving subthreshold activity at the soma, and characterizing such connections. For the second part, they found an effect of EPSCs on firing, but they barely did any quantification of its relevance due to the lack of a reference.

With the availability of Neuropixels probes, there is certainly use for their tool in in vivo applications, and their statistical analysis provides a reference for future studies.<br /> The relevance of excitatory and inhibitory currents on spiking remains to be seen in an updated version of the manuscript.

I feel that specifically Figures 6 and 7 lack relevant detail and a consistent representation that would allow the reader to establish links between the different panels. The analysis shows very detailed examples, but then jumps into analyses that show population averages over averaged responses, losing or ignoring the variability across trials. In addition, while their results themselves pass a statistical test, it is crucial to establish some measure of how relevant these results are. For that, I would really want to know how much spiking would actually be restricted by the constraints that would be posed by these results, i.e. would this be reflected in tiny changes in spiking probabilities, or are there times when spiking probabilities are necessarily high, or do we see times when we would almost certainly get a spike, but neurons can fire during other times as well.<br /> I would agree that a detailed, quantitative analysis of this question is beyond the scope of this paper, but a qualitative analysis is feasible and should be done. In the following, I am detailing what I would consider necessary to be done about these two Figures:

Figure 6C is indeed great, though I don't see why the authors would characterize synchrony as low. When comparing with Figure 4B, I'd think that some of these values are quite high. And it wouldn't help me to imagine error bars in panel 6D.<br /> Figure 6B is useful, but could be done better: The autocovariance of a shotnoise process is a convolution of the autocovariance of underlying point process and the autocovariance of the EPSC kernel. So one would want to separate those to obtain a better temporal resolution. But a shotnoise process has well defined peaks, and the time of these local maxima can be estimated quite precisely. Now if I would do a peak triggered average instead of the full convolution, I would do half of the deconvolution and obtain a temporally asymmetric curve of what is expected to happen around an EPSC. Importantly, one could directly see expected excitation after inhibition or expected inhibition after excitation, and this visualization could be much better and more intuitively compared to panel 6E.<br /> Panel D needs some variability estimate (i.e. standard deviation or interquartile range or even a probability density) for those traces.<br /> Figure 6E: Please use more visible colors. A sensitivity analysis to see traces for 2E/(2E+I) and E/(E+2I) would be great.<br /> Figure 6F: with an updated panel B, we should be able to have a slope for average inhibition after excitation for each of these cells. A second panel / third column showing those slopes would be of interest. It would serve as a reference for what could be expected from E-I interactions alone.<br /> Figure 6G: Could the authors provide an interquartile range here?

Figure 7A: it may be hard to squeeze in variability estimates here, but the information on whether and how much variance might be explained is essential. Maybe add another panel to provide a variability estimate? The variability estimate in panel 7B and 7D only reflect variability across connections, and it would be useful to add panels for the timecourses of the variability of g (or E/(E+I) respectively).

As a suggestion for further analysis, though I am well aware that this is likely beyond the scope of this manuscript, I'd suggest the following analysis:<br /> I would split the data into the high and low activity states. Then I would compute the average of E/(E+I) values for spikes. Assuming that spikes tend to happen for local maxima of E/(E+I) I would find local maxima for periods without spike such that their average is equal to the value for actual spikes. Finally, I would test for a systematic difference in either excitation or inhibition.<br /> If there is no difference, you can make the claim that synaptic input does not guarantee a spike, and compare to a global average of E/(E+I).

Reviewer #2 (Public Review):

In this paper, the authors seek to identify genes that contribute to gut inflammation by capitalizing on deep phenotyping data in a mouse genetic reference population fed a high-fat or chow diet and then integrating it with human genetic data on gut inflammatory diseases, such as inflammatory bowel disease (IBD) and Ulcerative Colitis (UC). To achieve this the authors performed genome-wide gene expression in the colon of 52 BXD strains of mice fed either a high-fat or chow diet. From this analysis, they observed significant variation in gene expression related to inflammation among the 52 BXD strains and differential gene expression of inflammatory genes fed a high-fat diet. Overlaying this data with existing mouse and human data of inflammatory gut disease identified a significant enrichment. Using the 52 BXD strains the authors were able to identify specific subsets of strains that were susceptible and resistant to gut inflammation and analysis of gene expression within the colon of these strains was enriched with mouse and human IBD. Furthermore, analysis of cytokine levels of IL-10 and IL-15 were analyzed and found to be increased in resistant BXD strains and increased in susceptible BXD strains.

Using the colon genome-wide gene expression data from the 52 BXD strains, the authors performed gene co-expression analysis and were able to find distinct modules (clusters) of genes that correlated with mouse UC and human IBD datasets. Using the two modules, termed HFD_M28 and HFD_M9 that correlated with mouse UC and human IBD, the authors performed biological interrogation along with transcription factor binding motif analysis to identify possible transcriptional regulators of the module. Next, they performed module QTL analysis to identify potential genetic regulators of the two modules and identified a genome-wide significant QTL for the HFD_M28 on mouse chromosome 16. This QTL contained 552 protein-coding genes and through a deduction method, 27 genes were prioritized. These 27 genes were then integrated with human genetic data on IBD and two candidate genes, EPHA6 and MUC4 were prioritized.

Overall, this paper provides a framework and elegant use of data from a mouse genetic reference population coupled with human data to identify two strong candidate genes that contribute to human IBD and UC diseases. In the future, it will be interesting to perform targeted studies with EPHA6 and MUC4 and understand their role in gut inflammatory diseases.

Reviewer #2 (Public Review):

This is a novel and interesting study in which the authors aimed to gain a better understanding of whether there is an optimum number of close friends to gain good mental well-being/functioning and its underlying neural mechanisms. They thoroughly examined how the number of close friendships contributes to mental health, cognition, (social) brain structure, and neural molecular processes in adolescents. They conducted multiple analyses on two large datasets to answer their research question(s) and support the results with visually attractive figures. I believe this paper is of added value to the literature as the evidence presently robustly points to the optimum number of 5 close friends in relation to mental health and cognition and related neurobiological mechanisms. This greatly advances the knowledge in the field of social and neurocognitive psychology.

The authors use a variety of measures to assess mental health, cognition, and neural mechanisms, which is a strength of the study. However, the theoretical background of these constructs should be elaborated on or unpacked to a greater extent in the introduction. Relatedly, the discussion could benefit from clearer main messages conveyed by individual paragraphs. It is currently hard to follow how the authors interpret their results in the context of existing literature.

Reviewer #2 (Public Review):

Transporters cycle between several conformational states; however, developing a unifying cycle for a single transporter is often difficult, as different homologs are often used to experimentally determine the structures of different conformations. The manuscript of Mitrovic et al. is a clever and inspiring combination of computational methods to reconstruct the transport cycle and free-energy landscape of a single sugar transporter. Using co-evolution and machine learning, the authors extracted state-specific residue contacts, many of which were previously unobserved, and potentially describe subtle yet important structural features. Using these contacts, they bias AlphaFold2 structure determination and MD simulations to accurately predict any conformation. These structures combined with enhanced sampling methods facilitate the inference of free-energy landscapes of the transport cycle. Notably, this work continues to push the limits of using and interpreting AlphaFold2 past static snapshots of highly dynamic proteins. This combination of techniques represents the forefront of structural biology, clearly demonstrating how static protein structures can be leveraged using bioinformatic and computational techniques to understand the biophysical mechanisms of proteins. Though the methodology is technically and theoretically exciting, it is as of yet unclear if this represents a substantial enough improvement over existing techniques for wider adoption. Nevertheless, this work represents an innovative combination of existing approaches to create a cohesive framework of the sugar transport cycle, and the authors provide detailed methods and supplementary information to recreate these approaches in other transporter families.

Reviewer #2 (Public Review):

Summary:

Here, the authors show that neutral lipids play a role in spermatogenesis. Neutral lipids are components of lipid droplets, which are known to maintain lipid homeostasis, and to be involved in non-gonadal differentiation, survival, and energy. Lipid droplets are present in the testis in mice and Drosophila, but not much is known about the role of lipid droplets during spermatogenesis. The authors show that lipid droplets are present in early differentiating germ cells, and absent in spermatocytes. They further show a cell autonomous role for the lipase brummer in regulating lipid droplets and, in turn, spermatogenesis in the Drosophila testis. The data presented show that a relationship between lipid metabolism and spermatogenesis is congruous in mammals and flies, supporting Drosophila spermatogenesis as an effective model to uncover the role lipid droplets play in the testis.

Strengths and weaknesses:

The authors do a commendably thorough characterization of where lipid droplets are detected in normal testes: located in young somatic cells, and early differentiating germ cells. They use multiple control backgrounds in their analysis, including w[1118], Canton S, and Oregon R, which adds rigor to their interpretations. The authors employ markers that identify which lipid droplets are in somatic cells, and which are in germ cells. The authors use these markers to present measured distances of somatic and germ cell-derived lipid droplets from the hub. Because they can also measure the distance of somatic and germ cells with age-specific markers from the hub, these results allow the authors to correlate position of lipid droplets with the age of cells in which they are present. This analysis is clearly shown and well quantified.

The quantification of lipid droplet distance from the hub is applied well in comparing brummer mutant testes to wild type controls. The authors measure the number of lipid droplets of specific diameters, and the spatial distribution of lipid droplets as a function of distance from the hub. These measurements quantitatively support their findings that lipid droplets are present in an expanded population of cells further from the hub in brummer mutants. The authors further quantify lipid droplets in germline clones of specified ages; the quantitative analysis here is displayed clearly, and supports a cell autonomous role for brummer in regulating lipid droplets in spermatocytes.

Data examining testis size and number of spermatids in brummer mutants clearly indicates the importance of regulating lipid droplets to spermatogenesis. The authors show beautiful images supported by rigorous quantification supporting their findings that brummer mutants have both smaller testes with fewer spermatids at both 29 and 25C. There is also significant data supporting defects in testis size for 14-day-old brummer mutant animals compared to controls. The comparison of number of spermatids at this age is not significant, which does not detract from the the story but does not support sperm development defects specifically caused by brummer loss at 14 days. Their analysis clearly shows an expanded region beyond the testis apex that includes younger germ cells, supporting a role for lipid droplets influencing germ cell differentiation during spermatogenesis.

The authors present a series of data exploring a cell autonomous role for brummer in the germline, including clonal analysis and tissue specific manipulations. The clonal data indicating increased lipid droplets in spermatocyte clones, and a higher proportion of brummer mutant GSCs at the hub are convincing and supported by quantitation. The authors also show a tissue specific rescue of the brummer testis size phenotype by knocking down mdy specifically in germ cells, which is also supported by statistically significant quantitation. The authors present data examining the number of spermatocyte and post-meiotic clones 14 days after clonal induction. While data they present is significant with a 95% confidence interval and a p value of 0.0496, its significance is not as robust as other values reported in the study, and it is unclear how much information can be gained from that specific result.

The authors do a beautiful job of validating where they detect brummer-GFP by presenting their own pseudotime analysis of publicly available single cell RNA sequencing data. Their data is presented very clearly, and supports expression of brummer in older somatic and germline cells of the age when lipid droplets are normally not detected. The authors also present a thorough lipidomic analysis of animals lacking brummer to identify triglycerides as an important lipid droplet component regulating spermatogenesis.

Impact:

The authors present data supporting the broad significance of their findings across phyla. This data represents a key strength of this manuscript. The authors show that loss of a conserved triglyceride lipase impacts testis development and spermatogenesis, and that these impacts can be rescued by supplementing diet with medium-chain triglycerides. The authors point out that these findings represent a biological similarity between Drosophila and mice, supporting the relevance of the Drosophila testis as a model for understanding the role of lipid droplets in spermatogenesis. The connection buttresses the relevance of these findings and this model to a broad scientific community.

Reviewer #2 (Public Review):

The manuscript illuminates the biological function of the Cac-1 "KER" region within the CAF-1 chromatin assembly factor 1. (This region has a high density of lysine, glutamic acid and arginine residues). The authors present a comprehensive study including quantitative EMSA analyses, analysis of mutants in-vivo, CD, and X-ray crystallography to identify the KER domain as a single alpha-helix element (SAH) that is largely responsible for the ability of the yCAF-1 complex to selectively binding ~40 bp dsDNA fragments over shorter ds oligos, thought to be a 'measuring' function that determines there is sufficient space for assembling H3/H4 tetramers after passage of the DNA replication complex. Moreover, they find that deletions or modifications of the KER domain contribute to yeast phenotypes consistent with a deficiency in chromatin assembly. The data in the paper is compelling, supports the conclusions and adds critical new information regarding how CAF-1 functions accomplishes its 'spacing' function in cooperation with DNA replication machinery to deposit H3/H4 dimers onto replicated DNA.

Reviewer #2 (Public Review):

The authors demonstrated that noradrenaline regulates Cav1.2 through PKC, which phosphorylates and activates Pyk2. Pyk2, in turn, autophosphorylates itself at Y402, which serves as a binding site for Src SH2 domain. Src will then phosphorylate Pyk2 at Y579 for full activation. Src also autophosphorylates itself at Y416. In this way, these two proteins generate a self-activating complex where Pyk activate Src, which then activates Pyk. Overall, this leads to an an activation of Cav1.2 and mediates noradrenaline-mediated augmentation of LTCC-mediated LTP.

Reviewer #2 (Public Review):

One of the key questions in circuit neuroscience is how learned information guides behavior. Modi et al. investigated this question in Drosophila's mushroom bodies (MBs), where olfactory memory traces are formed during pavlovian olfactory conditioning. They have used optogenetics to restrict the formation of memory traces in selective output compartments of the Kenyon cell (KC) axon terminals, the principal intrinsic neurons of the MB, and tested how flies use these 'minimal memories' during learned olfactory discrimination. They found that memory traces formed in some compartments support discrimination between similar odor pairs, whereas others do not. They then investigated the neural basis of this difference by comparing the responses of relevant output neurons (MBONs) to similar and dissimilar odor pairs. They discovered that MBONs' responses could predict behavioral outcomes if odor presentation profiles during calcium imaging mimic olfactory experience during behavior. This paper and previous works support the idea that flies use olfactory memory templates flexibly to suit their behavioral needs. However, one key difference between this paper and the previous works is the site of discrimination. While previous studies using intensity discrimination have pointed towards spike-latency and on and off responses of the KCs as the main mechanism behind discrimination, Modi et al. have not detected any response difference for similar odor pairs among the KCs. Therefore, they concluded that a hitherto unknown mechanism creates these context-specific responses at the MBONs. The findings will advance our understanding of how memories are recalled during behavior. However, the authors need to bolster their data by including some critical controls that are currently missing.

Reviewer #2 (Public Review):

In Rey et al., the authors goal was to characterize the development of a myelin-like (lacunar) expansion of glial membrane in Drosophila. Although myelin is largely considered a vertebrate innovation, there are a handful of invertebrate models that have been described with glial-derived "myelin," though these systems are not amenable to the same genetic control as Drosophila. To that end, the authors first newly-developed genetics and antibodies to characterize the presence of an axon initial segment (AIS) for adult Drosophila motor neurons that is present at the border between the central and peripheral nervous systems. They show that both sodium (Para) and potassium (Shal) channels, which are typically enriched at the AIS in mammalian neurons, are enriched at this border specifically on motor neurons. They then used multiple types of transmission electron microscopy to visualize this region and found that along with clustering of channels, there is an expansion of membranes from wrapping glia that is reminiscent of myelin. At times, this expansion spirally wraps around larger axons. Finally, they show that genetic ablation of wrapping glia results in an upregulation and redistribution of Para.

Major strengths of this manuscript include the creation of new genetic tools for visualization of subcellular features (e.g. channels) by both light microscopy and electron microscopy.

While this manuscript provides an interesting set of data, but suffers from a lack of quantification and annotation to allow the reader to judge whether this is a robust phenomenon. To increase the reader's confidence in these studies, substantially more quantification of the data is required.

Furthermore, to improve the accessibility of this manuscript, I have the following suggestions:

1. Please label the panels throughout the figures with an abbreviated genotype and what the fluorophores signify. Similarly, the presence of scale bars in uneven across the figures.

2. For panels where only one channel is shown, please show these in black and white, which is easier for the visually-impaired.

Overall, the description of "myelin" in Drosophila would open up the field of myelin biology to a new model system to study the molecular mechanisms that facilitated the evolution of this important glial structure. Thus, further analysis of the data would be advantageous.

Reviewer #2 (Public Review):

In this manuscript, Clay et al. investigate the underlying effects of reduced mRNA translation beneficial on protein aggregation and aging. They aim to test two pre-existing hypotheses: The selective translation model proposes that downregulation of overall translation increases the capacity of ribosomes to translate selected factors that in turn increase stress resistance against toxicity. The reduced folding load model suggests that during high mRNA translation rates, newly synthesized peptides and proteins can overwhelm the protein folding capacity of the cell and therefore cause protein toxicity. By generally lowering mRNA translation, lower loads of newly synthesized proteins should cause less protein folding stress and hence protein toxicity.

To understand how reduced mRNA translation mediates its beneficial effects in the context of the proposed models, the authors use different drugs established previously in other in vitro and in vivo systems to inhibit selected steps of translation. The systemic effects of translation initiation versus elongation inhibition in C. elegans are compared during heat shock, specific protein aggregation stresses and aging. These phenotypes are further tested for dependence on hsf-1, as contradictory data on the effect of translation inhibition during thermal stress in the context of hsf-1 dependency exist.

The data show that inhibition of translation initiation protects from heat stress and age-associated protein aggregation but on the contrary further sensitizes animals to protein toxicity induced by a misfunctioning proteasome. Further, inhibition of translation initiation increases lifespan in WT animals. The survival phenotypes observed during heat shock and regular lifespan assays are dependent of HSF-1, supporting the selective translation model. As stated in the manuscript, these findings themselves are not new, given that similar observations were made before using genetic models. Interestingly, the inhibition of translation elongation protects from heat stress, but, unlike initiation inhibition, also proteasome-misfunction-induced protein toxicity. Both phenotypes were observed to be independent of hsf-1. The authors further find that inhibiting elongation does not reduce protein aggregation in aged worms and does not prolong lifespan in wildtype animals. It does increase lifespan in short-lived hsf-1 mutants, where protein homeostasis is compromised. To a degree, these findings support the reduced folding load model. Overall, from these observations the authors summarize that the systemic consequences of lowering translation depend on the step in which translation is inhibited as well as the environmental context. The authors conclude that different ways to inhibit translation can protect from different insults by independent mechanisms.

Impact, strengths and weaknesses:

mRNA translation and its regulation is one of the most studied mechanisms connected to lifespan extension. However, gaps behind the protective effects of translation inhibition are so far unresolved, as stated by the authors. Therefore, testing existing hypotheses explaining the beneficial effects of translation inhibition is of great interest, not only for C. elegans researchers but a broad community working on the effects of misregulated translation during aging and disease. Overall, the conclusions made by the authors are generally supported by the data shown in this manuscript. However, some major gaps remain and need to be clarified and extended.

Reviewer #2 (Public Review):

The authors explore the role of bicarbonate-regulated soluble adenylate cyclase in modulating cardiac mitochondrial energy supply. In isolated rat mitochondria, they show that cyclic AMP (but not the permeable cAMP analog 8-Br-cAMP) increases ATP production via a Ca-independent mechanism at a location in the intermembrane space of the mitochondria, rather than in the matrix, as previously reported. Moreover, they show that inhibition of EPAC, but not PKA, inhibits the response. The effect required supplementing the mitochondria with GTP and GDP to facilitate activation of the EPAC effector GTPase Rap1. The study provides interesting new information about how the heart might adapt to changes in energy supply and demand through complementary regulatory processes involving both Ca and cyclic AMP.

The authors nicely demonstrate that soluble adenylate cyclase is localized to mitochondria. They argue, based on the effects of cyclic AMP, which is accessible to the mitochondrial intermembrane space (IMS) but not the matrix, that the signalling pathway is located in the IMS. They also find that EPAC/Rap1 is the likely downstream effector of cyclic AMP, through yet unknown targets regulating oxidative phosphorylation.

A weakness is that the components of signaling (sAC, EPAC, and rap1) are not definitively localized to a specific mitochondrial compartment using the superresolution imaging methods employed.