Reviewer #3 (Public Review):

Summary:

Using ex vivo electrophysiology and morphological analysis, Boi et al. investigate the electrophysiological and morphological properties of serotonergic and dopaminergic subpopulations in the dorsal raphe nucleus (DRN). They performed labor-intensive and rigorous electrophysiology with posthoc immunohistochemistry and neuronal reconstruction to delineate the two major cell classes in the DRN: DRN-DA and DRN-5HT, named according to their primary neurotransmitter machinery. They find that the dopaminergic (DRN-DA) and serotonergic (DRN-5HT) neurons are electrophysiologically and morphologically distinct, and are altered following striatal injection of the toxin 6-OHDA. However, these alterations were largely prevented in DRN-5HT neurons by pre-treatment with desipramine. These findings suggest an important interplay between catecholaminergic systems in healthy and parkinsonian conditions, as well as a relationship between neuronal structure and function.

Strengths:

Large, well-validated dataset that will be a resource for others.<br /> Complementary electrophysiological and anatomical characterizations.<br /> Conclusions are justified by the data.<br /> Relevant for basic scientists interested in DRN cell types and physiology<br /> Relevant for those interested in serotonin and/or DRN neurons in Parkinson's Disease

Weaknesses:

Given the scope of the author's questions and hypotheses, I did not identify any major weaknesses.

Reviewer #3 (Public Review):

Summary:

The authors have examined the 5-HT3 receptor using voltage clamp fluorometry, which enables them to detect structural changes at the same time as the state of receptor activation. These are ensemble measurements, but they enable an impressive scheme of the action of different agonists and antagonists to be built up. The growing array of structural snapshots of 5-HT3 receptors is used to good effect to understand the results.

Strengths:

The combination of rigorously tested fluorescence reporters with oocyte electrophysiology across a large panel of ligands is a solid development for this receptor type.

Weaknesses:

In their revision, the authors corrected all the weaknesses of the original submission.

Reviewer #3 (Public Review):

Summary:

Chang et al. investigated the mechanisms governing collagen fibrillogenesis, firstly demonstrating that cells within tail tendons are able to uptake exogenous collagen and use this to synthesize new collagen-1 fibrils. Using an endocytic inhibitor, the authors next showed that endocytosis was required for collagen fibrillogenesis and that this process occurs in a circadian rhythmic manner. Using knockdown and overexpression assays, it was then demonstrated that collagen fibril formation is controlled by vacuolar protein sorting 33b (VPS33b), and this VPS33b-dependent fibrillogenesis is mediated via Integrin alpha-11 (ITGA11). Finally, the authors demonstrated increased expression of VPS33b and ITGA11 at the gene level in fibroblasts from patients with idiopathic pulmonary fibrosis (IPF), and greater expression of these proteins in both lung samples from IPF patients and in chronic skin wounds, indicating that endocytic recycling is disrupted in fibrotic diseases.

Strengths:

The authors have performed a comprehensive functional analysis of the regulators of endocytic recycling of collagen, providing compelling evidence that VPS33b and ITGA11 are crucial regulators of this process.

Weaknesses:

Throughout the study, several different cell types have been used (immortalised tail tendon fibroblasts, NIHT3T cells, and HEK293T cells). In general, it is not clear which cells have been used for a particular experiment, and the rationale for using these different cell types is not explained. In addition, some experimental details are missing from the methods.

There is also a lack of functional studies in patient-derived IPF fibroblasts which means the link between endocytic recycling of collagen and the role of VPS33b and ITGA11 cannot be fully established.

Reviewer #4 (Public Review):

Summary:

Masala N et al showed interesting aberrant calcium microwaves in the hippocampus when synapsin promoter driven GCaMPs were expressed for a long period of time. These aberrant hippocampal Ca2+ micro-waves depend on the viral titre of the GECI. The microwave of Ca2+ was not observed when GECI was expressed only a sparse set of neurons.

Strengths:

These findings are important to wide neuroscience community especially when considering a great number of investigators are using similar approaches. Results look convincing and are consistent across several laboratories.

Weaknesses:

Synapsin promoter labels both excitatory pyramidal neurons and inhibitory neurons. To avoid aberrant Ca2+ microwave, a combination of Flex virus and CaMKII-Cre or Thy-1-GCaMP6s and 6f mice were tested. However, all these approaches limit the number of infected pyramidal neurons. While the comprehensive display of these results is appreciated, one additional important test would be more informative. To distinguish whether the microwave of Ca2+ is sufficiently caused via the expression of GCaMP in interneurons, or just a matter of pyramidal neuron density, testing Flex-GCaMP6 in interneuron specific mouse lines such as PV-Cre and SOM-Cre will provide further clarifications.

Reviewer #3 (Public Review):

Summary:

This paper aims to demonstrate the role of G-quadruplex DNA structures in the establishment of chromosome loops. The authors introduced an array of G4s spanning 275 bp, naturally found within a very well-characterized promoter region of the hTERT promoter, in an ectopic region devoid of G-quadruplex and annotated gene. As a negative control, they used a mutant version of the same sequence in which G4 folding is impaired. Due to the complexity of the region, 3 G4s on the same strand and one on the opposite strand, 12 point mutations were made simultaneously (G to T and C to A). Analysis of the 3D genome organization shows that the WT array establishes more contact within the TAD and throughout the genome than the control array. Additionally, a slight enrichment of H3K4me1 and p300, both enhancer markers, was observed locally near the insertion site. The authors tested whether the expression of genes located either nearby or up to 5 Mb away was up-regulated based on this observation. They found that four genes were up-regulated from 1.5 to 3-fold. An increased interaction between the G4 array compared to the mutant was confirmed by the 3C assay. For in-depth analysis of the long-range changes, they also performed Hi-C experiments and showed a genome-wide increase in interactions of the WT array versus the mutated form.

Strengths:

The experiments were well-executed and the results indicate a statistical difference between the G4 array inserted cell line and the mutated modified cell line.

Weaknesses:

The control non-G4 sequence contains 12 point mutations, making it difficult to draw clear conclusions. These mutations not only alter the formation of G4, but also affect at least three Sp1 binding sites that have been shown to be essential for the function of the hTERT promoter, from which the sequence is derived. The strong intermingling of G4 and Sp1 binding sites makes it impossible to determine whether all the observations made are dependent on G4 or Sp1 binding. As a control, the authors used Locked Nucleic Acid probes to prevent the formation of G4. As for mutations, these probes also interfere with two Sp1 binding sites. Therefore, using this alternative method has the same drawback as point mutations. This major issue should be discussed in the paper. It is also possible that other unidentified transcription factor binding sites are affected in the presented point mutants.

Reviewer #3 (Public Review):

Summary:

The authors study the function of HCN channels in L2/3 pyramidal neurons, employing somatic whole-cell recordings in acute slices of visual cortex in adult mice and a bevy of technically challenging techniques. Their primary claim is a non-uniform HCN distribution across the dendritic arbor with a greater density closer to the soma (roughly opposite of the gradient found in L5 PT-type neurons). The second major claim is that multiple sources of long-range excitatory input (cortical and thalamic) are differentially affected by the HCN distribution. They further describe an interesting interplay of NMDAR and HCN, serotonergic modulation of HCN, and compare HCN-related properties at 1, 2 and 6 weeks of age. Several results are supported by biophysical simulations.

Strengths:

The authors collected data from both male and female mice, at an age (6-10 weeks) that permits comparison with in vivo studies, in sufficient numbers for each condition, and they collected a good number of data points for almost all figure panels. This is all the more positive, considering the demanding nature of multi-electrode recording configurations and pipette-perfusion. The main strength of the study is the question and focus.

Weaknesses:

Unfortunately, in its present form, the main claims are not adequately supported by the experimental evidence: primarily because the evidence is indirect and circumstantial, but also because multiple unusual experimental choices (along with poor presentation of results) undermine the reader's confidence. Additionally, the authors overstate the novelty of certain results and fail to cite important related publications. Some of these weaknesses can be addressed by improved analysis and statistics, resolving inconsistent data across figures, reorganizing/improving figure panels, more complete methods, improved citations, and proofreading. In particular, given the emphasis on EPSPs, the primary data (for example EPSPs, overlaid conditions) should be shown much more.

However, on the experimental side, addressing the reviewer's concerns would require a very substantial additional effort: direct measurement of HCN density at different points in the dendritic arbor and soma; the internal solution chosen here (K-gluconate) is reported to inhibit HCN; bath-applied cesium at the concentrations used blocks multiple potassium channels, i.e. is not selective for HCN (the fact that the more selective blocker ZD7288 was used in a subset of experiments makes the choice of Cs+ as the primary blocker all the more curious); pathway-specific synaptic stimulation, for example via optogenetic activation of specific long-range inputs, to complement / support / verify the layer-specific electrical stimulation.

Reviewer #3 (Public Review):

Summary:

Authors suggest a new biomarker of chronic back pain with the option to predict the result of treatment. The authors found a significant difference in a fractional anisotropy measure in superior longitudinal fasciculus for recovered patients with chronic back pain.

Strengths:<br /> The results were reproduced in three different groups at different studies/sites.

Weaknesses:<br /> - The number of participants is still low.<br /> - An explanation of microstructure changes was not given.<br /> - Some technical drawbacks are presented.

Reviewer #3 (Public Review):

Summary:

Right-sided colorectal Cancer (CRC) is very different from left-sided CRC. Therefore it is important to model this cancer in mice and find new molecular targets. A broad set of data exists on FAK (Focal Adhesion Kinase) being important in colorectal cancer. However, this has focussed on APC mutant CRC which tends to be left-sided. BRAF mutation is common in right-sided CRC (and is rarely mutated with APC). Therefore the authors have tested whether FAK is important in this context. The authors show that FAK deletion surprisingly accelerates BRAF mutant CRC. Tumours arise in the proximal colon (which recapitulates BRAF mutant right-sided CRC). There are low for Lgr5 and high for foetal programmes. Mechanistically they suggest a pathway from FAK to NEDD4 to Lgr4 may underpin this phenotype.

Strengths:

Strong genetic data from FAK revealed that there is an acceleration of tumourigenesis and mice now develop proximal colon tumours and can be viewed as a good model of right-sided CRC.<br /> The expression data between humans and mice is strong.

Weaknesses:

The functional mechanism of how FAK loss promotes tumourigenesis is still quite correlative. An alternative hypothesis is that it drives inflammation in the proximal colon that drives tumourigenesis.

We still did not know the functional role for LGR4 (loss leads to a loss of paneth cells in homeostasis) so I'm not sure you can hypothesise a stem cell role.

Reviewer #3 (Public Review):

Summary:

The authors aimed to investigate the effectiveness of streptavidin imaging as an alternative to traditional antibody labeling for visualizing proteins within cellular contexts. They sought to address challenges associated with antibody accessibility and inconsistent localization by comparing the performance of streptavidin imaging with a TurboID-HA tandem tag across various protein localization scenarios, including phase-separated regions. They aimed to assess the reliability, signal enhancement, and potential advantages of streptavidin imaging over antibody labeling techniques.

Overall, the study provides a convincing argument for the utility of streptavidin imaging in cellular protein visualization. By demonstrating the effectiveness of streptavidin imaging as an alternative to antibody labeling, the study offers a promising solution to issues of accessibility and localization variability. Furthermore, while streptavidin imaging shows significant advantages in signal enhancement and preservation of protein interactions, the authors must consider potential limitations and variations in its application. Factors such as the fact that tagging may sometimes impact protein function, background noise, non-specific binding, and the potential for off-target effects may impact the reliability and interpretation of results. Thus, careful validation and optimization of streptavidin imaging protocols are crucial to ensure reproducibility and accuracy across different experimental setups.

Strengths:

- Streptavidin imaging utilizes multiple biotinylation sites on both the target protein and adjacent proteins, resulting in a substantial signal boost. This enhancement is particularly beneficial for several applications with diluted antigens, such as expansion microscopy or correlative light and electron microscopy.

- This biotinylation process enables the identification and characterization of interacting proteins, allowing for a comprehensive understanding of protein-protein interactions within cellular contexts.

Weaknesses:

- One of the key advantages of antibodies is that they label native, endogenous proteins, i.e. without introducing any genetic modifications or exogenously expressed proteins. This is a major difference from the approach in this manuscript, and it is surprising that this limitation is not really mentioned, let alone expanded upon, anywhere in the manuscript. Tagging proteins often impacts their function (if not their localization), and this is also not discussed.

- Given that BioID proximity labeling encompasses not only the protein of interest but also its entire interacting partner history, ensuring accurate localization of the protein of interest poses a challenge.

- The title of the publication suggests that this imaging technique is widely applicable. However, the authors did not show the ability to track the localization of several distinct proteins on the same sample, which could be an additional factor demonstrating the outperformance of streptavidin imaging compared with antibody labeling. Similarly, the work focuses only on small 2D samples. It would have been interesting to be able to compare this with 3D samples (e.g. cells encapsulated in an extracellular matrix) or to tissues.

Reviewer #3 (Public Review):

Summary:

Cellulose is a major component of the primary cell wall of growing cells and it is made by cellulose synthases (CESAs) organized into multi-subunit complexes in the plasma membrane. Previous results have resolved the structure of secondary cell wall CESAs, which are only active in a subset of cells. Here, the authors evaluate the structure of CESAs from soybeans (Glycine max, Gm) via cryo-EM and compare these structures to secondary cell wall CESAs. First, they expressed GmCESA1, GmCESA3, or GmCESA6 in insect cells, purified these proteins as both monomers and homotrimers and demonstrated their capacity to incorporate 3H-labelled glucose into the cellulase-sensitive product in a pH and divalent cation (e.g., Mg2+) -dependant fashion (Figure 1). Although CESA1, CESA3, and a CESA6-like isoform are essential for cellulose synthesis in Arabidopsis, in this study, monomers and homotrimers both showed catalytic activity, and there was more variation between individual isoforms than between their oligomerization states (i.e., CESA3 monomers and trimers showed similar activities, which were substantially different from CESA1 monomers or trimers).

They next use cryo-EM to solve the structure of each homotrimer to ~3.0 to 3.3 A (Figure 2). They compare this with PttCESA8 and find important similarities, such as the unidentified density at a positively-charged region near Arg449, Lys452, and Arg453, and differences, such as the position and relatively low resolution (suggesting higher flexibility) of TM7, which presumably creates a large lateral lipid-exposed channel opening, rather than the transmembrane pore in PttCESA8. Like PttCESA8, an oligosaccharide in the translocation channel was co-resolved with the protein structure. Neither the N-terminal domains nor the CSRs (a plant-specific insert into the cytosolic loop between TM2 and TM3) are resolved well.

Several previous models have proposed that the cellulose synthase complexes may be composed of multiple heterotrimers, but since the authors were able to isolate beta-glucan-synthesizing homotrimers, their results challenge this model. Using the purified trimers, the authors investigated how the CESA homotrimers might assemble into higher-order complexes. They detected interactions between each pair of CESA homotrimers via pull-down assays (Figure 3), although these same interactions were also detected among monomers (Supplemental Figure 4). Neither catalytic activity nor these inter-homotrimer interactions required the N-terminal domain (Figure 4). When populations of homotrimers were mixed, they formed larger aggregations in vitro (Figure 4) and displayed increased activity, compared to the predicted additive activity of each enzyme alone (Figure 5). Intriguingly, this synergistic behavior is observed even when one trimer is chemically inactivated before mixing (Supplemental Figure 6), suggesting that the synergistic effects are due to structural interactions.

Strengths:

The main strength of this manuscript is its detailed characterization of the structure of multiple CESAs, which complements previous studies of secondary cell wall CESAs. They provide a comprehensive comparison of these new structures with previously resolved CESA structures and discuss several intriguing similarities and differences. The synergistic activity observed when different homotrimers are mixed is a particularly interesting result. These results provide fundamental in vitro support for a cellulose synthase complex comprised of a hexamer of CESA homotrimers.

Weaknesses:

There are several weaknesses in the manuscript. The authors do not present any data to indicate that GmCESA1, GmCESA3, and GmCESA6 are primary cell wall CESAs (e.g. expression patterns, phylogenetic evidence). Furthermore, their evidence that these proteins make cellulose in vitro is limited to the beta-glucanase-sensitive digestion of the product. Previous reports characterizing CESA structures have used multiple independent methods: sensitivity and resistance of the product to various enzymes, linkage analysis, and importantly, TEM of the product to ensure that it makes genuine cellulose microfibrils, rather than amorphous beta-glucan. Without demonstrating that GmCESA1, GmCESA3, and GmCESA6 are genuinely synthesizing cellulose microfibrils (via TEM) and that they are primary cell wall CESAs (via expression patterns & phylogenetic evidence), it is difficult to place the results into context. Finally, the authors indicate that they were unable to isolate heterotrimers in vitro, but they do not present any evidence of these experiments, which is essential to evaluate their conclusion that these CESAs operate as homotrimers in vitro.

Reviewer #3 (Public Review):

The authors have investigated the effect of noncaloric monosaccharides on angiogenesis in the zebrafish embryo. These compounds are used as substitutes of sugars to sweeten beverages and they are commonly used by diabetic patients. The authors show that noncaloric monosaccharides and glucose similarly induce excessive blood vessel formation due to the increased formation of tip cells by endothelial cells. The authors show that this excessive angiogenesis involved the foxo1a-marcksl1a pathway.

A limitation of the study is that the mechanism of angiogenesis in the retinal circulation and in peripheral vasculature is certainly different.

This result suggests that these noncaloric monosaccharides share common side effects with glucose. Consequently, more caution should be taken with regard to the use of these artificial sweeteners. This work is of interest for better management of diabetes.

Reviewer #3 (Public Review):

This article is the first report to study the effects of T. pallidum on the neural development of an iSPC-derived brain organoid model. The study indicates that T. pallidum inhibits the differentiation of subNPC1B neurons into hindbrain neurons, hence affecting brain organoid neurodevelopment. Additionally, the TCF3 and notch signaling pathways may be involved in the inhibition of the subNPC1B-hindbrain neuron differentiation axis. While the majority of the data in this study support the conclusions, there are still some questions that need to be addressed and data quality needs to be improved. The study provides valuable insights for future investigations into the mechanisms underlying congenital neurodevelopment disability.

Reviewer #3 (Public Review):

Summary:

This study presents a solid framework for the metabolic modeling of microbial species and resources in the rhizosphere environment. It is an ambitious effort to tackle the huge complexity of the rhizosphere and reveal the plant-microbiota interactions therein. Considering previously published data by Berihu et al., going through a series of steps, the framework then finds associations between an apple tree disease state and both microbes and metabolites. The framework is well explained and motivated. I think that further work should be done to validate the method, both using synthetic data, with a known ground truth and following up on key findings experimentally.

Strengths:

- The manuscript is well written with a good balance between detail and readability. The framework steps are well-motivated and explained.

- The authors faithfully acknowledge the limitations of their approach and do not try to "over-sell" their conclusions.

- The presented framework has the potential for significant discovery if the hypotheses generated are followed up with experimental validation.

Weaknesses:

- When presenting a computational framework, best practices include running it on artificial (synthetic) data where the ground truth is known and therefore the precision and accuracy of the method may be assessed. This is not an optional step, the same way that positive/negative controls in lab experiments are not optional. Without this validation step, the manuscript is severely limited. The authors should ask themselves: what have we done to convince the reader that the framework actually works, at least on our minimal synthetic data?

Justification of claims and conclusions:

The claims and conclusions are sufficiently well justified since the limitations of this approach are acknowledged by the authors.

Reviewer #3 (Public Review):

Summary:

Authors performed a genome-wide CRISPR-based screen for synthetic lethal interactions in leukemic cells expressing a mutant form of PPM1D and identified SOD1. Loss of SOD1 or its inhibition with small molecule compounds reduced survival of the cells containing truncated PPM1D. Further analysis revealed that mitochondria are functionally deficient in PPM1D mutant cells resulting in increased levels of ROS. Surprisingly, expression profiling and reverse phase protein arrays revealed that PPM1D mutant cells did not respond appropriately to the increased levels of ROS. The precise molecular mechanism underlying this phenotype remains currently unclear, nevertheless the study convincingly shows that PPM1D mutant cells are vulnerable to oxidative stress.

Strengths:

Experimental procedures used in the study are appropriate and overall the presented data are very convincing. The study identified an important vulnerability of leukemic cells that carry PPM1D mutation and provides a fundamental background for testing SOD1 inhibitors in preclinical research. In the revised version of the manuscript, authors provide several new experiments that support their former conclusions. In particular, they showed that deletion of SOD1 in AML cells improved survival of the transplanted mice and this effect was more prominent when using cells carrying the mutant PPM1D. Further, they included an important control experiment that showed decreased SOD1 activity after treatment with ATN-224 inhibitor.

Weaknesses:

In the opinion of reviewer, there are no obvious weaknesses in this study. In broader view, the findings presented here using in vitro cultures will need to be validated in vivo by future research. Cell lines used in the study were generated by CRSIPR approaches in AML cells that have already been transformed. In addition, genome editing is inheritably connected with a risk of off target effects. It would therefore be great to identify AML samples carrying the PPM1D mutation that has been naturally selected during the transformation process.

Reviewer #3 (Public Review):

The authors comprehensively demonstrated the Cbfβ gene, which is involved in articular cartilage homeostasis, can promote articular cartilage regeneration and repair in osteoarthritis (OA) through regulating Hippo/YAP signaling TGF-β signaling, and canonical Wnt signaling. First, the authors demonstrated the deletion of Cbfβ can induce the OA phenotypes including decreased articular cartilage and osteoblasts, and increased osteoclasts and subchondral bone hyperplasia, and induce the early onset of OA. Additionally, the authors showed that the deficiency of Cbfβ in cartilage can increase canonical Wnt signaling and decrease TGF-β and Hippo signaling. Finally, the authors demonstrated that the overexpression of Cbfβ can inhibit Wnt signaling and enhance Hippo/YAP signaling in knee joints articular cartilage of ACLT-induced OA mice and protect against ACLT-induced OA. The manuscript is overall well-constructed, and the authors provided evidence to support their findings.

In Fig. 7I, it could be better to show the statistical analysis between normal and AAV-mediated Cbfβ ACLT mice groups.

In Fig. 9H-K, in the quantification analysis, the OARSI score in the DMM+AAV-YFP group is higher than in the sham group significantly. However, the SO staining results appear to show no significant difference between the DMM+AAV-luc-YFP group (Fig. 9I) and the sham group (Fig. 9H).

Reviewer #3 (Public Review):

The authors first characterize their mouse model of pancreatic cancer and show that CYRI-B mRNA is detectable in pancreatic lesions and that its amount increases over time. They also show that genetic deletion of CYRI-B accelerates pancreatic ductal adenocarcinoma (PDAC), leading to lower survival of mice. This is accompanied by higher levels of phospho-(i.e. activated)-JNK and -ERK, which are likely two of the factors driving cancer cell proliferation. Using in vivo transplantation, the authors further demonstrate that cancer cells depleted for CYRI-B exhibit decreased numbers of metastases in the mesentery, despite showing similar proliferation as control cells.<br /> Cancer cell migration can be driven by LPA, which binds LPAR1 at the surface of PDAC cells. Investigation of chemotactic migration of cancer cells towards fetal bovine serum as a source of LPA further shows that cancer cells depleted for CYRI-B and expressing GFP as control exhibit strongly reduced chemotactic migration, while cells re-expressing CYRI-B-GFP show normal chemotactic migration. Furthermore, this restored migration is blocked by using the LPAR1/3 inhibitor K116425, showing that CYRI-B is required for the chemotactic migration of PDAC cells in a gradient of serum LPA.

Using live cell imaging, the authors show that CYRI-B-GFP and LPAR1-mCherry localize to macropinocytic cups and to macropinosomes, indicating that LPAR1 can be internalized by PDAC cells through macropinocytosis. This notion is supported by immunofluorescence analyses showing that PDAC cells depleted for CYRI-B have reduced LPAR1-mCherry internalization upon stimulation with LPA, compared to cells rescued by CYRI-B-GFP expression. Collectively, the authors suggest that CYRI-B regulates macropinocytic uptake of LPAR1, thus regulating the chemotactic migration of PDAC cells towards LPA, which supports the metastasis of pancreatic cancer.

This is an interesting manuscript that makes a convincing case for the involvement of CYRI-B as a driver of PDAC. A particular strength is the expert use of different mouse models and derived cancer cell lines. The major conclusions are supported by the data presented. The results could be further strengthened by detecting CYRI-B protein (in addition to mRNA) in cancer lesions and also by staining endogenous CYRI-B and LPAR1 in the macropinocytosis experiments.

Reviewer #3 (Public Review):

The submitted paper by Hoving et al addresses the role of N-cadherin in Schwann cell collective cell migration and its previously unknown relationship with the slit/robo signaling pathway. The main conclusion is that N-cadherin has two distinct functions. One that is dependent on its classical role as a cell-cell junction protein promoting cell clustering and one that promotes cell repulsion and polarity independently of the formation of cell adhesion complexes. The second function is mediated by the Slit/Robo pathway. It is proposed that N-cadherin and Glypican-4 act together to present Slit2/3 at the surface of Schwann cells in order to trigger Robo signaling on neighboring cells.

The data about N-cadherin loss of function and the associated rescue experiments with the various truncated forms of N-cadherin are well substantiated by proper controls for efficiency and specificity. They show that the extracellular domain of Ncadherin is the one required for the repulsive effect. The experiments performed to distinguish the roles in adhesion and repulsion seem clear and conclusive. In addition, the fact the slit signal needs to be provided in a polarized manner for directional migration to occur is also clearly demonstrated in vitro and on slice assays. Overall the model that Ncadherin plays two different roles, a repulsive one via presentation of slit at the cell surface and a cell adhesion one via formation of adherens junctions, is well supported by the data and will be of interest beyond the subfield of the authors.

However, other parts of the manuscript seem weaker. If N-cadherin presentation of the Slit signal is so critical why are repulsion rates still very high in cells without N-cadherin? Same is observed with Glypican4 knockdowns. In both loss of function 50% of cell collisions lead to repulsion (compared to 70% amongst control cells). While significant such drop remains modest. The authors propose a cooperative role of Glypican-4 and N-cadherin at the cell surface as co-binding factors for Slit2/3 but they have not checked whether double knockdown of N-cad and Glypican4 might have a stronger effect. Could Glypican and N-cadherin present Slit at the cell surface independently in a somewhat redundant manner? Can Glypican and Slit interact physically in absence of N-cadherin? They also have not further analyzed the putative colocalization fo Ncad and Glypican at the cell surface.

The data supporting a role for N-cadherin in Slit's trafficking to the cell surface seem also circumstantial. While western blot data seem to indicate no change in Slit protein level after N-cad knockdown, immunostaining for Slit in such condition show a dramatic loss of Slit signal. These two independent data sets are difficult to reconcile and are not designed to address whether Slit reaches the cell surface in control or N-cadherin knockdown conditions.

If Slit signaling is so critical for repulsion why in double sit2/3 knockdown 40% of collisions still lead to repulsion. Also, no analysis of cell collision are provided upon Robo1/2 knockdown for comparison with Slit knockdowns. Altogether, these relatively mild effects of n-cad, slit or glypican knockdown on repulsion seem to indicate that other signals might contribute to contact-inhibition and polarization/repulsion of cells upon physical contact but this is unfortunately not discussed. All statements related to cell polarity stem from the overall cell morphology without being substantiated by actual polarity analysis (using markers such as detection of Rac-GTP or using a proxy such as the golgi-nucleus axis). The authors present the cell cluster generated after Sox2 expression and Sox2 + exposure to recombinant Slit2 as lacking polarity, however in one case cells do not present any flat membrane at their free edge whereas in the other case they do. This suggests a minimal cell polarity with a protrusive-like organization away from the contact. Finally, Robo1/2 siRNA knockdown are used but contrary to the other loss of functions it seems that controls for knockdown efficiency/specificity were not provided.

Therefore, while the study is overall well documented and based on solid data, some weaknesses exist.

The overall topic is clearly of broad interest as N-cadherin is protein essential in various biological settings from development to disease but the range of its biological functions remains to be fully explored. This study clearly adds to the current knowledge and how N-cadherin might act in vivo and in particular how it could mediate crosstalks between various signaling pathways.

Reviewer #3 (Public Review):

B cell receptors are produced through a combination of random V(D)J recombination and somatic hypermutation. Identifying clonal lineages - cells that descend from a common V(D)J rearrangement - is an important part of B cell repertoire analysis. Here, the authors developed a new method to identify clonal lineages from BCR data. This method builds off of prior advances in the field and uses both an adaptive clonal distance threshold and shared somatic hypermutation information to group B cells into clonal lineages.

The major strength of this paper is its thorough quantitative treatment of the subject and integration of multiple improvements into the clonal clustering process. By their simulation results, the method is both highly efficient and accurate.

The only notable weakness we identified is that much of the impact of the method will depend on its superiority to existing approaches, and this is not convincingly demonstrated by Fig. 4. In particular, little detail is given on how the other clonal clustering programs were run, and this can significantly impact their performance. More specifically:

(1) Scoper supports multiple methods for clonal clustering, including both adaptive CDR3 distance thresholds (Nouri and Kleinstein, 2018) and shared V-gene mutations (Nouri and Kleinstein, 2020). It is not clear which method was used for benchmarking. The specific functions and settings used should have been detailed and justified. Spectral clustering with shared V gene mutations would be the most comparable to the authors' method. Similar detail is needed for partis.<br /> (2) It is not clear how the adaptive thresholds and shared mutation analysis in the authors' method differ from prior approaches such as scoper and partis.<br /> (3) The scripts for performing benchmarking analyses, as well as the version numbers of programs tested, are not available.<br /> (4) Similar to above, P. 10 describes single linkage hierarchical clustering with a fixed threshold as a "crude method" that "suffers from inaccuracy as it loses precision in the case of highly-mutated sequences and junctions of short length." As far as we could tell, this statement is not backed up by either citations or analyses in the paper. It should not be difficult for the authors to test this though using their simulations, as this method is also implemented in scoper.

References<br /> Nouri N, Kleinstein SH. 2020. Somatic hypermutation analysis for improved identification of B cell clonal families from next-generation sequencing data. PLOS Comput Biol 16:e1007977. doi:10.1371/journal.pcbi.1007977<br /> Nouri N, Kleinstein SH. 2018. A spectral clustering-based method for identifying clones from high-throughput B cell repertoire sequencing data. Bioinformatics 34:i341-i349. doi:10.1093/bioinformatics/bty235

Reviewer #3 (Public Review):

Mohseni and Elhaik challenge the widespread use of PCA as an analytical and interpretive tool in the study of geometric morphometrics. The standard approach in geometric morphometrics analysis involves Generalised Procrustes Analysis (GPA) followed by Principal Component Analysis (PCA). Recent research challenges PCA outcomes' accuracy, robustness, and reproducibility in morphometrics analysis. In this paper, the authors demonstrate that PCA is unreliable for such studies. Additionally, they test and compare several Machine-Learning methods and present MORPHIX, a Python package of their making that incorporates the tools necessary to perform morphometrics analysis using ML methods.

Mohseni and Elhaik conducted a set of thorough investigations to test PCA's accuracy, robustness, and reproducibility following renewed recent criticism and publications where this method was abused. Using a set of 2 and 3D morphometric benchmark data, the authors performed a traditional analysis using GPA and PCA, followed by a reanalysis of the data using alternative classifiers and rigorous testing of the different outcomes.

In the current paper, the authors evaluated eight ML methods and compared their classification accuracy to traditional PCA. Additionally, common occurrences in the attempted morphological classification of specimens, such as non-representative partial sampling, missing specimens, and missing landmarks, were simulated, and the performance of PCA vs ML methods was evaluated.

The main problem with this manuscript is that it is three papers rolled into one, and the link doesn't work. The title promises a new Python package, but the actual text of the manuscript spends relatively little time on the Python package itself and barely gives any information about the package and what it includes or its usefulness. It is definitely not the focus of the manuscript. The main thrust of the manuscript, which takes up most of the text, is the analysis of the papionin dataset, which shows very convincingly that PCA underperforms in virtually all conditions tested. In addition, the manuscript includes a rather vicious attack against two specific cases of misuse of PCA in paleoanthropological studies, which does not connect with the rest of the manuscript at all.

If the manuscript is a criticism of PCA techniques, this should be reflected in the title. If it is a report of a new Python package, it should focus on the package. Otherwise, there should be two separate manuscripts here.

The criticism of PCA is valid and important. However, pointing out that it is problematic in specific cases and is sometimes misused does not justify labeling tens of thousands of papers as questionable and does not justify vilifying an entire discipline. The authors do not make a convincing enough case that their criticism of the use of PCA in analyzing primate or hominin skulls is relevant to all its myriad uses in morphometrics. The criticism is largely based on statistical power, but it is framed as though it is a criticism of geometric morphometrics in general.

Reviewer #3 (Public Review):

Summary:

Drug resistance is a perennial problem for malaria control and strategies to prevent the acquisition and spread of drug resistance mutations are desperately needed. One strategy is to identify drug resistance mutations that arise in blood-stage parasites, but cannot be readily spread to a new human host. Since malaria parasites must survive and replicate in mosquitoes in order to be transmitted, mutations with elevated mosquito-stage fitness defects will not spread efficiently. Buchanan and coworkers focus on the drug azithromycin and its known role of inhibiting the ribosomes found in parasite apicoplast organelles. Apicoplast organelles are known to have elevated metabolic activity in mosquito stage parasites and azithromycin resistance mutations could interfere with mosquito stage parasite development and parasite transmission.

To address this hypothesis, azithromycin-resistant P. berghei and P. falciparum parasites were generated and analyzed for transmission defects. All lines had mutations in the apicoplast ribosomal protein Rpl4 consistent with the known role of azithromycin inhibiting the 50S ribosomal subunit. Overall, the three lines (3 berghei and one falciparum) had phenotypes that should limit parasite transmission, however, detailed characterization showed that there were surprising differences between the two parasite species and even between the P. berghei lines. The P. berghei lines produced fewer oocysts and sporozoites with aberrant apicoplast morphology compared to wild-type controls. Sporozoites from azithromycin-resistant lines appeared to have motility defects and typically were not able to infect mice (one strain produced infections when 10,000 sporozoites were injected, but not when 1,000 were).

By contrast, the azithromycin-resistant P. falciparum strain did not display any mosquito-stage phenotypes and produced motile sporozoites with intact apicoplast organelles. These sporozoites, however, developed abnormally in a humanized mouse model with reduced liver-stage nuclear division and abnormal apicoplast morphology. These defects combined with a five-fold lower prevalence suggest that azithromycin-resistant P. falciparum parasites experience significant fitness costs during liver stage development (at least those harboring the G76V mutation).

Strengths:

This work was carefully conducted and transparently presented. It provides a comprehensive view of how parasite development is impacted by azithromycin resistance mutations during the mosquito and liver stages in P. berghei and P. falciparum. It adds a new dimension to the growing literature on the transmissibility of drug-resistant parasites, by showing that mutations in the apicoplast genome can impact transmission.

Weaknesses:

Whether these liver-stage defects in P. falciparum are severe enough to completely block subsequent blood-stage infection remains to be seen and would require experiments with humanized mice continuously grafted with human red blood cells - a difficult and expensive model system.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors describe a novel mode of release of small extracellular vesicles. These small EVs are released via the rupture of the membrane of so-called amphiectosomes that resemble "morphologically" Multivesicular Bodies.

These structures have been initially described by the authors as released by colorectal cancer cells (https://doi.org/10.1080/20013078.2019.1596668). In this manuscript, they provide experiments that allow us to generalize this process to other cells. In brief, amphiectosomes are likely released by ectocytosis of amphisomes that are formed by the fusion of multivesicular endosomes with autophagosomes. The authors propose that their model puts forward the hypothesis that LC3 positive vesicles are formed by "curling" of the autophagosomal membrane which then gives rise to an organelle where both CD63 and LC3 positive small EVs co-exist and would be released then by a budding mechanism at the cell surface that appears similar to the budding of microvesicles /ectosomes. Very correctly the authors make the distinction from migrasomes because these structures appear very similar in morphology.

Strengths:

The findings are interesting despite that it is unclear what would be the functional relevance of such a process and even how it could be induced. It points to a novel mode of release of extracellular vesicles.

Weaknesses:

This reviewer has comments and concerns concerning the interpretation of the data and the proposed model. In addition, in my opinion, some of the results in particular micrographs and immunoblots (even shown as supplementary data) are not of quality to support the conclusions.

Reviewer #3 (Public Review):

Summary:

White et al. described laser-induced wound healing of the Drosophila pupal notum. They found that the epithelial monolayer is dynamically induced to form syncytia by cell-cell fusion as an important part of repair. They reveal two processes: cell shrinking and border breakage that occur as part of syncytia formation. Expression of GFP in the cytoplasms of some epithelial cells reveals that cytoplasmic contents mix following injury and the GFP rapidly diffuses between cells. Using live imaging they observe that syncytia expand towards the wound, maintain their positions close to the leading edge, and apparently displace smaller cells. They propose that syncytia redistribute cellular components towards the wound facilitating repair and show that labelled actin becomes concentrated at the leading edge.

Strengths:

The manuscript is interesting and on an important and emerging topic of wound healing in a genetically tractable organism. The manuscript is very well written.

Weaknesses:

There are three major issues that the authors must address:

(1) Is cell-cell fusion sufficient to enhance/facilitate wound healing?

(2) Characterization of "border breakdown"; Is this phenomenon disassembly of apical junctions following membrane fusion?

(3) Are cells really shrinking or is it only the apical domains that "shrink" as the cells join the syncytium?

Reviewer #3 (Public Review):

Summary:

In this manuscript, Weng et al. detect a neuron-specific transcriptome that regulates aging. The authors first profile neuron-specific responses during aging at a time point where a loss in memory function is present. They discover signatures unique to neurons which validate their pipeline and reveal the loss of neuron identity with age. For example, old neurons reduce the expression of genes related to synaptic function and neuropeptide signaling and increase the expression of chromatin regulators, insulin peptides, and glycoproteins. The authors discover the detrimental effect of selected upregulated genes (utx-1, ins-19, and nmgp-1) by knocking them down in the whole body and detecting improvement of short memory functions. They then use their pipeline to test neuronal profiles of long-lived insulin/IGF mutants. They discover that genes related to stress response pathways are upregulated upon longevity (e.g. dod-24, F08H9.4) and that they are required for improved neuron function in long-lived individuals.

Strengths:

Overall, the manuscript is well-written, and the experiments are well-described. The authors take great care to explain their reasoning for performing experiments in a specific way and guide the reader through the interpretation of the results, which makes this manuscript an enjoyable and interesting read. Using neuron-specific transcriptomic analysis in aged animals the authors discover novel regulators of learning and memory, which underlines the importance of cell-specific deep sequencing. The time points of the transcriptomic profiling are elegantly chosen, as they coincide with the loss of memory and can be used to specifically reveal gene expression profiles related to neuron function. The authors showcase on the dod-24 example how powerful this approach is. In long-lived insulin/IGF-1 receptor mutants body-wide dod-24 expression differs from neuron-specific profiles. Importantly, the depletion of dod-24 has an opposing effect on lifespan and learning memory. The dataset will provide a useful resource for the C. elegans and aging community.

Weaknesses:

While this study nicely describes the neuron-specific profiles, the authors do not test the relevance in a tissue-specific way. It remains unclear if modifying the responses only in neurons has implications for either memory or potentially for lifespan. The authors point to this in the text and refer to tissue-specific datasets. However, it is possible that the tissue-specific profile changes with age. The authors should consider mining publicly available cell-specific aging datasets and performing neuron-specific RNAi to test the functional relevance of the neuron-specific response. This would strengthen the importance of cell-specific profiling.

Reviewer #3 (Public Review):

Summary:

The authors have created DHCR24 knockin mice and noted changes in the sperm sterol composition. Concurrently, alterations in the quantity, motility, and function of the sperm in DHCR24 knockin mice were identified.

Strengths:

The manuscript offers an intriguing perspective on how disruptions in sperm sterol composition can lead to sperm abnormalities.

Weaknesses:

From the current data, several issues remain to be clarified, including the fertility test results, which merit a more detailed presentation to ascertain whether differences stem from individual variability or overall changes. The authors suggest an increase in ROS in the sperm of DHCR24 knockin mice, leading to sperm damage, which also requires further confirmation. Moreover, the quality of some data requires verification or improvement, such as the morphological analysis of testicular sections and the OCR experiments.

Reviewer #3 (Public Review):

The author presents a novel theory and computational model suggesting that grid cells do not encode space, but rather encode non-spatial attributes. Place cells in turn encode memories of where those specific attributes occurred. The theory accounts for many experimental results and generates useful predictions for future studies. The model's simplicity and potential explanatory power will interest others in the field, though there are a number of concerns that should first be addressed.

A crucial assumption of the model is that the content of experience must be constant in space. It's difficult to imagine a real-world example that satisfies this assumption. Odors and sounds are used as examples. While they are often more spatially diffuse than an objects on the ground, odors and sounds have sources that are readily detectable. Animals can easily navigate to a food source or to a vocalizing conspecific. This assumption is especially problematic because it predicts that all grid cells should become silent when their preferred non-spatial attribute (e.g. a specific odor) is missing. I'm not aware of any experimental data showing that grid cells become silent. On the contrary, grid cells are known to remain active across all contexts that have been tested, including across sleep/wake states. Unlike place cells, grid cells do not seem to turn off. Since grid cells are active in all contexts, their preferred attribute must also be present in all contexts, and therefore they would not convey any information about the specific content of an experience.

The proposed novelty of this theory is that other models all assume that grid cells encode space. This isn't quite true of models based on continuous attractor networks, the discussion of which is notably absent. More specifically, these models focus on the importance of intrinsic dynamics within the entorhinal cortex in generating the grid pattern. While this firing pattern is aligned to space during navigation and therefore can be used as a representation of that space, the neural dynamics are preserved even during sleep. Similarly, it is because the grid pattern does not strictly encode physical space that grid-like signals are also observed in relation to other two-dimensional continuous variables.

The use of border cells or boundary vector cells as the main (or only) source of spatial information in the hippocampus is not well supported by experimental data. Border cells in the entorhinal cortex are not active in the center of an environment. Boundary-vector cells can fire farther away from the walls but are not found in the entorhinal cortex. They are located in the subiculum, a major output of the hippocampus. While the entorhinal-hippocampal circuit is a loop, the route from boundary-vector cells to place cells is much less clear than from grid cells. Moreover, both border cells and boundary-vector cells (which are conflated in this paper) comprise a small population of neurons compared to grid cells.

Reviewer #3 (Public Review):

In this manuscript, Berger et al. study how internal energy storage influence learning and memory. Since in Drosophila melanogaster, octopamine (OA) is involved in the regulation of energy homeostasis they focus on the roles of OA. To do so they use the tyramine-β-hydroxylase (Tbh) mutant that is lacking the neurotransmitter OA and study short term memory (STM), long-term memory (LTM) and anesthesia-resistant memory (ARM). They show that the duration of starvation affects the magnitude of both short- and long-term memory. In addition, they show that OA has a suppressive effect on learning and memory. In terms of energy storage, they show that internal glycogen storage influences how long sucrose is remembered, and high glycogen suppresses memory. Finally, they show that insulin-like signaling in octopaminergic neurons, which is also related to internal energy storage, suppresses learning and memory.

The revised version of the manuscript is greatly improved, and I thank the authors for taking the comment seriously. This is an important study that extends our knowledge on OA activity in learning and memory and the effects the metabolic state has on learning and memory. The authors nicely use the genetic tools available in flies to try and unravel the complex circuitry of metabolic state level, OA activity and learning and memory. The overall take-home message of the manuscript is clear and supported by the data presented.

Reviewer #3 (Public Review):

The manuscript by Mahapatra and Takahashi addresses the role of presynaptic release site clearance during sustained synaptic activity. The authors characterize the effects of pharmacologically interfering with SV endocytosis (pre-incubation with Dynasore or Pitstop-2) on synaptic short-term plasticity (STP) at two different CNS synapses (calyx of Held synapses and hippocampal SC to CA1 synapses) using patch-clamp recordings in acute slices under experimental conditions designed to closely mimic a physiological situation (37{degree sign}C and 1.3 mM external [Ca2+]). Endocytosis blocker-induced changes in STP and in the recovery from short-term depression (STD) are compared to those seen after pharmacologically inhibiting actin filament assembly (pre-incubation with Latrunculin-B or the selective Cdc42 GTPase inhibitor ML-141). Presynaptic capacitance (Cm) recordings in calyx terminals were used to establish the effects of the pharmacological maneuvers on SV endocytosis.<br /> Latrunculin-B and ML-141 neither affect SV endocytosis (assayed by Cm recordings) nor EPSC recovery following conditioning trains, but strongly enhances STD at calyx synapses. No changes in STP were observed at Latrunculin-B- or ML-141-treated SC to CA1 synapses.

Dynasore and Pitstop-2 slow down endocytosis, limit the total amount of exocytosis in response to long stimuli, enhance STD in response to 100 Hz stimulation, but profoundly accelerate EPSC recovery following conditioning 100 Hz trains at calyx synapses. At SC to CA1 synapses, Dynasore and Pitstop-2 reduce the extend of facilitation and lower relative steady-state EPSCs suggesting a change in the facilitation-depression balance in favor of the latter.

The authors use state-of-the art techniques and their data, which is clearly presented, leads to authors to conclude that endocytosis is universally important for clearance of release sites while the importance of scaffold protein-mediated site clearance is limited to 'fast synapses'.

Unfortunately, and perhaps not completely unexpected in view of the pharmacological tools chosen, there are several observations which remain difficult to understand:

(1) Blocking site clearance affects release sites that have previously been used, i.e. sites at which SV fusion has occurred and which therefore need to be cleared. Calyces use at most 20% of all release sites during a single AP, likely fewer at 1.3 mM external [Ca2+]. Even if all those 20% of release sites become completely unavailable due to a block of release site clearance, the 2nd EPSC in a train should not be reduced by >20% because ~80% of the sites cannot be affected. However, ~50% EPSC reduction was observed (Fig. 2B1, lower right panel) raising the possibility that Dynasore does more than specifically interfering with SVs endocytosis (and possibly Pitstop as well). Non-specific effects are also suggested by the observed two-fold increase in initial EPSC size in SC to CA1 synapses after Dynasore pre-incubation.

(2) More severe depression was observed at calyx synapses after blocking endocytosis which the authors attribute to a presynaptic mechanism affecting pool replenishment. When probing EPSC recovery after conditioning 100 Hz trains, a speed up was observed mediated by an "unknown mechanism" which is "masked in 2 mM [Ca2+]". These two observations, deeper synaptic depression during 100 Hz but faster recovery from depression following 100 Hz, are difficult to align and no attempt was made to find an explanation.

(3) To reconcile previous data reporting a block of Ca2+-dependent recovery (CDR) by Dynasore or Latrunculin (measured at 2 mM external [Ca2+]) with the data presented here (using 1.3 mM external [Ca2+]) reporting no effect or a speed up of recovery from depression, the authors postulate that "CDR may operate only when excessive Ca2+ enters during massive presynaptic activation" (page 10 line 244). While that is possible, such explanation ignores plenty of calyx studies demonstrating fiber stimulation-induced CDR and elucidating molecular pathways mediating fiber stimulation-induced CDR, and it also completely dismisses the strong change in recovery time course after 10 Hz conditioning (single exponential) as compared to 100 Hz conditioning (double exponential with a pronounced fast component).

Strong presynaptic stimuli such as those illustrated in Figs. 1B,C induce massive exocytosis. The illustrated Cm increase of 2 to 2.5 pF represents fusion of 25,000 to 30,000 SVs (assuming a single SV capacitance of 80 aF) corresponding to a 12 to 15% increase in whole terminal membrane surface (assuming a mean terminal capacitance of ~16 pF). Capacitance measurements can only be considered reliable in the absence of marked changes in series and membrane conductance. Documentation of the corresponding conductance traces is therefore advisable for such massive Cm jumps and merely mentioning that the first 450 ms after stimulation were skipped during analysis or referring to previous publications showing conductance traces is insufficient.<br /> All bar graphs in Figures 1 through 6 and Figures S3 through S6 compare three or even four (Fig. 5C) conditions, i.e. one control and at least two treatment data sets. It appears as if repeated t-tests were used to run multiple two-group comparisons (i.e. using the same control data twice for two different comparisons). Either a proper multiple comparison test should be used or a Bonferroni correction or similar multiple-comparison correction needs to be applied.

Finally, the terminology of contrasting "fast-signaling" (calyx synapses) and "slow-plastic" (SC synapses) synapses seems to imply that calyx synapses lack plasticity, as does the wording "conventional bouton-type synapses involved in synaptic plasticity" (page 11, line 251). I assume, the authors primarily refer to the maximum frequencies these two synapse types typically transmit (fast-signaling vs slow-signaling)?

Reviewer #3 (Public Review):

Summary:

This study concerns how macaque visual cortical area MT represents stimuli composed of more than one speed of motion.

Strengths:

The study is valuable because little is known about how the visual pathway segments and preserves information about multiple stimuli. The study presents compelling evidence that (on average) MT neurons represent the average of the two speeds, with a bias that accentuates the faster of the two speeds. An additional strength of the study is the inclusion of perceptual reports from both humans and one monkey participant performing a task in which they judged whether the stimuli involved one vs two different speeds. Ultimately, this study raises intriguing questions about how exactly the response patterns in visual cortical area MT might preserve information about each speed, since such information could potentially be lost in an average response as described here, depending on assumptions about how MT activity is evaluated by other visual areas.

Weaknesses:

My main concern is that the authors are missing an opportunity to make clear that the divisive normalization, while commonly used to describe neural response patterns in visual areas (and which fits the data here), fails on the theoretical front as an explanation for how information about multiple stimuli can be preserved. Thus, there is a bit of a disconnect between the goal of the paper - how does MT represent multiple stimuli? - and the results: mostly averaging responses which, while consistent with divisive normalization, would seem to correspond to the perception of a single intermediate speed. This is in contrast to the psychophysical results which show that subjects can at least distinguish one from two speeds. The paper would be strengthened by grappling with this conundrum in a head-on manner.

Reviewer #3 (Public Review):

Summary:

In order to study the factors and neural dynamics that lead to the suppression of irrelevant information in the brain, the authors trained artificial neural networks in the execution of a task that involved the discrimination of complex stimuli with three main features: color, shape, and width. Specific combinations of color and shape led to a reward, but the temporal structure made color dynamically irrelevant at the beginning of the trial, and then it became relevant once the shape was presented. On the other hand, the width of the stimulus was always irrelevant. Importantly, non-human primates were also trained to execute this task (in a previous study by the authors) and the activity from neural populations from the dorsolateral Prefrontal Cortex (dlPFC) was recorded, allowing to compare the coding of information by the artificial neural network model with what happens in biological neural populations.

The authors changed systematically the amount of noise present in the neural network model, as well as limiting the firing rate of the artificial neurons to simulate the limitations imposed by high metabolic costs in biological neurons. They found that models with medium and low noise, as well as medium and low metabolic cost, developed information encoding patterns that resembled the patterns observed throughout learning in the dlPFC, as follows: early in the learning process, color information was strongly represented during the whole trial, as well as shape and width, whereas the color/shape combination significance (XOR operation) was weakly encoded. Late in learning, color information was initially suppressed (while it was deemed irrelevant) and became more prominent during the shape presentation. Width information coding decreased, and the XOR operation result became more strongly encoded.

Subthreshold activity dynamics were studied by training artificial networks consisting of 2 neurons, with the aim of understanding how dynamically irrelevant information is suppressed and then encoded more strongly at a different time during the trial. Under medium noise and medium metabolic cost, color information is suppressed by the divergence of the activity away from the level that triggers spikes. The authors claim that this subthreshold dynamic explains the suppression of irrelevant information in biological neural networks.

Strengths:

The study leverages the power of computational models to simulate biological networks and do manipulations that are difficult (if not impossible) to perform in vivo. The analyses of the activity of the network model are neat and thorough and provide a clear demonstration of how noise and metabolic costs may affect the information coding in the brain. The mathematical analyses are rigorous and nicely documented.

Weaknesses:

The study does not leverage the fact that they have access to the activity of individual neurons both on a neural network model and in neural recordings. The model/brain comparison results are limited to the decodability of different pieces of information during the execution of the task at different stages of learning. It would have been useful if the authors had shown response profiles of individual neurons, both biological and artificial, to strengthen the claim that the activity patterns are similar. Perhaps showing that the firing rates vary in a similar way in the large models (like they do for the 2-neuron model) would have been informative. For instance, it is possible that suppression is not occurring in the dlPFC, but that the PFC receives input with this information already suppressed. If suppression indeed happens in the PFC, response profiles associated with this process may be observed.

There is no way to say that the 2-neuron models are in any way informative of what happens in brain neurons, or even larger artificial networks since the sources of sensory input, noise, and inhibition will differ between biological and artificial networks. And because the firing patterns are not shown for large networks, it is not clear if some non-coding artificial neurons will become broadly inhibitory but maintain a relatively high firing rate (to mention only one possibility).

Reviewer #3 (Public Review):

Summary

In their manuscript, Vickers and McCormick have demonstrated the potential of leveraging mesoscale two-photon calcium imaging data to unravel complex behavioural motifs in mice. Particularly commendable is their dedication in providing detailed surgical preparations and corresponding design files, a contribution that will greatly benefit the broader neuroscience community as a whole. The quality of the data is high and examples are available to the community. More importantly, the authors have acquired activity-clustered neural ensembles at an unprecedented spatial scale to further correlate with high level behaviour motifs identified by B-SOiD. Such an advancement marks a significant contribution to the field. While the manuscript is comprehensive and the analytical strategy proposed is promising, some technical aspects warrant further clarification. Overall, the authors have presented an invaluable and innovative approach, effectively laying a solid foundation for future research in correlating large scale neural ensembles with behavioural. The implementation of a custom sound insulator for the scanner is a great idea and should be something implemented by others.

This is a methods paper, but there is no large diagram (in the main figures) that shows how all the parts are connected, communicating and triggering between each other. This is described in the methods and now supplemental figure, but a visual representation would greatly benefit the readers looking to implement something similar as a main figure but I guess they can find it in the methods. No stats for the results shown in Figure 6e, it would be useful to know which of these neural densities for all areas show a clear statistical significance across all the behaviors. While I understand that this is a methods paper, it seems like the authors are aware of the literature surrounding large neuronal recordings during mouse behavior. Indeed, in line 178-179 the authors mention how a significant portion of the variance in neural activity can be attributed to changes in "arousal or self-directed movement even during spontaneous behavior." Why then did the authors not make an attempt at a simple linear model that tries to predict the activity of their many thousands of neurons by employing the multitude of regressors at their disposal (pupil, saccades, stimuli, movements, facial changes, etc). These models are straightforward to implement, and indeed it would benefit this work if the model extracts information on par with what it's known from the literature. We also realize such a model could be done in the future.

Reviewer #3 (Public Review):

Summary:

The authors established a new virtual reality place preference task. On the task, rats, which were body-restrained on top of a moveable Styrofoam ball and could move through a circular virtual environment by moving the Styrofoam ball, learned to navigate reliably to a high-reward location over a low-reward location, using allocentric visual cues arranged around the virtual environment.

The authors also showed that functional inhibition by bilateral microinfusion of the GABA-A receptor agonist muscimol, which targeted the dorsal or intermediate hippocampus, disrupted task performance. The impact of functional inhibition targeting the intermediate hippocampus was more pronounced than that of functional inhibition targeting the dorsal hippocampus.

Moreover, the authors demonstrated that the same manipulations did not significantly disrupt rats' performance on a virtual reality task that required them to navigate to a spherical landmark to obtain reward, although there were numerical impairments in the main performance measure and the absence of statistically significant impairments may partly reflect a small sample size (see comments below).

Overall, the study established a new virtual-reality place preference task for rats and established that performance on this task requires the dorsal to intermediate hippocampus. They also established that task performance is more sensitive to the same muscimol infusion (presumably - doses and volumes used were not clearly defined in the manuscript, see comments below) when the infusion was applied to the intermediate hippocampus, compared to the dorsal hippocampus, although this does not offer strong support for the authors claim that dorsal hippocampus is responsible for accurate spatial navigation and intermediate hippocampus for place-value associations (see comments below).

Strengths:

(1) The authors established a new place preference task for body-restrained rats in a virtual environment and, using temporary pharmacological inhibition by intra-cerebral microinfusion of the GABA-A receptor agonist muscimol, showed that task performance requires dorsal to intermediate hippocampus.

(2) These findings extend our knowledge about place learning tasks that require dorsal to intermediate hippocampus and add to previous evidence that, for some place memory tasks, the intermediate hippocampus may be more important than other parts of the hippocampus, including the dorsal hippocampus, for goal-directed navigation based on allocentric place memory.

(3) The hippocampus-dependent task may be useful for future recording studies examining how hippocampal neurons support behavioral performance based on place information.

Weaknesses:<br /> (1) The new findings do not strongly support the authors' suggestion that the dorsal hippocampus is responsible for accurate spatial navigation and the intermediate hippocampus for place-value associations.

The authors base this claim on the differential effects of the dorsal and intermediate hippocampal muscimol infusions on different performance measures. More specifically, dorsal hippocampal muscimol infusion significantly increased perimeter crossings and perimeter crossing deviations, whereas dorsal infusion did not significantly change other measures of task performance, including departure direction and visits to the high-value location. However, these statistical outcomes offer only limited evidence that dorsal hippocampal infusion specifically affected the perimeter crossing, without affecting the other measures. Numerically the pattern of infusion effects is quite similar across these various measures: intermediate hippocampal infusions markedly impaired these performance measures compared to vehicle infusions, and the values of these measures after dorsal hippocampal muscimol infusion were between the values in the intermediate hippocampal muscimol and the vehicle condition (Figures 5-7). Moreover, I am not so sure that the perimeter crossing measures really reflect distinct aspects of navigational performance compared to departure direction and hit rate, and, even if they did, which aspects this would be. For example, in line 316, the authors suggest that 'departure direction and PCD [perimeter crossing deviation] [are] indices of the effectiveness and accuracy of navigation, respectively'. However, what do the authors mean by 'effectiveness' and 'accuracy'? Accuracy typically refers to whether or not the navigation is 'correct', i.e. how much it deviates from the goal location, which would be indexed by all performance measures.

So, overall, I would recommend toning down the claim that the findings suggest that the dorsal hippocampus is responsible for accurate spatial navigation and the intermediate hippocampus for place-value associations.

(2) The claim that the different effects of intermediate and dorsal hippocampal muscimol infusions reflect different functions of intermediate and dorsal hippocampus rests on the assumption that both manipulations inhibit similar volumes of hippocampal tissue to a similar extent, but at different levels along the dorso-ventral axis of the hippocampus. However, this is not a foregone conclusion (e.g., drug spread may differ depending on the infusion site or drug effects may differ due to differential expression of GABA-A receptors in the dorsal and intermediate hippocampus), and the authors do not provide direct evidence for this assumption. Therefore, a possible alternative account of the weaker effects of dorsal compared to intermediate hippocampal muscimol infusions on place-preference performance is that the dorsal infusions affect less hippocampal volume or less markedly inhibit neurons within the affected volume than the intermediate infusions. I would recommend that the authors briefly consider this issue in the discussion. Moreover, from the Methods, it is not clear which infusion volume and muscimol concentration were used for the different infusions (see below, 4.a.), and this must be clarified.

(3) It is good that the authors included a comparison/control study using a spherical beacon-guided navigation task, to examine the specific psychological mechanisms disrupted by the hippocampal manipulations. However, as outlined below (4.b.), the sample size for the comparison study was lower than for the main study, and the data in Figure 8 suggest that the comparison task may be affected by the hippocampal manipulations similarly to the place-preference task, albeit less markedly. This would raise the question as to which mechanisms that are common to the two tasks may be affected by hippocampal functional inhibition, which should be considered in the discussion.

(4) Several important methodological details require clarification:<br /> a. Drug infusions (from line 673):<br /> - '0.3 to 0.5 μl of either phosphate-buffered saline (PBS) or muscimol (MUS) was infused into each hemisphere'; the authors need to clarify when which infusion volume was used and why different infusion volumes were used.<br /> - I could not find the concentration of the muscimol solution that was used. The authors must clarify this and also should include a justification of the doses used, e.g. based on previous studies.<br /> - Please also clarify if the injectors and dummies were flush with the guides or by which distance they protruded from the guides.<br /> b. Sample sizes: The authors should include sample size justifications, e.g. based on considerations of statistical power, previous studies, practical considerations, or a combination of these factors. Importantly, the smaller sample size in the control study using the spherical beacon-guided navigation task (n=5 rats) limits comparability with the main study using the place-preference task (n=8). Numerically, the findings on the control task (Figure 8) look quite similar to the findings on the place-preference task, with intermediate hippocampal muscimol infusions causing the most pronounced impairment and dorsal hippocampal muscimol infusions causing a weaker impairment. These effects may have reached statistical significance if the same sample size had been used in the place-preference study.<br /> c. Statistical analyses: Why were the data of the intermediate and dorsal hippocampal PBS infusion conditions averaged for some of the analyses (Figure 5; Figure 6B and C; Figure 7B and C; Figure 8B) but not for others (Figure 6A and Figure 7A)?

Reviewer #3 (Public Review):

A mechanical model of C. elegans, embedded in a resistive force environment, is used to calculate input torque patterns required to generate output curvature patterns and coordinates, corresponding to a number of different locomotion behaviors in C. elegans.

Strengths:

The use of a mechanical model to study a variety of locomotor sequences and the grounding in empirical data are strengths. The matching of speeds (though requiring adjusted drag coefficients) is a strength.

Weaknesses:

The paper lacks evidence of numerical validation or comparison with the results and tools in the literature. E.g. is it surprising that the uniform torque distribution yields maximal speed? What is the relation between input and output data? How does the input-output relation depend on the parameters of the model? What novel model predictions are made?

In particular, if validated, the breakdown of drag forces and torque distributions during forward locomotion and turning behaviors may be interesting to compare to predictions by other tools, and to empirical measurement. One caveat is that the worm touches itself during such turns, and even crosses over itself in delta turns, and so the estimated drag coefficients and the resultant mechanical forces are likely incorrect.

Reviewer #3 (Public Review):

In Okholm et al., the authors evaluate the functional impact of circHIPK3 in bladder cancer cells. By knocking down circHIPK3 and performing an RNA-seq analysis, the authors found thousands of deregulated genes which look unaffected by miRNAs sponging function and that are, instead, enriched for a 11-mer motif. Further investigations showed that the 11-mer motif is shared with the circHIPK3 and able to bind the IGF2BP2 protein. The authors validated the binding of IGF2BP2 and demonstrated that IGF2BP2 KD antagonizes the effect of circHIPK3 KD and leads to the upregulation of genes containing the 11-mer. Among the genes affected by circHIPK3 KD and IGF2BP2 KD, resulting in downregulation and upregulation respectively, the authors found the STAT3 gene, which also consistently has concomitant upregulation of one of its targets TP53. The authors propose a mechanism of competition between circHIPK3 and IGF2BP2 triggered by IGF2BP2 nucleation, potentially via phase separation.

Strengths:

Although the number of circRNAs continues to grow, this field lacks many instances of detailed molecular investigations. The presented work critically addresses some of the major pitfalls in the field of circRNAs, and there has been a careful analysis of aspects frequently poorly investigated. Experiments involving use of time-point knockdown followed by RNA-seq, investigation of miRNA-sponge function of circHIPK3, identification of 11-mer motif, identification and validation of IGF2BP2, and the analysis of copy number ratio between circHIPK3 and IGF2BP2 in assessing the potential ceRNA mode of action are thorough and convincing.

Weaknesses:

It is unclear why the authors used certain bladder cancer cells versus non-bladder cells in some experiments. The efficacy of certain experiments (specifically rescue experiments) and some control conditions is still questionable. Overall, the presented study adds some further knowledge in describing circHIPK3 function, its capability to regulate some downstream genes, and its interaction and competition for IGF2BP2.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

The behavioral task used in this study is best suited for investigating elapsed time perception, rather than interval timing. Timing bisection tasks are often employed to study interval timing in humans and animals. The main results from unit recording (opposing slopes of D1/D2 cell firing rate, as shown in Figure 3D) appear to be very sensitive to a couple of outlier cells, and the predictive power of ensemble recording seems to be only slightly above chance levels. In the optogenetic experiment, the laser was kept on for too long (18 seconds) at high power (12 mW). This has been shown to cause adverse effects on population activity (for example, through heating the tissue) that are not necessarily related to their function during the task epochs. Given the systemic delivery of pharmacological interventions, it is difficult to conclude that the effects are specific to the dorsomedial striatum. Future studies should use the local infusion of drugs into the dorsomedial striatum.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors discover that nuclear volume decreases after mitotic exit following cell confinement in a manner that scales with the extent of confinement. This adaptation appears to protect the cells from adverse outcomes of critical confinement such as nuclear blebs and DNA damage. The evidence to support these claims is strong.

The authors also provide a model in which argue that what they call the "apparent nuclear surface area" is modulated by confinement through a mechanism regulated by cPLA2 and myosin II activities. Here there are weaknesses in that the manuscript relies on a single approach, measurements are indirect, and alternative models are not explored. Similarly, additional considerations need to be addressed so that the reader can interpret the data presented - for example whether cell volume is also changing coincident with nuclear volume changes, and whether other aspects of cell physiology such as cytokinesis are altered.

Considerations that could support the manuscript further:

One essential consideration that goes unaddressed is whether the nuclear volume alone is changing under compression (resulting in a higher nuclear to cytoplasmic ratio) or if the cell volume is changing and the nuclear volume is following suit (no change in the N:C ratio). Depending on which of these is the case, the overall model would likely shift. In particular, interpreting the effect of disrupting myosin II activity given its different distribution at the cortex in response to the higher confinement would be influenced by which of these conditions are at play.

A key approach used and interpreted by the investigators is an assessment of the folding of the "inner lamin envelope", which they derive from an image analysis routine of lamin staining that they developed and argue reflects "nuclear envelope tension". I am not convinced of the robustness of this approach or what it mechanistically reveals. It may or may not reflect the contour of the inner nuclear membrane, which (perhaps) is the most relevant to the authors' interpretation of nuclear envelope tension. Given the major contribution of this data to the model, which is based on the "unfolding" of the nuclear envelope, an orthogonal approach (e.g. electron microscopy - which one needs to truly address the high-frequency undulations of the nuclear envelope) is needed to support the larger conclusions.

The authors argue that nuclear tension is lost after mitosis in the confined devices because nuclear volume has decreased. While a smaller nuclear volume might indeed translate to less compressive force from the device on the nucleus, one would imagine that the chromosomes still have to be accommodated and that confining them in a smaller volume could increase the tension. Although arguable, the potential alternative possibilities suggest that actual measurements of nuclear envelope tension are needed to robustly test the model. The authors cite the observation that blebs are less prevalent after mitosis as additional support for this model, but this is expected as nuclear envelope breakdown and reformation will "reset" the nuclear contour while the appearance of blebs at mitotic entry is essential a "memory" of all blebs and ruptures over the entire preceding cell cycle.

Representative images for the pharmacological perturbations other than blebbistatin are notably absent - only the analyzed data are presented in the manuscript or the supplemental material. How these perturbations (e.g. to cPLA2) also affect the cortex is important to interpret the data given the point raised above. Orthogonal approaches would also strengthen the conclusions (for example, the statement that "nuclear adaptation observed during mitosis requires nuclear tension sensing through cPLA2" requires more evidence to be convincing - it is not sufficiently supported by the data presented). Even if this is the case, the authors acknowledge that cPLA2 is likely not the answer to the adaption observed under the lower degrees of confinement. Thus, the mechanisms underlying the adaptive changes to nuclear volume remain enigmatic.

One more consideration that seems to go without comment is that the cells under confinement do not appear to successfully complete cytokinesis (Fig. 5b). At a minimum this seems like a major perturbation to cell physiology and needs to be more fully discussed by the authors as playing a role in the observed changes in nuclear volume.

Reviewer #3 (Public Review):

The authors report on the engineering of an induced Pluripotent Stem Cell (iPSC) line that harbours a single copy of a split mNeonGreen, mNG2(1-10). This cell line is subsequently used to take endogenous protein with a smaller part of mNeonGreen, mNG2(11), enabling complementation of mNG into a fluorescent protein that is then used to visualize the protein. The parental cell is validated and used to construct several iPSC line with endogenously tagged proteins. These are used to visualize and quantify endogenous protein localisation during mitosis.

I see the advantage of tagging endogenous loci with small fragments, but the complementation strategy has disadvantages that deserve some attention. One potential issue is the level of the mNG2(1-10). In addition, this may probably not work for organelle-resident proteins, where the mNG2(11) tag is localised in a membrane enclosed compartment.

Overall the tools and resources reported in this paper will be valuable for the community that aims to study proteins at endogenous levels.

Reviewer #3 (Public Review):

Summary:

Kokinovic et al. presents an interesting paper that addresses an important gap in knowledge about the differences in the development of direct and indirect pathway striatal neurons in the striosome and matrix compartments. The division of the striatum into 4 distinct populations, striosome-dSPNs, striosome-iSPNs, matrix-dSPNs, and matrix-iSPNs is important, but rarely done. This study records all four populations across early development and shows differences in action potential characteristics and intrinsic properties. They also suppress striosome activity during postnatal development and evaluate the characteristics of adult dopaminergic neurons in control and previously striosome-quieted conditions.

Strengths:

The striatal electrophysiology is beautifully and carefully done and shows important developmental differences between neural subtypes.

The idea to test the striatonigral connection is a good idea.

Weaknesses:

The authors didn't actually test the striatonigral connection. The experiments they do instead don't convincingly show that the striosomal or even striatal connection to the dopaminergic neurons is altered after postnatal striosome suppression.

Major concerns:

(1) mIPSCs are measured and are reduced after chemogenetic suppression of striosomal neurons during development. This is an interesting finding, but these mIPSCs could be coming from any inhibitory input onto the SNc neurons. It is unlikely that most of the mIPSCs are coming from the striosomal inputs. The GPe is much more likely to be the source of these mIPSCs than the striatum because the GPe inputs form synapses nearer the soma and have a higher probability of release (Evans et al., 2020). dSPNs inhibit GPe neurons through a non-canonical pathway (Cui et al., 2021; Spix et al., 2021) and striosomes also inhibit the SNr (McGregor et al., 2019). The striatum has the potential to disinhibit SNc neurons through both the SNr or the GPe (Evans, 2022), and modification of the striosome-SNr or striosome-GPe connections during development could be what is causing the mIPSC changes. To claim that the striosome-SNc connection is altered, a direct test of this connection is necessary.

(2) The dopaminergic neurons recorded seem to be randomly selected, but the striosomes do not inhibit all SNc dopamine neurons. They selectively inhibit the ventral tier SNc neurons (Evans et al., 2020). In the present manuscript, it is impossible to know which subpopulation of SNc neurons was recorded, so it is impossible to tell whether the dopaminergic neurons recorded are the ones expected to receive striosomal input.

(3) Very similarly, the striosomes selectively wrap around the "SNr dendrite" of SNc neurons that participate in striosome-dendron bouquets (Crittenden et al., 2016). However, not all SNc neurons have prominent SNr dendrites (Henny et al., 2012). In the morphological images of Supplemental Figure 3, it looks like the recorded cells sometimes have an SNr dendrite and sometimes don't (but it is hard to tell because the medial-lateral rostral-caudal axis is not labeled in the images). The presence or absence of the "SNr dendrite" is a strong determinant of whether an individual dopaminergic neuron receives striosomal inhibition or not (Evans et al., 2020). As above, not knowing whether the neurons recorded have SNr dendrites makes it impossible to know whether they should be receiving striosomal input at all.

(4) It's quite interesting that the dendron-bouquet structure is intact even after striosomal activity suppression, as cannabinoid receptor knockout greatly disrupts the structural integrity of bouquets (Crittenden et al., 2022). However, going along with point 3, the gephyrin puncta analysis only at the somas is very limiting. The striosome-SNc relevant puncta would be primarily on the SNr dendrite. Gephyrin density on the SNr dendrites or in bouquets would be much more informative than density on the soma.

(5) The authors claim that "CNO didn't affect the shape of the DA neuron dendritic tree", but more information about the morphological analysis should be added. It is not clear how the sholl analysis was conducted or whether a full 3D reconstruction was made. This claim seems to be based on only one dendritic measurement (sholl analysis), but many other dendritic or morphological features could be altered.

Crittenden, J.R., Tillberg, P.W., Riad, M.H., Shima, Y., Gerfen, C.R., Curry, J., Housman, D.E., Nelson, S.B., Boyden, E.S., & Graybiel, A.M. (2016) Striosome-dendron bouquets highlight a unique striatonigral circuit targeting dopamine-containing neurons. Proc. Natl. Acad. Sci. U.S.A., 113, 11318-11323.<br /> Crittenden, J.R., Yoshida, T., Venu, S., Mahar, A., & Graybiel, A.M. (2022) Cannabinoid Receptor 1 Is Required for Neurodevelopment of Striosome-Dendron Bouquets. eNeuro, 9, ENEURO.0318-21.2022.<br /> Cui, Q., Du, X., Chang, I.Y.M., Pamukcu, A., Lilascharoen, V., Berceau, B.L., García, D., Hong, D., Chon, U., Narayanan, A., Kim, Y., Lim, B.K., & Chan, C.S. (2021) Striatal Direct Pathway Targets Npas1+ Pallidal Neurons. J Neurosci, 41, 3966-3987.<br /> Evans, R.C. (2022) Dendritic involvement in inhibition and disinhibition of vulnerable dopaminergic neurons in healthy and pathological conditions. Neurobiol Dis, 172, 105815.<br /> Evans, R.C., Twedell, E.L., Zhu, M., Ascencio, J., Zhang, R., & Khaliq, Z.M. (2020) Functional Dissection of Basal Ganglia Inhibitory Inputs onto Substantia Nigra Dopaminergic Neurons. Cell Rep, 32, 108156.<br /> Henny, P., Brown, M.T.C., Northrop, A., Faunes, M., Ungless, M.A., Magill, P.J., & Bolam, J.P. (2012) Structural correlates of heterogeneous in vivo activity of midbrain dopaminergic neurons. Nat. Neurosci., 15, 613-619.<br /> McGregor, M.M., McKinsey, G.L., Girasole, A.E., Bair-Marshall, C.J., Rubenstein, J.L.R., & Nelson, A.B. (2019) Functionally Distinct Connectivity of Developmentally Targeted Striosome Neurons. Cell Rep, 29, 1419-1428.e5.<br /> Spix, T.A., Nanivadekar, S., Toong, N., Kaplow, I.M., Isett, B.R., Goksen, Y., Pfenning, A.R., & Gittis, A.H. (2021) Population-specific neuromodulation prolongs therapeutic benefits of deep brain stimulation. Science, 374, 201-206.

Reviewer #3 (Public Review):

Summary:

The receptor binding domain of SARS-Cov-2 spike protein contains two N-glycans which have been conserved by the variants observed in these last 4 years. Through the use of extensive molecular dynamics, the authors demonstrate that even if glycosylation is conserved, the stabilization role of glycans at N343 differs among the strains. They also investigate the effect of this glycosylation on the binding of RBD towards sialylated gangliosides, as a function of evolution.

Strengths:

The molecular dynamics characterization is well performed and demonstrates differences in the effect of glycosylation as a factor of evolution. The binding of different strains to human gangliosides shows variations of strong interest. Analyzing the structure function of glycans on SARS-Cov-2 surface as a function of evolution is important for the surveillance of novel variants since it can influence their virulence.

Weaknesses:

The article is difficult to read, with no sufficient efforts of clarification for non-glycobiology audiences. The presentation of previous knowledge about RBD glycosylation and its effect on structure is very difficult to follow and should be reorganized. The choice of the nature of the biantennary glycan at N343 is not rationalized. A major weakness is the absence of data supporting the proposed binding site for ganglioside.

Reviewer #3 (Public Review):

Summary:

This work aims to demonstrate how recent advances in thermal stability assays can be utilised to screen chemical libraries and determine the compound mechanism of action. Focusing on 96 compounds with known mechanisms of action, they use the PISA assay to measure changes in protein stability upon treatment with a high dose (10uM) in live K562 cells and whole cell lysates from K562 or HCT116. They intend this work to showcase a robust workflow that can serve as a roadmap for future studies.

Strengths:

The major strength of this study is the combination of live and whole cell lysates experiments. This allows the authors to compare the results from these two approaches to identify novel ligand-induced changes in thermal stability with greater confidence. More usefully, this also enables the authors to separate the primary and secondary effects of the compounds within the live cell assay.

The study also benefits from the number of compounds tested within the same framework, which allows the authors to make direct comparisons between compounds.

These two strengths are combined when they compare CHEK1 inhibitors and suggest that AZD-7762 likely induces secondary destabilisation of CRKL through off-target engagement with tyrosine kinases.

Weaknesses:

One of the stated benefits of PISA compared to the TPP in the original publication (Gaetani et al 2019) was that the reduced number of samples required allows more replicate experiments to be performed. Despite this, the authors of this study performed only duplicate experiments. They acknowledge this precludes the use of frequentist statistical tests to identify significant changes in protein stability. Instead, they apply an 'empirically derived framework' in which they apply two thresholds to the fold change vs DMSO: absolute z-score (calculated from all compounds for a protein) > 3.5 and absolute log2 fold-change > 0.2. They state that the fold-change threshold was necessary to exclude non-specific interactors. While the thresholds appear relatively stringent, this approach will likely reduce the robustness of their findings in comparison to an experimental design incorporating more replicates. Firstly, the magnitude of the effect size should not be taken as a proxy for the importance of the effect. They acknowledge this and demonstrate it using their data for PIK3CB and p38α inhibitors (Figures 2B-C). They have thus likely missed many small, but biologically relevant changes in thermal stability due to the fold-change threshold. Secondly, this approach relies upon the fold-changes between DMSO and compound for each protein being comparable, despite them being drawn from samples spread across 16 TMT multiplexes. Each multiplex necessitates a separate MS run and the quantification of a distinct set of peptides, from which the protein-level abundances are estimated. Thus, it is unlikely the fold changes for unaffected proteins are drawn from the same distribution, which is an unstated assumption of their thresholding approach. The authors could alleviate the second concern by demonstrating that there is very little or no batch effect across the TMT multiplexes. However, the first concern would remain. The limitations of their approach could have been avoided with more replicates and the use of an appropriate statistical test. It would be helpful if the authors could clarify if any of the missed targets passed the z-score threshold but fell below the fold-change threshold.

The authors use a single, high, concentration of 10uM for all compounds. Given that many of the compounds likely have low nM IC50s, this concentration will often be multiple orders of magnitude above the one at which they inhibit their target. This makes it difficult to assess the relevance of the off-target effects identified to clinical applications of the compounds or biological experiments. The authors acknowledge this and use ranges of concentrations for follow-up studies (e.g. Figure 2E-F). Nonetheless, this weakness is present for the vast bulk of the data presented.

The authors claim that combining cell-based and lysate-based assays increases coverage (Figure 3F) is not supported by their data. The '% targets' presented in Figure 3F have a different denominator for each bar. As it stands, all 49 targets quantified in both assays which have a significant change in thermal stability may be significant in the cell-based assay. If so, the apparent increase in % targets when combining reflects only the subsetting of the data. To alleviate this lack of clarity, the authors could update Figure 3F so that all three bars present the % targets figure for just the 60 compounds present in both assays.

Aims achieved, impact and utility:

The authors have achieved their main aim of presenting a workflow that serves to demonstrate the potential value of this approach. However, by using a single high dose of each compound and failing to adequately replicate their experiments and instead applying heuristic thresholds, they have limited the impact of their findings. Their results will be a useful resource for researchers wishing to explore potential off-target interactions and/or mechanisms of action for these 96 compounds, but are expected to be superseded by more robust datasets in the near future. The most valuable aspect of the study is the demonstration that combining live cell and whole cell lysate PISA assays across multiple related compounds can help to elucidate the mechanisms of action.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Long et al. demonstrated that the deletion of SUL1, which encodes a sulfate transporter localized on the plasma membrane, extends the replicative lifespan in S. cerevisiae. The authors further investigated the mechanism underlying this lifespan extension. They found that, unlike sul1∆ mutants, other mutants that have been shown to have a deficiency in sulfate transport cannot extend lifespan, from which they concluded that it is unlikely that SUL1 deletion extends lifespan by impairing sulfate intake. The authors then performed a series of characterizations on sul1∆ mutants and found that consistent with previous studies, PKA activity is downregulated when SUL1 is deleted. The authors demonstrated that SUL1 deletion promotes the nuclear localization of Msn2, as well as autophagy, which are known downstream signals of the PKA pathway. In addition, the authors show that MSN2 and ATG8 are indispensable for the lifespan extension in sul1∆ cells. Altogether, this manuscript suggests that SUL1 deletion extends lifespan by affecting PKA activity.

Strengths:

This study reported an interesting phenotype that the deletion of SUL1, but not SUL2, promotes lifespan extension in budding yeast. The authors performed some characterizations on sul1∆ mutants and epistatic studies to demonstrate that this lifespan extension requires MSN2 and ATG8, which further support the importance of the PKA pathway in regulating lifespan.

Weaknesses:

However, one of the major findings in this paper that SUL1 deletion extends lifespan independently of its role in sulfate uptake was merely based on lifespan measurements on sul2∆, SUL1E427Q, and met3∆ mutants, which cannot exclude the possibility that yeast lifespan is affected by sulfate intake. In addition, the strength of evidence for whether SUL1 deletion extends lifespan through affecting PKA activity is incomplete. It has been shown that Sul1 and Sul2 have redundant functions in both sulfate transport and PKA activation (Kankipati et al. 2015). However, in this manuscript, as shown by the authors, the deletion of SUL2 does not extend the lifespan compared with sul1∆ mutants. Without a further characterization on why deletion of SUL1, but not SUL2, extends lifespan, it is likely that SUL1 deletion extends lifespan independently of either sulfate transport or PKA activation.

Reviewer #3 (Public Review):

Summary

The authors of this work aim to address the challenge of accurately and efficiently identifying protein binding sites from sequences. They recognize that the limitations of current methods, including reliance on multiple sequence alignments or experimental protein structure, and the under-explored geometry of the structure, which limit the performance and genome-scale applications. The authors have developed a multi-task network, GPSite, that predicts binding residues for a range of biologically relevant molecules, including DNA, RNA, peptides, proteins, ATP, HEM, and metal ions, using sequence embeddings from protein language models and ESMFold-predicted structures. The reported results showed to be superior to current sequence-based and structure-based methods in terms of accuracy and efficiency.

Strengths<br /> (1) The GPSite model's ability to predict binding sites for a wide variety of molecules, including DNA, RNA, peptides, and various metal ions.<br /> (2) Based on the presented results, GPSite outperforms state-of-the-art methods in several benchmark datasets in terms of accuracy and efficiency.<br /> (3) GPSite adopts predicted structure instead of native structures as input, enabling the model to be applied to a wider range of scenarios where native structures are rare.<br /> (4) The low computational cost of GPSite is beneficial, which enables rapid genome-scale binding residue annotations, indicating the model's potential for large-scale downstream applications and discoveries.

Weaknesses

There are no major weaknesses after the revision.

Reviewer #3 (Public Review):

Summary:

How the microbial composition of the human body is influenced by and influences disease progression is an important topic. For people with COVID-19, symptomatic progression and deterioration can be difficult to predict. This manuscript attempts to associate the nasal and fecal microbiomes of COVID-19 patients with the severity of disease symptoms, with the goal of identifying microbial markers that can predict disease outcomes. However, the value of this work is held back by unclear methods and data presentation.

Strengths:

Analysis of microbiomes from two distinct anatomical locations and across three distinct patient groups is a substantial undertaking. How these microbiomes influence and are influenced by COVID-19 disease progression is an important question. In particular, the putative biomarker identified here could be of clinical value with additional research.

Weaknesses:

The methods and statistics used for several figures and comparisons are unclear or used in non-standard ways. For instance: the description of the Bray-Curtis test for Figure 1 is inaccurate and conflicts between the text and figure legend; the method used to compare the relative abundance of genera in Figure 2 is not clear; and it is not stated how the "total amount" of detected bacteria is inferred from the data presented in Figures 2C and 2D.

The description of results for Figure 1 is overstated or unclear for both the alpha diversity among disease groups and the overlap for nasal samples.

The most abundant phyla from nasal samples cumulatively account for less than 1% of abundance and it is unclear why this would be expected or how it compares to other work. Relatedly, the potential biological relevance of the very small proportional changes among phyla in the nasal samples is also not clear.

There is no real discussion of how the identified biomarkers might work in practice. While some microbes are detected in one condition but not others, it is unclear whether these organisms are expected to already exist below the detection threshold and then increase in abundance along with disease severity, or if they are picked up from the environment. For instance, would the presence of these 'severe' - associated microbes in patients with mild or moderate disease justify additional treatment to prevent disease progression?

The authors use the term "nasopharyngeal-faecal axis", but there is no substantial discussion of how these two microbiomes interact to influence disease progression, or how they are jointly affected to yield useful biomarkers. With one exception, correlation values between nasal and fecal microbes range from negligible to modest. It is unclear, then, how much parallel influence disease has on these microbiomes.

Silent weapons for quiet wars<br /> Operations Research Technical Manual<br /> TW-SW7905.1

Welcome Aboard

This publication marks the 25th anniversary of the Third World War, called the "Quiet War", being conducted using subjective biological warfare, fought with "silent weapons".<br /> This book contains an introductory description of this war, its strategies, and its weaponry.<br /> May 1979 #74-1120

Security

It is patently impossible to discuss social engineering or the automation of a society, i.e., the engineering of social automation systems (silent weapons) on a national or worldwide scale without implying extensive objectives of social control and destruction of human life, i.e., slavery and genocide.<br /> This manual is in itself an analog declaration of intent. Such a writing must be secured from public scrutiny. Otherwise, it might be recognized as a technically formal declaration of domestic war. Furthermore, whenever any person or group of persons in a position of great power and without full knowledge and consent of the public, uses such knowledge and methodologies for economic conquest - it must be understood that a state of domestic warfare exists between said person or group of persons and the public.<br /> The solution of today's problems requires an approach which is ruthlessly candid, with no agonizing over religious, moral or cultural values.<br /> You have qualified for this project because of your ability to look at human society with cold objectivity, and yet analyze and discuss your observations and conclusions with others of similar intellectual capacity without the loss of discretion or humility. Such virtues are exercised in your own best interest. Do not deviate from them.

https://ia802300.us.archive.org/10/items/silent-weapons-for-quiet-wars_202110/Silent%20Weapons%20for%20Quiet%20Wars.pdf

Reviewer #3 (Public Review):

This study explores sensory prediction errors in the sensory cortex. It focuses on the question of how these signals are shaped by non-hierarchical interactions, specifically multimodal signals arising from same-level cortical areas. The authors used 2-photon imaging of mouse auditory cortex in head-fixed mice that were presented with sounds and/or visual stimuli while moving on a ball. First, responses to pure tones, visual stimuli, and movement onset were characterized. Then, the authors made the running speed of the mouse predictive of sound intensity and/or visual flow. Mismatches were created through the interruption of sound and/or visual flow for 1 second while the animal moved, disrupting the expected sensory signal given the speed of movement. As a control, the same sensory stimuli triggered by the animal's movement were presented to the animal decoupled from its movement. The authors suggest that auditory responses to the unpredicted silence reflect mismatch responses. That these mismatch responses were enhanced when the visual flow was congruently interrupted, indicates the cross-modal influence of prediction error signals.

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

Reviewer #3 (Public Review):

Summary:

In this article, Hermannova et al catalog the changes in ribosome association with mRNAs when the eukaryotic translation initiation factor 3 is disrupted by knocking down subunits of the multisubunit protein. They find that RNAs relying on TOP motifs for translation, such as ribosomal protein RNAs, and RNAs encoding proteins that modify other proteins in the ER or components of the lysosome are upregulated. In contrast, proteins encoding components of MAP kinase cascades are downregulated when subunits of eIF3 are knocked down.

Strengths:

The authors use ribosome profiling of well-characterized mutants lacking subunits of eIF3 and assess the changes in translation that take place. They supplement the ribosome association studies with western blotting to determine protein level changes of affected transcripts. They analyze what is being encoded by the transcripts undergoing translation changes, which is important for understanding more broadly how translation initiation factor levels affect cancer cell translatomes.

Weaknesses:

(1) The data are presented as a catalog of effects, and the paper would be strengthened if there were a clear model tying the various effects together or linking individual subunit knockdown to cancerous phenotypes. It is unclear what the hypothesis is for cells having more MAPK activity with less of the MAPK proteins being translated, so the main findings of the paper become observational without context.

(2) The conclusions drawn are presented as very generalized other than in the last paragraph, but the experiments were only done in Hela cells. Since conclusions are being made about how translation changes affect MAP kinase signaling and there is mention in the abstract that dysregulation of these subunits is observed in cancer, at least one other cell line would need to be analyzed to provide evidence that the effects of subunit knockdown aren't cell-line specific.

(3) It is also unclear how replicates were performed and how many replicates were performed for several experiments. Biological replicates are mentioned, but what the authors did for biological replicates isn't defined and the description of the collection of cells for polysome/ribosome footprint/RNA seq samples makes it unclear whether the "biological replicates" are samples from separate transfections (true biological replicates) or different aliquots or wells from a single transfection (technical replicates) being run over a separate gradient. If using technical replicates, the data comparing the effects of knocking down D vs E vs H subunits are substantially weakened because subunit-specific differences could be the result of non-specific events that occurred in a transfection. It's also notable that while the pooled siRNAs will increase the potency of knockdown, it is possible that one or more of the siRNAs could have off-target effects, and analyzing individual siRNAs would be better for ensuring effects are specific.

(4) Many of the changes in protein levels reported by Western are subtle. Data from all western blots making claims of quantitative differences should really be quantified relative to nontreated over-loading control or total protein quantified from the gel, and presented with a degree of error from biological replicates to make conclusions about differences in protein levels between samples.

Reviewer #3 (Public Review):

This study investigates subtelomeric repetitive sequences in the budding yeast Saccharomyces cerevisiae, known as Y' and X-elements. Taking advantage of yeast strain SY12 that contains only 3 chromosomes and six telomeres (normal yeast strains contain 32 telomeres) the authors are able to generate a strain completely devoid of Y'- and X-elements.

Strengths:

They demonstrate that the SY12 delta XY strain displays normal growth, with stable telomeres of normal length that were transcriptionally silenced, a key finding with wide implications for telomere biology. Inactivation of telomerase in the SY12 and SY12 delta XY strains frequently resulted in survivors that had circularized all three chromosomes, hence bypassing the need for telomeres altogether. They show that survivors with fused chromosomes and so-called atypical survivors arise independently of the central recombination protein Rad52. The SY12 and SY12 delta XY yeast strains can become a useful tool for future studies of telomere biology. The conclusions of this manuscript are well supported by the data and are valuable for researchers studying telomeres.

Weaknesses:

A weakness of the manuscript is the analysis of telomere transcriptional silencing. They state: "The results demonstrated a significant increase in the expression of the MPH3 and HSP32 upon Sir2 deletion, indicating that telomere silencing remains effective in the absence of X and Y'-elements". However, for the SY12 strain, their analyses indicate that the difference between the WT and sir2 strains is nonsignificant. In addition, a striking observation is that the SY12 strain (with only three chromosomes) express much less of both MPH3 and HSP32 than the parental strain BY4742 (16 chromosomes), both in the presence and absence of Sir2.

Reviewer #3 (Public Review):

Summary:

This valuable study by Semenova and colleagues describes a large cross-sectional cohort of 115 individuals on ART. Participants contributed a single blood sample which underwent IPDA, and 25-color flow with various markers (pre and post-stimulation). The authors then used clustering, decision tree analyses, and machine learning to look for correlations between these immunophenotypic markers and several measures of HIV reservoir volume. They identified two distinct clusters that can be somewhat differentiated based on total HIV DNA level, intact HIV DNA level, and multiple T cell cellular markers of activation and exhaustion.

The conclusions of the paper are supported by the data but the relationships between independent and dependent variables in the models are correlative with no mechanistic work to determine causality. It is unclear in most cases whether confounding variables could explain these correlations. If there is causality, then the data is not sufficient to infer directionality (ie does the immune environment impact the HIV reservoir or vice versa or both?). In addition, even with sophisticated and appropriate machine learning approaches, the models are not terribly predictive or highly correlated. For these reasons, the study is very much hypothesis-generating and will not impact cure strategies or HIV reservoir measurement strategies in the short term.

Strengths:

The study cohort is large and diverse in terms of key input variables such as age, gender, and duration of ART. Selection of immune assays is appropriate. The authors used a wide array of bioinformatic approaches to examine correlations in the data. The paper was generally well-written and appropriately referenced.

Weaknesses:

(1) The major limitation of this work is that it is highly exploratory and not hypothesis-driven. While some interesting correlations are identified, these are clearly hypothesis-generating based on the observational study design.

(2) The study's cross-sectional nature limits the ability to make mechanistic inferences about reservoir persistence. For instance, it would be very interesting to know whether the reservoir cluster is a feature of an individual throughout ART, or whether this outcome is dynamic over time.

(3) A fundamental issue is that I am concerned that binarizing the 3 reservoir metrics in a 50/50 fashion is for statistical convenience. First, by converting a continuous outcome into a simple binary outcome, the authors lose significant amounts of quantitative information. Second, the low and high reservoir outcomes are not actually demonstrated to be clinically meaningful: I presume that both contain many (?all) data points above levels where rebound would be expected soon after interruption of ART. Reservoir levels would also have no apparent outcome on the selection of cure approaches. Overall, dividing at the median seems biologically arbitrary to me.

(4) The two reservoir clusters are of potential interest as high total and intact with low % intact are discriminated somewhat by immune activation and exhaustion. This was the most interesting finding to me, but it is difficult to know whether this clustering is due to age, time on ART, other co-morbidity, ART adherence, or other possible unmeasured confounding variables.

(5) At the individual level, there is substantial overlap between clusters according to total, intact, and % intact between the clusters. Therefore, the claim in the discussion that these 2 cluster phenotypes may require different therapeutic approaches seems rather speculative. That said, the discussion is very thoughtful about how these 2 clusters may develop with consideration of the initial insult of untreated infection and / or differences in immune recovery.

(6) The authors state that the machine learning algorithms allow for reasonable prediction of reservoir volume. It is subjective, but to me, 70% accuracy is very low. This is not a disappointing finding per se. The authors did their best with the available data. It is informative that the machine learning algorithms cannot reliably discriminate reservoir volume despite substantial amounts of input data. This implies that either key explanatory variables were not included in the models (such as viral genotype, host immune phenotype, and comorbidities) or that the outcome for testing the models is not meaningful (which may be possible with an arbitrary 50/50 split in the data relative to median HIV DNA volumes: see above).

(7) The decision tree is innovative and a useful addition, but does not provide enough discriminatory information to imply causality, mechanism, or directionality in terms of whether the immune phenotype is impacting the reservoir or vice versa or both. Tree accuracy of 80% is marginal for a decision tool.

(8) Figure 2: this is not a weakness of the analysis but I have a question about interpretation. If total HIV DNA is more predictive of immune phenotype than intact HIV DNA, does this potentially implicate a prior high burden of viral replication (high viral load &/or more prolonged time off ART) rather than ongoing reservoir stimulation as a contributor to immune phenotype? A similar thought could be applied to the fact that clustering could only be detected when applied to total HIV DNA-associated features. Many investigators do not consider defective HIV DNA to be "part of the reservoir" so it is interesting to speculate why these defective viruses appear to have more correlation with immunophenotype than intact viruses.

(9) Overall, the authors need to do an even more careful job of emphasizing that these are all just correlations. For instance, HIV DNA cannot be proven to have a causal effect on the immunophenotype of the host with this study design. Similarly, immunophenotype may be affecting HIV DNA or the correlations between the two variables could be entirely due to a separate confounding variable.

(10) In general, in the intro, when the authors refer to the immune system, they do not consistently differentiate whether they are referring to the anti-HIV immune response, the reservoir itself, or both. More specifically, the sentence in the introduction listing various causes of immune activation should have citations. (To my knowledge, there is no study to date that definitively links proviral expression from reservoir cells in vivo to immune activation as it is next to impossible to remove the confounding possible imprint of previous HIV replication.) Similarly, it is worth mentioning that the depletion of intact proviruses is quite slow such that provial expression can only be stimulating the immune system at a low level. Similarly, the statement "Viral protein expression during therapy likely maintains antigen-specific cells of the adaptive immune system" seems hard to dissociate from the persistence of immune cells that were reactive to viremia.

(11) Given the many limitations of the study design and the inability of the models to discriminate reservoir volume and phenotype, the limitations section of the discussion seems rather brief.

Reviewer #3 (Public Review):

The manuscript "Theory of active self-organization of dense nematic structures in the actin cytoskeleton" analysis self-organized pattern formation within a two-dimensional nematic liquid crystal theory and uses microscopic simulations to test the plausibility of some of the conclusions drawn from that analysis. After performing an analytic linear stability analysis that indicates the possibility of patterning instabilities, the authors perform fully non-linear numerical simulations and identify the emergence of stripe-like patterning when anisotropic active stresses are present. Following a range of qualitative numerical observations on how parameter changes affect these patterns, the authors identify, besides isotropic and nematic stress, also active self-alignment as an important ingredient to form the observed patterns. Finally, microscopic simulations are used to test the plausibility of some of the conclusions drawn from continuum simulations.

The paper is well written, figures are mostly clear and the theoretical analysis presented in both, main text and supplement, is rigorous. Mechano-chemical coupling has emerged in recent years as a crucial element of cell cortex and tissue organization and it is plausible to think that both, isotropic and anisotropic active stresses, are present within such effectively compressible structures. Even though not yet stated this way by the authors, I would argue that combining these two is of the key ingredients that distinguishes this theoretical paper from similar ones. The diversity of patterning processes experimentally observed is nicely elaborated on in the introduction of the paper, though other closely related previous work could also have been included in these references (see below for examples).

To introduce the continuum model, the authors exclusively cite their own, unpublished pre-print, even though the final equations take the same form as previously derived and used by other groups working in the field of active hydrodynamics (a certainly incomplete list: Marenduzzo et al (PRL, 2007), Salbreux et al (PRL, 2009, cited elsewhere in the paper), Jülicher et al (Rep Prog Phys, 2018), Giomi (PRX, 2015),...). To make better contact with the broad active liquid crystal community and to delineate the present work more compellingly from existing results, it would be helpful to include a more comprehensive discussion of the background of the existing theoretical understanding on active nematics. In fact, I found it often agrees nicely with the observations made in the present work, an opportunity to consolidate the results that is sometimes currently missed out on. For example, it is known that self-organised active isotropic fluids form in 2D hexagonal and pulsatory patterns (Kumar et al, PRL, 2014), as well as contractile patches (Mietke et al, PRL 2019), just as shown and discussed in Fig. 2. It is also known that extensile nematics, \kappa<0 here, draw in material laterally of the nematic axis and expel it along the nematic axis (the other way around for \kappa>0, see e.g. Doostmohammadi et al, Nat Comm, 2018 "Active Nematics" for a review that makes this point), consistent with all relative nematic director/flow orientations shown in Figs. 2 and 3 of the present work.

The results of numerical simulations are well-presented. Large parts of the discussion of numerical observations - specifically around Fig. 3 - are qualitative and it is not clear why the analysis is restricted to \kappa<0. Some of the observations resonate with recent discussions in the field, for example the observation of effectively extensile dynamics in a contractile system is interesting and reminiscent of ambiguities about extensile/contractile properties discussed in recent preprints (https://arxiv.org/abs/2309.04224). It is convincingly concluded that, besides nematic stress on top of isotropic one, active self-alignment is a key ingredient to produce the observed patterns.

I compliment the authors for trying to gain further mechanistic insights into this conclusion with microscopic filament simulations that are diligently performed. It is rightfully stated that these simulations only provide plausibility tests and, within this scope, I would say the authors are successful. At the same time, it leaves open questions that could have been discussed more carefully. For example, I wonder what can be said about the regime \kappa>0 (which is dropped ad-hoc from Fig. 3 onward) microscopically, in which the continuum theory does also predict the formation of stripe patterns - besides the short comment at the very end? How does the spatial inhomogeneous organization the continuum theory predicts fit in the presented, microscopic picture and vice versa?

Overall, the paper represents a valuable contribution to the field of active matter and, if strengthened further, might provide a fruitful basis to develop new hypothesis about the dynamic self-organisation of dense filamentous bundles in biological systems.

Reviewer #3 (Public Review):

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

This is an exciting and novel discovery. I am not an expert in sequencing technologies or sequencing data analysis, so I will limit my comments purely to biology and not technical points. The PI is a leader in applying innovative sequencing methods to studying mRNA decay.

One aspect that appeared overlooked is that poly(A) tail shortening per se does lead to decapping. It is shortening below a certain threshold of 8-10 As that triggers decapping. Therefore, I found the conclusion that poly(A) tail shortening is not required for stress-induced decay to be somewhat premature. For a robust test of this hypothesis, the authors should consider performing their analysis in conditions where CNOT7/8 is knocked down with siRNA.

Similarly, as XRN1 requires decapping to take place, it necessitates the experiment where a dominant-negative DCP2 mutant is over-expressed.

Are G3BP1/2 stress granules required for stress-induced decay or simply sites for storage? This part seems unclear. A very worthwhile test here would be to assess in XRN1-null background.

Finally, the authors speculate that the mechanism of stress-induced decay may have evolved to relieve translational load during stress. But why degrade the 5' end when removing the cap may be sufficient? This returns to the question of assessing the role of decapping in this mechanism.

Reviewer #3 (Public Review):

Summary:

In this study, the authors set out to understand the mechanisms behind chlorophyll biosynthesis in rice, focusing in particular on the role of OsNF-YB7, an ortholog of Arabidopsis LEC1, which is a positive regulator of chlorophyll (Chl) biosynthesis in Arabidopsis. They showed that OsNF-YB7 loss-of-function mutants in rice have chlorophyll-rich embryos, in contrast to Arabidopsis LEC1 loss-of-function mutants. This contrasting phenotype led the authors to carry out extensive molecular studies on OsNF-YB7, including in vitro and in vivo protein interaction studies, gene expression profiling, and protein-DNA interaction assays. The evidence provided well supported the core arguments of the authors, emphasising that OsNF-YB7 is a negative regulator of Chl biosynthesis in rice embryos by mediating the expression of OsGLK1, a transcription factor that regulates downstream Chl biosynthesis genes. In addition, they showed that OsNF-YB7 interacts with OsGLK1 to negatively regulate the expression of OsGLK1, demonstrating the broad involvement of OsNF-YB7 in rice Chl biosynthetic pathways.

Strengths:

This study clearly demonstrated how OsNF-YB7 regulates its downstream pathways using several in vitro and in vivo approaches. For example, gene expression analysis of OsNF-YB7 loss-of-function and gain-of-function mutants revealed the expression of selected downstream chl biosynthetic genes. This was further validated by EMSA on the gel. The authors also confirmed this using luciferase assays in rice protoplasts. These approaches were used again to show how the interaction of OsNF-YB7 and OsGLK1 regulates downstream genes. The main idea of this study is very well supported by the results and data.

Weaknesses:

From an evolutionary perspective, it is interesting to see how two similar genes have come to play opposite roles in Arabidopsis and rice. It would have been more interesting if the authors had carried out a cross-species analysis of AtLEC1 and OsNF-YB7. For example, overexpressing AtLEC1 in an osnf-yb7 mutant to see if the phenotype is restored or enhanced. Such an approach would help us understand how two similar proteins can play opposite roles in the same mechanism within their respective plant species.

Reviewer #3 (Public Review):

This study from the Flores group aims at understanding neuronal circuit changes during adolescence which is an ill-defined, transitional period involving dramatic changes in behavior and anatomy. They focus on DA innervation of the prefrontal cortex, and their interaction with the guidance cue Netrin-1. They propose DA axons in the PFC increase in the postnatal period, and their density is reduced in a Netrin 1 knockdown, suggesting that Netrin abets the development of this mesocortical pathway. In such mice impulsivity gauged by a go-no go task is reduced. They then provide some evidence that Unc5c is developmentally regulated in DA axons. Finally they use an interesting hamster model, to study the effect of light hours on mesocortical innervation, and make some interesting observations about the timing of innervation and Unc5c expression, and the fact that females housed in winter day length conditions display an accelerated innervation of the prefrontal cortex.

Comments on the revision. Several points were addressed; some remain to be addressed.

4. It's not clear to me that TH doesnt stain noradrenergic axons in the PFC. See Islam and Blaess, 2021, and references therein.

6. The Netrin knockdown data provided is from a previous study/samples.

8. While the authors make the argument that the behavior is linked to DA, they still haven't formally tested it, in my opinion.

13. Fig 3, UNc 5c levels are not yet quantified. Furthermore, I agree with the previous reviewer that Unc5C knockdown would corroborate key aspects of the model.

New - Developmental trajectory of prefrontal TH-positive axons from early adolescence to adulthood is similar in male and female rats, (Willing Juraska et al., 2017). This needs discussion.

Reviewer #3 (Public Review):

This study from the Flores group aims at understanding neuronal circuit changes during adolescence which is an ill-defined, transitional period involving dramatic changes in behavior and anatomy. They focus on DA innervation of the prefrontal cortex, and their interaction with the guidance cue Netrin-1. They propose DA axons in the PFC increase in the postnatal period, and their density is reduced in a Netrin 1 knockdown, suggesting that Netrin abets the development of this mesocortical pathway. In such mice impulsivity gauged by a go-no go task is reduced. They then provide some evidence that Unc5c is developmentally regulated in DA axons. Finally they use an interesting hamster model, to study the effect of light hours on mesocortical innervation, and make some interesting observations about the timing of innervation and Unc5c expression, and the fact that females housed in winter day length conditions display an accelerated innervation of the prefrontal cortex.

Comments on the revision. Several points were addressed; some remain to be addressed.

4. It's not clear to me that TH doesnt stain noradrenergic axons in the PFC. See Islam and Blaess, 2021, and references therein.

6. The Netrin knockdown data provided is from a previous study/samples.

8. While the authors make the argument that the behavior is linked to DA, they still haven't formally tested it, in my opinion.

13. Fig 3, UNc 5c levels are not yet quantified. Furthermore, I agree with the previous reviewer that Unc5C knockdown would corroborate key aspects of the model.

New - Developmental trajectory of prefrontal TH-positive axons from early adolescence to adulthood is similar in male and female rats, (Willing Juraska et al., 2017). This needs discussion.

Reviewer #3 (Public Review):

Summary:

This interesting manuscript presents a comparison of biophysical properties, TEM appearances, and phosphorylation patterns of brain-derived synuclein fibrils from 3 subjects each with Parkinson Disease (PD), Parkinson Disease with Dementia (PDD), Dementia with Lewy bodies (DLB) and Multiple System Atrophy (MSA), the effects of studying these brain-derived fibrils in a Seeding Aggregation Assay (SAA), and a comparison of the seeded and resultant fibers. The results are not unexpected.

Strengths:

The work explores an important question. Namely, what is the fidelity of synuclein fibrils produced during an SAA reaction to the starting material if that material has been extracted from the brains of deceased patients with synucleinopathies.

Weaknesses:

The work suffers from several methodological flaws

The experiments are missing two important controls. 1) what to fibrils generated by different in vitro fibril preparations made from recombinant synclein protein look like; and 2) the use of CSF from the same patients whose brain tissue was used to assess whether CSF and brain seeds look and behave identically. The latter is perhaps the most important question of all - namely how representative are CSF seeds of what is going on in patients' brains?

In their discussion the authors do not comment on the obvious differences in the conditions leading to the formation of seeds in the brain and in the artificial conditions of the seeding assay. Why should the two sets of conditions be expected to yield similar morphologies, especially since the extracted fibrils are subjected to harsh conditions for solubilization and re-suspension.

Finally, the key experiment was not performed - would the resultant seeds from SAA preparations from the different nosological entities produce different pathologies when injected into animal brains? But perhaps this is the subject of a future manuscript.

Furthermore, the authors comment on phosphorylation patterns, stating that the resultant seeds are less heavy phosphorylated than the original material. Again, this should not be surprising, since the SAA assay conditions are not known to contain the enzymes necessary to phosphorylate synuclein. The discussion of PTMs is limited to pS-129 phosphorylation. What about other PTMs? How does the pattern of PTMs affect the seeding pattern.

Lastly, the manuscript contains no data on how the diagnostic categories were assigned at autopsy. This information should be included in the supplementary material.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Wang et al. performed a study looking at vascular changes in response to anesthesia in awake mice using ultrasound localization microscopy (ULM). The authors report a reduction of vascularity and blood flow velocity in the awake state. In addition, they demonstrate the reproducibility of ULM measurements in time.

Strengths:

Demonstration that high-quality, state-of-the-art ULM images can be performed using cranial windows in awake animals.<br /> Demonstration that repeated imaging in time produces comparable images.

Weaknesses:

It is unclear whether multiple animals were used in the statistical analysis.<br /> Generalizations are sometimes drawn from what seems to be the analysis of a single vessel.<br /> The description of the statistical analysis is mostly qualitative.<br /> Some terms used are insufficiently defined.<br /> Additional limitations should be included in the discussion.<br /> Some technical details are lacking.

Without information about whether the results obtained come from multiple animals, it is difficult to conclude that the authors generally achieved their aim. They do achieve it in a single animal.

The results that are shown are interesting and could have an impact on the ULM community and beyond. In particular, the experimental setup they used along with the high reproducibility they report could become very important for the use of ULM in larger animal cohorts.

Reviewer #3 (Public Review):

Summary:

The protein kinase, Aurora B, is a critical regulator of mitosis and cytokinesis in eukaryotes, exhibiting a dynamic localisation. As part of the Chromosomal Passenger Complex (CPC), along with the Aurora B activator, INCENP, and the CPC localisation module comprised of Borealin and Survivin, Aurora B travels from the kinetochores at metaphase to the spindle midzone at anaphase, which ensures its substrates are phosphorylated in a time- and space-dependent manner. In the kinetoplastid parasite, T. brucei, the Aurora B orthologue (AUK1), along with an INCENP orthologue known as CPC1, and a kinetoplastid-specific protein CPC2, also displays a dynamic localisation, moving from the kinetochores at metaphase, to the spindle midzone at anaphase, to the anterior end of the newly synthesised flagellum attachment zone (FAZ) at cytokinesis. However, the trypanosome CPC lacks orthologues of Borealin and Survivin, and T. brucei kinetochores also have a unique composition, being comprised of dozens of kinetoplastid-specific proteins (KKTs). Of particular importance for this study are KKT7 and the KKT8 complex (comprising KKT8, KKT9, KKT11, and KKT12). Here, Ballmer and Akiyoshi seek to understand how the CPC assembles and is targeted to its different locations during the cell cycle in T. brucei.

Strengths & Weaknesses:

Using immunoprecipitation and mass-spectrometry approaches, Ballmer and Akiyoshi show that AUK1, CPC1, and CPC2 associate with two orphan kinesins, KIN-A and KIN-B, and with the use of endogenously expressed fluorescent fusion proteins, demonstrate for the first time that KIN-A and KIN-B display a dynamic localisation pattern similar to other components of the CPC, providing compelling evidence for KIN-A and KIN-B being bona fide CPC proteins.

They then demonstrate, by using RNAi to deplete individual components, that the CPC proteins have hierarchical interdependencies for their localisation to the kinetochores at metaphase. These experiments appear to have been well performed.

Ballmer and Akiyoshi then go on to determine the kinetochore localisation domains of KIN-A and KIN-B. Using ectopically expressed GFP-tagged truncations, they show that coiled coil domains within KIN-A and KIN-B, as well as a disordered C-terminal tail present only in KIN-A, but not the N-terminal motor domains of KIN-A or KIN-B, are required for kinetochore localisation. These data are strengthened by immunoprecipitating CPC complexes and crosslinking them prior to mass spectrometry analysis (IP-CLMS), a state-of-the-art approach, to determine the contacts between the CPC components. Structural predictions of the CPC structure are also made using AlphaFold2, suggesting that coiled coils form between KIN-A and KIN-B, and that KIN-A/B interact with the N termini of CPC1 and CPC2. Experimental results showing that CPC1 and CPC2 are unable to localise to kinetochores if they lack their N-terminal domains are consistent with these predictions. Altogether these data provide compelling evidence of the protein domains required for CPC kinetochore localisation and CPC protein interactions and indicate that both KIN-A and KIN-B have a role to play.

Next, using a mixture of RNAi depletion and LacI-LacO recruitment experiments, the authors show that kinetochore proteins KKT7 and KKT9 are required for AUK1 to localise to kinetochores (other KKT8 complex components were not tested here) and that all components of the KKT8 complex are required for KIN-A kinetochore localisation. Further, both KKT7 and KKT8 were able to recruit AUK1 to an ectopic locus in S phase, and KKT7 recruited KKT8 complex proteins, indicating it is upstream of KKT8, in line with previous work showing kinetochore localization of KKT7 is unaffected by disruption of the KKT8 complex. This leads to the conclusion that the KKT8 complex is likely the main kinetochore receptor of the CPC.

Further IP-CLMS experiments, in combination with recombinant protein pull down assays and structural predictions, suggested that within the KKT8 complex, there are two subcomplexes of KKT8:KKT12 and KKT9:KKT11, and that KKT7 interacts with KKT9:KKT11 to recruit the remainder of the KKT8 complex. The authors also assess the interdependencies between KKT8 complex components for localisation and expression, showing that all four subunits are required for the assembly of a stable KKT8 complex and present AlphaFold2 structural modelling data to support the two subcomplex model. In general, these data are of high quality and convincing, although it is a shame that data showing the effects of KKT8, KKT9 and KKT12 depletion on KKT11 localisation and abundance could not be presented alongside the reciprocal experiments in Fig S4I-L.

The authors also convincingly show that AlphaFold2 predictions of interactions between KKT9:KKT11 and a conserved domain (CD1) in the C-terminal tail of KIN-A are correct, with CD1 and a second conserved domain, CD2, identified through sequence analysis, acting synergistically to promote KIN-A kinetochore localisation at metaphase, but not being required for KIN-A to move to the central spindle at anaphase. They then hypothesise that the kinesin motor domain of KIN-A (but not KIN-B which is predicted to be inactive based on non-conservation of residues key for activity) determines its central spindle localisation at anaphase through binding to microtubules. In support of this hypothesis, the authors show that KIN-A, but not KIN-B can bind microtubules in vitro and in vivo. However, ectopically expressed GFP-NLS fusions of full length KIN-A or KIN-A motor domain did not localise to the central spindle at anaphase. The authors suggest this is due to the GFP fusion disrupting the ATPase activity of the motor domain, although they provide no evidence that this is the case. Instead, they replace endogenous KIN-A with a predicted ATPase-defective mutant (G210A), showing that while this still localises to kinetochores, the kinetochores were frequently misaligned at metaphase, and that it no longer concentrates at the central spindle (with concomitant mis-localisation of AUK1), causing cells to accumulate at anaphase. From these data, the authors conclude that KIN-A ATPase activity is required for chromosome congression to the metaphase plate and its central spindle localisation at anaphase. While these data are highly suggestive that KIN-A possesses ATPase activity, and that this activity is essential for its function, definitive biochemical evidence of KIN-A's ATPase activity is still lacking.

Impact:

Overall, this work uses a wide range of cutting edge molecular and structural predictive tools to provide a significant amount of new and detailed molecular data that shed light on the composition of the unusual trypanosome CPC and how it is assembled and targeted to different cellular locations during cell division. Given the fundamental nature of this research, it will be of interest to many parasitology researchers as well as cell biologists more generally, especially those working on aspects of mitosis and cell division, and those interested in the evolution of the CPC.

Reviewer #3 (Public Review):

Sang et al. successfully demonstrate that a set of single sensory neurons in the pharynx of Drosophila promotes avoidance of food with high salt concentrations, complementing previous findings on Ir7c neurons with an additional internal sensing mechanism. The experiments are well-conducted and presented, convincingly supporting their important findings and extending the understanding of internal sensing mechanisms.

The authors convincingly demonstrate the avoidance phenotype using different behavioral assays, thus comprehensively analyzing different aspects of the behavior. The experiments are straightforward and well-contextualized within existing literature.

Reviewer #3 (Public Review):

Summary:

The authors found two endosomal fusion modes by live cell imaging of endosomes in yolk sac lateral endoderm cells of 8.5-day-old embryonic mice and described the fusion modes by mathematical models and simulations. They also showed that actin polymerization is involved in the regulation of one of the fusion modes.

Strengths:

The strength of this study is that the authors' claims are well supported by beautiful live cell images and theoretical models. By using specialized cells, yolk sac visceral endoderm cells, the live images of endosomal fusion, localization of actin-related molecules, and validation data from multiple inhibitor experiments are clear.

Weaknesses:

This study does not include any assessment of whether the two types of endosome fusions claimed by the authors occur in general cells, so the article is limited to showing a phenomenon specific to yolk sac lateral endoderm cells. Also, the study does not show the physiological importance of the two types of fusion. There are some unclear points in the method of image analysis and some of the descriptions in the text are not logical.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #3 (Public Review):

Summary:

This manuscript details the characterization of ClpL from L. monocytogenes as a potent and autonomous AAA+ disaggregase. The authors demonstrate that ClpL has potent and DnaK-independent disaggregase activity towards a variety of aggregated model substrates, and that this disaggregase activity appears to be greater than that observed with the canonical DnaK/ClpB co-chaperone. Furthermore, LmClpL appears to have greater thermostability as compared to LmDnaK, suggesting that ClpL-expressing cells may be able to withstand more severe heat stress conditions. Interestingly, LmClpL can provide thermotolerance to E. coli that have been genetically depleted of either ClpB or in cells expressing a mutant DnaK103. The authors further characterized the mechanisms by which ClpL interacts with protein aggregates, identifying that the N-terminal domain of ClpL is essential for disaggregase function. Lastly, by EM and mutagenesis analysis the authors report that ClpL can exist in a variety of larger macromolecular complexes, including dimer or trimers of hexamers/heptamers, and they provide evidence that the N-terminal domains of ClpL prevent dimer ring formation, thus promoting an active and substrate-binding ClpL complex. Throughout this manuscript the authors compare LmClpL to ClpG, another potent and autonomous disaggregase found in gram-negative bacteria that has been reported on previously, demonstrating that these two enzymes share homologous activity and qualities. Taken together this report clearly establishes ClpL as a novel and autonomous disaggregase.

Analysis:

The work presented in this report amounts to a significant body of novel and significant work that will be of interest to protein chaperone community. Furthermore, by providing examples of how ClpL can provide in vivo thermotolerance to both E. coli and L. gasseri the authors have expanded the significance of this work and provides novel insight into potential mechanisms responsible for thermotolerance in food-borne pathogens. The figures are clearly depicted, well-labeled, and easy to understand, and the manuscript is well-written. Experimentally the work was performed to a high standard with excellent controls, aiding in the ability for the audience to understand the major findings and conclusions. Additionally, the authors have effectively and efficiently expanded on their work through the peer review process, further increasing the understandability and significance of their work. Overall, the data presented, and analysis thereof, support the authors' conclusions, and thus this study represents an important addition to our understanding of molecular chaperone biochemistry. Lastly, this study establishes new avenues for research into autonomous disaggregates, their role in in vivo thermotolerance, and the mechanisms by which AAA+ chaperones recognize and interact with substrate proteins.

Reviewer #3 (Public Review):

Summary:

In this article, Gao et. al. uses single-molecule FRET (smFRET) and position-specific labelling of RNA (PLOR) to dissect the folding and behavioral ligand sensing of the Guanidine-IV riboswitch in the presence and absence of the ligand guanidine and the cation Mg2+. The results provided valuable information on the mechanistic aspects of the riboswitch, including the confirmation of the kissing loop present in the structure as essential for folding and riboswitch activity. Co-transcriptional investigations of the system provided key information on the ligand-sensing behavior and ligand-binding window of the riboswitch. A plausible folding model of the Guanidine-IV riboswitch was proposed as a final result. The evidence presented here sheds additional light on the mode of action of transcriptional riboswitches.

Strengths:

The investigations were very thorough, providing data that supports the conclusions. The use of smFRET and PLOR to investigate RNA folding has been shown to be a valuable tool for the understanding of folding and behavior properties of these structured RNA molecules. The co-transcriptional analysis brought important information on how the riboswitch works, including the ligand-sensing and the binding window that promotes the structural switch. The fact that investigations were done with the aptamer domain, aptamer domain + terminator/anti-terminator region, and the full-length riboswitch were essential to inform how each domain contributes to the final structural state if in the presence of the ligand and Mg2+.

Weaknesses:

The system has its own flaws when compared to physiological conditions. The RNA polymerase used (the study uses T7 RNA polymerase) is different from the bacterial RNA polymerase, not only in complexity, but also in transcriptional speed, which can directly interfere with folding and ligand-sensing. Additionally, rNTPs concentrations were much lower than physiological concentrations during transcription, likely causing a change in the polymerase transcriptional speed. These important aspects and how they could interfere with results are important to be addressed to the broad audience. Another point of consideration to be aware of is that the bulky fluorophores attached to the nucleotides can interfere with folding to some extent.

Reviewer #3 (Public Review):

Summary:

Zeng and Stanley show that in yeast, intron-lariat intermediates that accumulated due to defects in pre-mRNA splicing, are transported to the cytoplasm using the canonical mRNA export pathway. Moreover, they demonstrate that export requires the nuclear basket, a sub-structure of the nuclear pore complex previously implicated with the retention of immature mRNAs. These observations are important as they put into question a longstanding model that the main role of the nuclear basket is to ensure nuclear retention of immature or faulty mRNAs.

Strengths:

The authors elegantly combine genetic, biochemical, and single-molecule resolution microscopy approaches to identify the cellular pathway that mediates the cytoplasmic accumulation of lariat intermediates. Cytoplasmic accumulation of such splicing intermediates had been observed in various previous studies but how these RNAs reach the cytoplasm had not yet been investigated. By using smFISH, the authors present compelling, and, for the first time, direct evidence that these intermediates accumulate in the cytoplasm and that this requires the canonical mRNA export pathway, including the RNA export receptor Mex67 as well as various RNA-binding proteins including Yra1, Npl3 and Nab2. Moreover, they show that the export of lariat intermediates, but not mRNAs, requires the nuclear basket (Mlp1) and basket-associated proteins previously linked to the mRNP rearrangements at the nuclear pore. This is a surprising and important observation with respect to a possible function of the nuclear basket in mRNA export and quality control, as it challenges a longstanding model that the role of the basket in mRNA export is primarily to act as a gatekeeper to ensure that immature mRNAs are not exported. As discussed by the authors, their finding suggests a role for the basket in promoting the export of certain types of RNAs rather than retention, a model also supported by more recent studies in mammalian cells. Moreover, their findings also collaborate with a recent paper showing that in yeast, not all nuclear pores contain a basket (PMID: 36220102), an observation that also questioned the gatekeeper model of the basket, as it is difficult to imagine how the basket can serve as a gatekeeper if not all nuclear pore contain such a structure.

Weaknesses:

One weakness of this study is that all their experiments rely on using synthetic splicing reporter containing a lacZ gene that produces a relatively long transcript compared to the average yeast mRNA.

The rationale for using a reporter containing the brG (G branch point) resulting in more stable lariat intermediates due to them being inefficient substrates for the debranching enzyme Dbr1 could be described earlier in the manuscript, as this otherwise only becomes clear towards the end, what is confusing.

Discussion of their observation in the context that, in yeast, not all pores contain a basket would be useful.

Reviewer #3 (Public Review):

Summary:

The authors provide the first image analysis by cryoET of toroids assembled by FtsZ crosslinked by ZapD. Previously toroids of FtsZ alone have been imaged only in projection by negative stain EM. The authors attempt to distinguish ZapD crosslinks from the underlying FtsZ filaments. I did not find this distinction convincing, especially because it seems inconsistent with the 1:1 stoichiometry demonstrated by pelleting. I was intrigued by one image showing straight filament pairs, which may suggest a new model for how ZapD crosslinks FtsZ filaments.

Strengths:

(1) The first image analysis of FtsZ toroids by cryoET.<br /> (2) The images are accompanied by pelleting assays that convincingly establish a 1:1 stoichiometry of FtsZ:ZapD subunits.<br /> (3) Fig. 5 shows an image of a pair of FtsZ filaments crosslinked by ZapD. This seems to have higher resolution than the toroids. Importantly, it suggests a new model for the structure of FtsZ-ZapD that resolves previously unrecognized conflicts. (This is discussed below under weaknesses, because it is so far only supported by a single image.)

Weaknesses:

This paper reports a study by cryoEM of polymers and bundles assembled from FtsZ plus ZapD. Although previous studies by other labs have focused on straight bundles of filaments, the present study found toroids mixed with these straight bundles, and they focused most of their study on the toroids. In the toroids they attempt to delineate FtsZ filaments and ZapD crosslinks. A major problem here is with the stoichiometry. Their pelleting assays convincingly established a stoichiometry of 1:1, while the mass densities identified as ZapD are sparse and apparently well below the number of FtsZ (FtsZ subunits are not resolved in the reconstructions, but the continuous sheets or belts seem to have a lot more mass than the identified crosslinks.) Apart from the stoichiometry I don't find the identification of crosslinks to be convincing. It is missing an important control - cryoET of toroids assembled from pure FtsZ, without ZapD.

However, if I ignore these and jump to Fig. 5, I think there is an important discovery that resolves controversies in the present study as well as previous ones, controversies that were not even recognized. The controversy is illustrated by the Schumacher 2017 model (their Fig. 7), which is repeated in a simplified version in Fig. 1a of the present mss. That model has a two FtsZ filaments in a plane facing ZapD dimers which bridge them. In this planar model the C-terminal linker, and the ctd of FtsZ that binds ZapD facing each other and the ZapD in the middle, with. The contradiction arises because the C-terminus needs to face the membrane in order to attach and generate a bending force. The two FtsZ filaments in the planar model are facing 90{degree sign} away from the membrane. A related contradiction is that Houseman et al 2016 showed that curved FtsZ filaments have the C terminus on the outside of the curve. In a toroid the C termini should all be facing the outside. If the paired filaments had the C termini facing each other, they could not form a toroid because the two FtsZ filaments would be bending in opposite directions.

Fig. 5 of the present mss seems to resolve this by showing that the two FtsZ filaments and ZapD are not planar, but stacked. The two FtsZ filaments have their C termini facing the same direction, let's say up, toward the membrane, and ZapD binds on top, bridging the two. The spacing of the ctd binding sites on the Zap D dimer is 6.5 nm, which would fit the ~8 nm width of the paired filament complex observed in the present cryoEM (Fig S13). In the Schumacher model the width would be about 20 nm. Importantly, the stack model has the ctd of each filament facing the same direction, so the paired filaments could attach to the membrane and bend together (using ctd's not bound by ZapD). Finally, the new arrangement would also provide an easy way for the complex to extend from a pair of filaments to a sheet of three or four or more.

A problem with this new model from Fig. 5 is that it is supported by only a single example of the paired FtsZ-ZapD complex. If this is to be the basis of the interpretation, more examples should be shown. Maybe examples could be found with three or four FtsZ filaments in a sheet.

What then should be done with the toroids? I am not convinced by the identification of ZapD as "connectors." I think it is likely that the ZapD is part of the belts that I discuss below, although the relative location of ZapD in the belts is not resolved. It is likely that the resolution in the toroid reconstructions of Fig. 4, S8,9 is less than that of the isolated pf pair in Fig. 5c.

Importantly, If the authors want to pursue the location of ZapD in toroids, I suggest they need to compare their ZapD-containing toroids with toroids lacking ZapD. Popp et al 2009 have determined a variety of solution conditions that favor the assembly of toroids by FtsZ with no added protein crosslinker. It would be very interesting to investigate the structure of these toroids by the present cryoEM methods, and compare them to the FtsZ-ZapD toroids. I suspect that the belts seen in the ZapD toroids will not be found in the pure FtsZ toroids, confirming that their structure is generated by ZapD.

Reviewer #3 (Public Review):

Summary:

In this paper, Wang et al. provide the most comprehensive description and comparison of the expression of the different genes required to synthesize, transport, and recycle the most common neurotransmitters (Glutamate, Acetylcholine, GABA, Serotonin, Dopamine, Octopamine, and Tyramine) used by hermaphrodite and male C. elegans. This paper will be a seminal reference in the field. Building and contrasting observations from previous studies using fosmid, multicopy reporters, and single-cell sequencing, they now describe CRISPR/Cas-9-engineered reporter strains that, in combination with the multicolor pan-neuronal labeling of all C. elegans neurons (NeuroPAL), allows rigorous elucidation of neurotransmitter expression patterns. These novel reporters also illuminate previously unappreciated aspects of neurotransmitter biology in C. elegans, including sexual dimorphism of expression patterns, co-transmission, and the elucidation of cell-specific pathways that might represent new forms of neurotransmission.

Strengths:

The authors set out to establish neurotransmitter identities in C. elegans males and hermaphrodites via varying techniques, including integration of previous studies, examination of expression patterns, and generation of endogenous CRISPR-labeled alleles. Their study is comprehensive, detailed, and rigorous, and achieves the aims. It is an excellent reference for the field, particularly those interested in biosynthetic pathways of neurotransmission and their distribution in vivo, in neuronal and non-neuronal cells.

Weaknesses:

No weaknesses were noted. The authors do a great job linking their characterizations with other studies and techniques, giving credence to their findings. As the authors note, there are sexually dimorphic differences across animals and varying expression patterns of enzymes. While it is unlikely there will be huge differences in the reported patterns across individual animals, it is possible that these expression patterns could vary developmentally, or based on physiological or environmental conditions. It is unclear from the study how many animals were imaged for each condition, and if the authors noted changes across individuals during development (could be further acknowledged in the discussion?)

Reviewer #3 (Public Review):

In the submitted manuscript, Dong and colleagues set out to dissect the role of the Rab10 small GTPase on the intracellular trafficking and exocytosis of dense core vesicles (DCVs). While the authors have already shown that Rab3 plays a central role in the exocytosis of DVC in mammalian neurons, the roles of several other Rab-members have been identified genetically, but their precise mechanism of action in mammalian neurons remains unclear. In this study, the authors use a carefully designed and thoroughly executed series of experiments, including live-cell imaging, functional calcium-imaging, proteomics, and electron microscopy, to identify that DCV secretion upon Rab10 depletion in adult neurons is primarily a result of dysregulated protein synthesis and, to a lesser extent, disrupted intracellular calcium buffering. Given that the full deletion of Rab10 has a deleterious effect on neurons and that Rab10 has a major role in axonal development, the authors cautiously employed the knock-down strategy from 7 DIV, to focus on the functional impact of Rab10 in mature neurons. The experiments in this study were meticulously conducted, incorporating essential controls and thoughtful considerations, ensuring rigorous and comprehensive results.

Reviewer #3 (Public Review):

Summary

The authors have made simultaneous recordings of the responses of large numbers of neurons from the primary visual cortex of macaque monkeys using optical two-photon imaging of calcium signals from the superficial layers of the cortex. Recordings were made to compare the responses of the cortical neurons under normal binocular viewing of a flat screen with both eyes open and monocular viewing of the same screen with one eye's view blocked by a translucent filter. The screen displayed visual stimuli comprising small contrast patches of Gabor function distributions of luminance, a stimulus that is known to excite cortical neurons.

Strengths

This is an important data set, given the large number of neurons recorded. The authors present a simple model to explain binocular combination of neuronal signals from the right and left eyes. The work advances the use of two-photon imaging in the cerebral neocortex. The research design adds valuable information to our understanding of the organization of binocular vision in macaque monkeys, which are the only realistic animal model of human vision for the study of binocular interactions.

Limitations and Weaknesses

(1) Given that these recordings are made optically, these results reflect primarily activations of neurons in the superficial layers of the cortex. This limitation arises from the usual constraints (depth of cortex, degree of myelination) on optical imaging in the macaque cortex. This means that the sample of neurons forming this data set is not fully representative of the population of binocular neurons within the visual cortex. This limitation is important in comparing the outcome of these experiments with the results from other studies of binocular combination, which have used single-electrode recording. Electrode recording will result in a sample of neurons that is drawn from many layers of the cortex, rather than just the superficial layers, noting that electrode recordings also carry different risks of sampling bias.

(2) Single neuron recording of binocular neurons in the primary visual cortex has shown that these neurons often have some spontaneous activity. Assessment of this spontaneous level of firing is important for accurate model fitting [1]. The present imaging approach works exclusively with differential measurements of neuronal signals, so assessment of the level of spontaneous activity is not feasible.

(3) The arrangements for visual stimulation and comparison of binocular and monocular responses mean that the stereoscopic disparity of the binocular stimuli is always at zero or close to zero. The consequence is that the experimental design does not test the cortical response over a range of different binocular depths.

The animal's fixation point is in the centre of a single display that is viewed binocularly. The fixation point is, by definition, at zero disparity.. Provided that the animals accurately converged their eyes on the binocular fixation point, then the disparity of the visual stimuli across the whole display will always be at or close to zero. However, we already know from earlier work that neurons in the visual cortex exhibit a range of selectivity for binocular disparity. Some neurons have their peak response at non-zero disparities, representing binocular depths nearer than the fixation depth or beyond it.

There are also other neurons whose response is maximally suppressed by disparities at the depth of the fixation point (so-called Tuned Inhibitory [TI] neurons). The simple model and analysis presented in the paper for the summation of monocular responses to predict binocular responses will perform adequately for neurons that are tuned to zero disparity, so-called tuned excitatory neurons [TE], but is necessarily compromised when applied to neurons that have other, different tuning profiles for binocular disparity. Specifically, when neurons are stimulated binocularly with a non-preferred disparity, the binocular response may be lower than the monocular response [2, 3]. The same limitation applies to another recent paper [4].

This more realistic view of binocular responses needs to be considered further to gain a full picture of the operation of the visual cortex in responding to binocular depth

Citations

1. Prince, S.J.D., Pointon, A.D., Cumming, B.G., and Parker, A.J., (2002). Quantitative analysis of the responses of V1 neurons to horizontal disparity in dynamic random-dot stereograms. Journal of Neurophysiology, 87: 191-208.

2. Prince, S.J.D., Cumming, B.G., and Parker, A.J., (2002). Range and mechanism of encoding of horizontal disparity in macaque V1. Journal of Neurophysiology, 87: 209-221.

3. Poggio, G.F. and Fischer, B., (1977). Binocular interaction and depth sensitivity in striate and prestriate cortex of behaving rhesus monkey. Journal of Neurophysiology, 40: 1392-1405 doi 10.1152/jn.1977.40.6.1392.

4. B. A. Mitchell, K. Dougherty, J. A. Westerberg, B. M. Carlson, L. Daumail, A. Maier, et al. (2022) Stimulating both eyes with matching stimuli enhances V1 responses.<br /> iScience 2022 Vol. 25 Issue 5 DOI: 10.1016/j.isci.2022.104182

Reviewer #3 (Public Review):

Strengths:

The authors present differences between ADHD and TD children in biological motion processing, and this question has not received as much attention as equivalent processing capabilities in autism. They use a task that appears well controlled. They raise some interesting mechanistic possibilities for differences in local and global motion processing, which are distinctions worth exploring. The group differences will therefore be of interest to those studying ADHD, as well as other developmental conditions, and those examining biological motion processing mechanisms in general.

Weaknesses:

The data are not strong enough to support claims about differences between global and lobal processing wrt social communication skills and age. The mechanistic possibilities for why these abilities may dissociate in such a way are interesting, but the crucial tests of differences between correlations do not present a clear picture. Further empirical work would be needed to test the authors' claims. Specifics:

The authors state frequently that it was the local BM task that related to social communication skills (SRS) and not the global tasks. However, the results section shows a correlation between SRS and all three tasks. The only difference is that when looking specifically within the ADHD group, the correlation is only significant for the local task. The supplementary materials demonstrate that tests of differences between correlations present an incomplete picture. Currently they have small samples for correlations, so this is unsurprising.

Theoretical assumptions. The authors make some statements about local vs global biological motion processing that should still be made more tentatively. They assume that local processing is specifically genetically whereas global processing is a product of experience. These data in newborn chicks are controversial and confounded - I cannot remember the specifics but I think there an upper vs lower visual field complexity difference here.

Readability. The manuscript needs very careful proofreading and correction for grammar. There are grammatical errors throughout.

"Il résulte donc de ce qui précède, qu’en l’absence d’obstacle juridique, l’organe délibératif de l’EPLE est parfaitement libre d’adopter le principe d’une répartition de l’année scolaire en deux semestres, au lieu de trois trimestres. Une fois cette résolution arrêtée, il conviendra également de modifier en conséquence le règlement intérieur de l’établissement."

Reviewer #3 (Public Review):

Summary:

This study aimed to investigate whether the development of functional connectivity (FC) is modulated by early physical growth and whether these might impact cognitive development in childhood. This question was investigated by studying a large group of infants (N=204) assessed in Gambia with fNIRS at 5 visits between 5 and 24 months of age. Given the complexity of data acquisition at these ages and following data processing, data could be analyzed for 53 to 97 infants per age group. FC was analyzed considering 6 ensembles of brain regions and thus 21 types of connections. Results suggested that: i) compared to previously studied groups, this group of Gambian infants have different FC trajectory, in particular with a change in frontal inter-hemispheric FC with age from positive to null values; ii) early physical growth, measured through weight-for-length z-scores from birth on, is associated with FC at 24 months. Some relationships were further observed between FC during the first two years and cognitive flexibility at 4-5 years of age, but results did not survive corrections for multiple comparisons.

Strengths:

The question investigated in this article is important for understanding the role of early growth and undernutrition on brain and behavioral development in infants and children. The longitudinal approach considered is highly relevant to investigate neurodevelopmental trajectories. Furthermore, this study targets a little-studied population from a low-/middle-income country, which was made possible by the use of fNIRS outside the lab environment. The collected dataset is thus impressive and it opens up a wide range of analytical possibilities.

Weaknesses:

- Analyzing such a huge amount of collected data at several ages is not an easy task to test developmental relationships between growth, FC, and behavioral capacities. In its present form, this study and the performed analyses lack clarity, unity and perhaps modeling, as it suggests that all possible associations were tested in an exploratory way without clear mechanistic hypotheses. Would it be possible to specify some hypotheses to reduce the number of tests performed? In particular, considering metrics at specific ages or changes in the metrics with age might allow us to test different hypotheses: the authors might clarify what they expect specifically for growth-FC-behaviour associations. Since some FC measures and changes might be related to one another, would it be reasonable to consider a dimensionality reduction approach (e.g., ICA) to select a few components for further correlation analyses?

- It seems that neurodevelopmental trajectories over the whole period (5-24 months) are little investigated, and considering more robust statistical analyses would be an important aspect to strengthen the results. The discussion mentions the potential use of structural equation modelling analyses, which would be a relevant way to better describe such complex data.

- Given the number of analyses performed, only describing results that survive correction for multiple comparisons is required. Unifying the correction approach (FDR / Bonferroni) is also recommended. For the association between cognitive flexibility and FC, results are not significant, and one might wonder why FC at specific ages was considered rather than the change in FC with age. One of the relevant questions of such a study would be whether early growth and later cognitive flexibility are related through FC development, but testing this would require a mediation analysis that was not performed.

- Growth is measured at different ages through different metrics. Justifying the use of weight-for-length z-scores would be welcome since weight-for-age z-scores might be a better marker of growth and possible undernutrition (this impacting potentially both weight and length). Showing the distributions of these z-scores at different ages would allow the reader to estimate the growth variability across infants.

- Regarding FC, clarifications about the long-range vs short-range connections would be welcome, as well as drawing a summary of what is expected in terms of FC "typical" trajectory, for the different brain regions and connections, as a marker of typical development. For instance, the authors suggest that an increase in long-range connectivity vs a decrease in short-range is expected based on previous fNIRS studies. However anatomical studies of white matter growth and maturation would suggest the reverse pattern (short-range connections developing mostly after birth, contrarily to long-range connections prenatally).

The authors test associations between FC and growth, but making sense of such modulation results is difficult without a clearer view of developmental changes per se (e.g., what does an early negative FC mean? Is it an increase in FC when the value gets close to 0? In particular, at 24m, it seems that most FC values are not significantly different from 0, Figure 2B). Observing positive vs negative association effects depending on age is quite puzzling. It is also questionable, for some correlation analyses with cognitive flexibility, to focus on FC that changes with age but to consider FC at a given age.

- The manuscript uses inappropriate terms "to predict", "prediction" whereas the conducted analyses are not prediction analyses but correlational.

Reviewer #3 (Public Review):

Summary:

Garcia et al., investigated whether the human left superior frontal sulcus (SFS) is involved in integrating evidence for decisions across either perceptual and/or value-based decision-making. Specifically, they had 20 participants perform two decision-making tasks (with matched stimuli and motor responses) in an fMRI scanner both before and after they received continuous theta burst transcranial magnetic stimulation (TMS) of the left SFS. The stimulation thought to decrease neural activity in the targeted region, led to reduced accuracy on the perceptual decision task only. The pattern of results across both model-free and model-based (Drift diffusion model) behavioural and fMRI analyses suggests that the left SLS plays a critical role in perceptual decisions only, with no equivalent effects found for value-based decisions. The DDM-based analyses revealed that the role of the left SLS in perceptual evidence accumulation is likely to be one of decision boundary setting. Hence the authors conclude that the left SFS plays a domain-specific causal role in the accumulation of evidence for perceptual decisions. These results are likely to add importance to the literature regarding the neural correlates of decision-making.

Strengths:

The use of TMS strengthens the evidence for the left SFS playing a causal role in the evidence accumulation process. By combining TMS with fMRI and advanced computational modelling of behaviour, the authors go beyond previous correlational studies in the field and provide converging behavioural, computational, and neural evidence of the specific role that the left SFS may play.

Sophisticated and rigorous analysis approaches are used throughout.

Weaknesses:

Though the stimuli and motor responses were equalised between the perception and value-based decision tasks, reaction times (according to Figure 1) and potential difficulty (Figure 2) were not matched. Hence, differences in task difficulty might represent an alternative explanation for the effects being specific to the perception task rather than domain-specificity per se.

No within- or between-participants sham/control TMS condition was employed. This would have strengthened the inference that the apparent TMS effects on behavioural and neural measures can truly be attributed to the left SFS stimulation and not to non-specific peripheral stimulation and/or time-on-task effects.

No a priori power analysis is presented.

Reviewer #3 (Public Review):

Summary:

In this very important study by Dantzer et al., 'Emerging role of oncogenic b-catenin in exosome biogenesis as a driver of immune escape in hepatocellular carcinoma' the authors define a role for oncogenic b-catenin on exosome biology and explore the link between reduce exosome secretion and tumor immune cell evasion. Using transcriptional and proteomic analysis of hepatocellular carcinoma cells with either oncogenic or wildtype b-catenin the authors find that oncogenic b-catenin negatively regulates exosome biogenesis.

The authors can provide compelling evidence that oncogenic b-catenin in different hepatocellular carcinoma cells negatively regulates exosome biogenesis and secretion, by downregulation of, amongst others, SDC4 and RAB27A, two proteins involved in exosome biogenesis. The authors corroborate these results by inducing b-catenin activation using CHIR99021 in a hepatocarcinoma cell line with non-oncogenic bCatenin (Huh7 cells). The authors can further demonstrate convincingly that a reduction in exosome release by hepatocarcinoma spheroids leads to a reduction in immune cell infiltration into the tumor spheroid.

Strengths:

This is a very important and well-conceived study, that appeals to a readership beyond the field of hepatocarcinoma. The authors demonstrate a compelling link between oncogenic bCatenin and exosome biogenesis. Their results are convincing and with well-designed control experiments. The authors included various complementary lines of investigation to verify their findings.

Weaknesses:

One limitation of this study is that the mechanistic relationship of exosome release and how they affect immune cells remains to be elucidated. In this context, the authors conclusions rest on the assumption that hepatocarcinoma immune evasion is based exclusively on the reduced number of exosomes. However, the authors do not analyze exosome composition between exosomes of wild type and oncogenic background, which could be different.

Reviewer #3 (Public Review):

Summary:

This study aims to understand gene regulation of the plant bacterial pathogen Pseudomonas syringae. Although the function of some TFs has been characterized in this strain, a global picture of the gene regulatory network remains elusive. The authors conducted a large-scale ChIP-seq analysis, covering 170 out of 301 TFs of this strain, and revealed gene regulatory hierarchy with functional validation of some previously uncharacterized TFs.

Strengths:

- This study provides one of the largest ChIP-seq datasets for a single bacterial strain, covering more than half of its TFs. This impressive resource enabled comprehensive systems-level analysis of the TF hierarchy.

- This study identified novel gene regulation and function with validations through biochemical and genetic experiments.

- The authors attempted on broad analyses including comparisons between different bacterial strains, providing further insights into the diversity and conservation of gene regulatory mechanisms.

Weaknesses:

(1) Some conclusions are not backed by quantitative or statistical analyses, and they are sometimes overinterpreted.

(2) Some figures and analyses are not well explained, and I was not able to understand them.

(3) The Method section lacks depth, especially in data analyses. It is strongly recommended that the authors share their analysis codes so that others can reproduce the analyses.

Reviewer #3 (Public Review):

When members of two related but diverged species mate, the resulting hybrids can produce offspring where parts of one species' genome replace those of the other. These "introgressions" often create regions with a much greater density of sequence differences than are normally found between members of the same species. Previous studies have shown that increased sequence differences, when heterozygous, can reduce recombination during meiosis specifically in the region of increased difference. However, most of these studies have focused on crossover recombination, and have not measured noncrossovers. The current study uses a pair of Saccharomyces uvarum crosses: one between two natural isolates that, while exhibiting some divergence, do not contain introgressions; the other is between two fermentation strains that, when combined, are heterozygous for 9 large regions of introgression that have much greater divergence than the rest of the genome. The authors wished to determine if introgressions differently affected crossovers and noncrossovers, and, if so, what impact that would have on the gene shuffling that occurs during meiosis.

While both crossovers and noncrossovers were measured, assessing the true impact of increased heterology (inherent in heterozygous introgressions) is complicated by the fact that the increased marker density in heterozygous introgressions also increases the ability to detect noncrossovers. The authors used a relatively simple correction aimed at compensating for this difference, and based on that correction, conclude that, while as expected crossovers are decreased by increased sequence heterology, counter to expectations noncrossovers are substantially increased. They then show that, despite this, genetic shuffling overall is substantially reduced in regions of heterozygous introgression. However, it is likely that the correction used to compensate for the effect of increased sequence density is defective, and has not fully compensated for the ascertainment bias due to greater marker density. The simplest indication of this potential artifact is that, when crossover frequencies and "corrected" noncrossover frequencies are taken together, regions of introgression often appear to have greater levels of total recombination than flanking regions with much lower levels of heterology. This concern seriously undercuts virtually all of the novel conclusions of the study.

Until this methodological concern is addressed, the work will not be a useful contribution to the field.

Reviewer #3 (Public Review):

While I am not a specialist in this field, I do have some knowledge of the subject matter and the computational aspects involved.

The authors employ simple machine learning techniques (such as SVM) for the following purposes:

a. Prediction of aversive valence.<br /> b. Predicting anti-repellent chemicals.<br /> c. Predicting calcium mobilization.

The approach is commonplace in chemoinformatics literature.

Weaknesses:

- All the above models are presented discretely, making it difficult to discern experiment design principles and connectedness.<br /> - The ML work is rudimentary, lacking adequate details. Chemoinformatics has reached great heights, and SVM does not seem contemporary.<br /> - There is significant existing research on finding repellents.

Strengths:

- Authors attempt to make a case for calcium mobilization in the context of repellency. This aspect sounds interesting but is not surprising.<br /> - Behavioral profiling of repellents could be useful.

Reviewer #3 (Public Review):

Summary:<br /> This work focuses on accessibility of scientific images for individuals with color vision deficiencies, particularly deuteranopia. The research involved an analysis of images from eLife published in 2012-2022. The authors manually reviewed nearly 5,000 images, comparing them with simulated versions representing the perspective of individuals with deuteranopia, and also evaluated several methods to automatically detect such images including training a machine-learning algorithm to do so, which performed the best. The authors found that nearly 13% of the images could be challenging for people with deuteranopia to interpret. There was a trend toward a decrease in problematic images over time, which is encouraging.

Strengths:<br /> The manuscript is well organized and written. It addresses inclusivity and accessibility in scientific communication, and reinforces that there is a problem and that in part technological solutions have potential to assist with this problem.

The number of manually assessed images for evaluation and training an algorithm is, to my knowledge, much larger than any existing survey. This is a valuable open source dataset beyond the work herein.

The sequential steps used to classify articles follow best practices for evaluation and training sets.

Weaknesses:<br /> I do not see any major issues with the methods. The authors were transparent with the limitations (the need to rely on simulations instead of what deuteranopes see), only capturing a subset of issues related to color vision deficiency, and the focus on one journal that may not be representative of images in other journals and disciplines.

Reviewer #3 (Public Review):

Summary:

This paper uses indirect immunofluorescence, superresolution fluorescence microscopy, and X-ChIP to demonstrate radial distribution profiles of all histone H1 somatic variants with the exception of histone H1.1. The results support earlier work from chromatin immunoprecipitation experiments that revealed biases for active versus repressed states of chromatin. The previous studies provided some support for the subtle sequence variation found primarily within the C-terminus of histone H1 variants conferred preferences in the type of DNA (e.g. methylated DNA) or chromatin bound. The current study significantly strengthens that argument. Importantly, this was shown across multiple cell lines and reveals conserved properties of localization of histone H1 variants.

Strengths:

The strength of the manuscript is the combined use of quantitative analysis of indirect immunofluorescence and X-ChIP. The results generally support the polar organization of the genome and a corresponding distribution of histone H1 variants that reflect this polar organization. AT-rich chromatin is positioned near the lamina and is found to be enriched in H1.2, H1.3, and H1.5. H1.4 and H1.X were more biased towards the GC-rich intranuclear chromatin.

There is emerging functional evidence for variant-specific properties to histone H1 subtypes. This work provides an important building block in understanding how different histone H1 variants may have specific functional consequences. The histone H1 variant that is most abundant in most cell types, H1.2, was found to decrease the area of the immunofluorescent slice that was chromatin-free when depleted, suggesting a more important role in global chromatin organization.

Weaknesses:

While histone H1 variants may show biases in their distributions, it is unlikely that these are more than biases. That is, it is unlikely that specific H1 variants are unable to bind to nucleosomes in regions where they are depleted. Fluorescence recovery after photobleaching experiments have demonstrated differences in binding affinity but the capacity to bind a range of chromatin structures, including highly acetylated chromatin, for histone H1 variants. Thus, it is critical in assessing this data to have accurate quantitative information on the relative abundance of the different histone variants amongst the cell lines tested here. The paper relies upon quantification by immunoblotting.

Another uncertainty in both the ChIP and immunofluorescence datasets is the accessibility of the epitope. This weakness is highlighted by the apparent loss of H1.2 and H1.4 in mitotic chromosomes that is revealed to be false by the detection of the phosphorylated species. The distributions relative to the surface of chromosomes in mitosis and the depletion of H1.2, H1.3, and H1.5 from the central regions of interphase nuclei reveals an unusual dissipation of the staining that is suggestive of antibody accessibility problems. The overall image quality of the immunofluorescence images is poor, further complicating analysis.

Reviewer #3 (Public Review):

In this study, Yang et al. address a fundamental question of the role of dorsal striatum in neural coding of gait. The authors study the respective role of D1 and D2 MSNs by linking their balanced activity to detailed gait parameters. In addition, they put in parallel the striatal activity related to whole-body measures such as initiation/cessation of movement or body speed. They are using an elegant combination of high-resolution single-limb motion tracking, identification of bouts of movements and electrophysiological recordings of striatal neurons to correlate those different parameters. Subpopulations of striatal output neurons (D1 and D2 expressing neurons) are identified in neural recordings with optogenetic tagging. Those complementary approaches show that a subset of striatal neurons have phase-locked activity to individual limbs. In addition, more than a third of MSNs appear to encode all three aspects of motor behavior addressed here, initiation/cessation of movement, body speed and gait. This activity is balanced between D1 and D2 neurons, with a higher activity of D1 neurons only for movement initiation. Finally, alterations of gait, and the associated striatal activity, is studied in a mouse model of Parkinson's Disease, using 6-OHDA lesions in the medial forebrain bundle (MFB). In the 6OHDA mice, there is an imbalance toward D2 activity.

Strengths:

The study combines elegant approaches to correlate cell-specific striatal activity with specific aspects of motion and how it is affected in a PD model. The results are convincing, and the methodology supports the conclusions presented here.

Weaknesses:

All the data were not fully exploited or explained in the first version of the manuscript and the present version has been significantly improved.

There is a long-standing debate on the respective role of D1 and D2 MSNs on the control of movement. This study goes beyond prior work by providing detailed quantification of individual limb kinematics, in parallel of whole-body motion, and showing high proportion of MSNs to be phase-locked to precise gait cycle and also encoding whole-body motion. The temporal resolution used here highlights preferential activity of D1 MSN at the movement starts, where previous studies described a more balanced involvement. Finally they reveal neural mechanisms of dopamine depletion induced gait alterations, with a preponderant phase-locked activity of D2 neurons.

Reviewer #3 (Public Review):

Summary:

Male fertility depends on both sperm and seminal plasma, but the functional effect of seminal plasma on sperm has been relatively understudied. The authors investigate the testosterone-dependent synthesis of seminal plasma and identify oleic acid as a key factor in enhancing sperm fertility.

Strengths:

The evidence for changes in cell proliferation and metabolism of seminal vesicle epithelial cells and the identification of oleic acid as a key factor in seminal plasma is solid.

Weaknesses:

The evidence that oleic acids enhance sperm fertility in vivo needs more experimental support, as the main phenotypic effect in vitro provided by the authors remains simply as an increase in the linearity of sperm motility, which does not necessarily correlate with enhanced sperm fertility.

Reviewer #3 (Public Review):

Summary:

The authors aimed to study the activation of gliogenesis and the role of newborn astrocytes in a post-ischemic scenario. Combining immunofluorescence, BrdU-tracing, and genetic cellular labelling, they tracked the migration of newborn astrocytes (expressing Thbs4) and found that Thbs4-positive astrocytes modulate the extracellular matrix at the lesion border by synthesis but also degradation of hyaluronan. Their results point to a relevant function of SVZ newborn astrocytes in the modulation of the glial scar after brain ischemia. This work's major strength is the fact that it is tackling the function of SVZ newborn astrocytes, whose role is undisclosed so far.

Strengths:

The article is innovative, of good quality, and clearly written, with properly described Materials and Methods, data analysis, and presentation. In general, the methods are designed properly to answer the main question of the authors, being a major strength. Interpretation of the data is also in general well done, with results supporting the main conclusions of this article.

Weaknesses:

However, there are some points of this article that still need clarification to further improve this work.

- As a first general comment, is it possible that the increase in Thbs4-positive astrocytes can also happen locally close to the glia scar, through the proliferation of local astrocytes or even from local astrocytes at the SVZ? As it was shown in published articles most of the newborn astrocytes in the adult brain actually derive from proliferating astrocytes, and a smaller percentage is derived from NSCs. How can the authors rule out a contribution of local astrocytes to the increase of Thbs4-positive astrocytes? The authors also observed that only about one-third of the astrocytes in the glial scar derived from the SVZ.

- It is known that the local, GFAP-reactive astrocytes at the scar can form the required ECM. The authors propose a role of Thbs4-positive astrocytes in the modulation, and perhaps maintenance, of the ECM at the scar, thus participating in scar formation likewise. So, this means that the function of newborn astrocytes is only to help the local astrocytes in the scar formation and thus contribute to tissue regeneration. Why do we need specifically the Thbs4-positive astrocytes migrating from the SVZ to help the local astrocytes? Can you discuss this further?

- The authors observed that the number of BrdU- and DCX-positive cells decreased 15 dpi in all OB layers (Fig. S5). They further suggest that ischemia-induced a change in the neuroblasts ectopic migratory pathway, depriving the OB layers of the SVZ newborn neurons. Are the authors suggesting that these BrdU/DCX-positive cells now migrate also to the ischemic scar, or do they die? In fact, they see an increase in caspase-3 positive cells in the SVZ after ischemia, but they do not analyse which type of cells are dying. Alternatively, is there a change in the fate of the cells, and astrogliogenesis is increased at the expense of neurogenesis? The authors should understand which cells are Cleaved-caspase-3 positive at the SVZ and clarify if there is a change in cell fate. Also please clarify what happens to the BrdU/DCX-positive cells that are born at the SVZ but do not migrate properly to the OB layers.

- The authors showed decreased Nestin protein levels at 15 dpi by western blot and immunostaining shows a decrease already at 7div (Figure 2). These results mean that there is at least a transient depletion of NSCs due to the promotion of astrogliogenesis. However, the authors show that at 30dpi there is an increase of slow proliferating NSCs (Figure 3). Does this mean, that there is a reestablishment of the SVZ cytogenic process? How does it happen, more specifically, how NSCs number is promoted at 30dpi? Please explain how are the NSCs modulated throughout time after ischemia induction and its impact on the cytogenic process.

- The authors performed a classification of Thbs4-positive cells in the SVZ according to their morphology. This should be confirmed with markers expressed by each of the cell subtypes.

- In Figure S6, the authors quantified HABP spots inside Thbs4-positive astrocytes. Please show a higher magnification picture to show how this quantification was done.

Reviewer #3 (Public Review):

In this manuscript, the authors investigated the effects of deletion of the ER-plasma membrane/Golgi tethering proteins tricalbins (Tcb1-3) on vacuolar morphology to demonstrate the role of membrane contact sites (MCSs) in regulating vacuolar morphology in Saccharomyces cerevisiae. Their data show that tricalbin deletion causes vacuolar fragmentation possibly in parallel with TORC1 pathway. In addition, their data reveal that levels of various lipids including ceramides, long-chain base (LCB)-1P, and phytosphingosine (PHS) are increased in tricalbin-deleted cells. The authors find that exogenously added PHS can induce vacuole fragmentation and by performing analyses of genes involved in sphingolipid metabolism, they conclude that vacuolar fragmentation in tricalbin-deleted cells is due to the accumulated PHS in these cells. Importantly, exogenous PHS- or tricalbin deletion-induced vacuole fragmentation was suppressed by loss of the nucleus vacuole junction (NVJ), suggesting the possibility that PHS transported from the ER to vacuoles via the NVJ triggers vacuole fission. Of note, the authors find that hyperosmotic shock increases intracellular PHS levels, suggesting a general role of PHS in vacuole fission in response to physiological vacuolar division-inducing stimuli.

This work provides valuable insights into the relationship between MCS-mediated sphingolipid metabolism and vacuole morphology. The conclusions of this paper are mostly supported by their results, but inclusion of direct evidence indicating increased transport of PHS from the ER to vacuoles via NVJ in response to vacuolar division-inducing stimuli would have strengthened this study.

There is another weakness in their claim that the transmembrane domain of Tcb3 contributes to the formation of the tricalbin complex which is sufficient for tethering ER to the plasma membrane and the Golgi complex. Their claim is based only on the structural simulation, but not on by biochemical experiments such as co-immunoprecipitation and pull-down.

Reviewer #3 (Public Review):

Summary: Irisin has previously been demonstrated to be a muscle-secreted factor that affects skeletal homeostasis. Through the use of different experimental approaches, such as genetic knockout models, recombinant Irisin treatment, or different cell lines, the role of Irisin on skeletal homeostasis has been revealed to be more complex than previously thought and this warrants further examination of its role. Therefore, the current study sought to rigorously examine the effects of global Irisin knockout (KO) in male and female mouse bone. Authors demonstrated that in calcium-demanding settings, such as lactation or low-calcium diet, female Irisin KO mice lose less bone compared to wildtype (WT) female mice. Interestingly male Irisin KO mice exhibited worse skeletal deterioration compared to WT male mice when fed low-calcium diet. When examined for transcriptomic profiles of osteocyte-enriched cortical bone, authors found that Irisin KO altered the expression of osteocytic osteolysis genes as well as steroid and fatty acid metabolism genes in males but not in females. These data support authors' conclusion that Irisin regulates skeletal homeostasis in a sex-dependent manner.

Strengths:

The major strength of the study is rigorous examination of the effects of Irisin deletion in the settings of skeletal maturity and increased calcium demands in female and male mice. Since many of the common musculoskeletal disorders are dependent on sex, examining both sexes in the preclinical setting is crucial. Had the investigators only examined females or males in this study, the conclusion from each sex would have contradicted each other regarding the role of Irisin on bone. Also, the approaches are thorough and comprehensive that assess the functional (mechanical testing), morphological (microCT, BSEM, and histology), and cellular (RNA-seq) properties of bone. Transcriptomic data deposited to NCBI GEO data repository will be a valuable resource to musculoskeletal researchers who aim to further assess the affects of Irisin on skeleton.

Weaknesses:

One of the weaknesses of this study is a lack of detailed mechanistic analysis of why Irisin has sex-dependent role on skeletal homeostasis. However, the osteocyte transcriptome comparisons between LC females vs. LC males lay a foundation for such future mechanistic studies.

Another weakness is authors did not present data that convincingly demonstrate that Irisin secretion is altered in the skeletal muscle between female vs. male WT mice in response to calcium restriction. The supplement skeletal muscle data only present functional and electrophysiological outcomes. Since Itgav or Itgb5 were not different in any of the experimental groups, it is assumed that the changes in the level of Irisin is responsible for the phenotypes observed in WT mice. Assessing Irisin expression will further strengthen the conclusion based on observing skeletal changes that occur in Irisin KO male and female mice.

Reviewer #3 (Public Review):

Summary:

The authors show that a GAN can learn to reproduce the distribution of outputs of a neuron endowed with Oja's plasticity rule throughout its learning process by learning a plasticity rule. The GAN does not, however, learn Oja's rule. Indeed, the plasticity dynamics it infers can differ dramatically from the true dynamics. The authors propose this approach as a way to uncover families of putative plasticity rules consistent with observed activity patterns in biological systems.

Oja's rule was a great choice for the comparison because it makes explicit, I think, the limitations of this approach. As is well known, Oja's rule allows a (linear) neuron to learn the first principal component of its inputs; the synaptic weights converge to the first eigenvector of the input covariance. After this learning process, the response of a neuron to a particular input sample measures the weighted angle between that input and that principal component.

The other, meta-learned plasticity rules that the authors' GAN uncovers notably do not learn the same computation as Oja's rule (Figure 2). This is, to me, the central finding of the paper and fleshed out nicely. It seems to me that this may be because the objective of the GAN is only to reproduce the marginal output statistics of the neuron. It is, if I understand correctly, blind to the input samples, the inputs' marginal statistics, and to correlations between the input and output. I wonder if a GAN that also had some knowledge of the correlation between input and outputs might be more successful at learning the underlying true dynamics.

The focus on reproducing output statistics has some similarity to some types of experiments (e.g., in vivo recordings) but also seems willfully blind to other aspects of these experiments. In my experience, experimentalists are well aware that the circuits they record receive external inputs. Those inputs are often recorded (perhaps in separate experiments or studies). The point being that I'm not sure that this is an entirely fair comparison to the field.

Finally, the plasticity models studied by theoreticians are not only constructed by intuition and hand-tuning. They also draw, often heavily, on biological data and principles. Oja's rule, for example, is simply the combination of Hebbian learning with a homeostatic constraint on the total synaptic weight amplitude (under the choice of a Euclidean norm).

To me, this study very nicely exposes the caveats and risks associated with a blind machine-learning approach to model specification in biology and highlights the need for understanding underlying biological mechanisms and principles. I agree with the authors that heterogeneity and degeneracy in plasticity rules deserve much more attention in the field.

Reviewer #3 (Public Review):

Summary:

This work provides insights into predictive coding models of visual cortex processing. These models predict that visual cortex neurons will show elevated responses when there are unexpected changes to learned sequential stimulus patterns. This model is currently controversial, with recent publications providing conflicting evidence. In this work, the authors test two types of unexpected pattern variations in layer 2/3 of the mouse visual cortex. They show that pattern omission evokes elevated responses, in favor of a predictive coding model, but find no evidence for prediction errors with substituted patterns, which conflicts with both prior results in L4, and with the expectations of a predictive coding model. They also report that with sequence training, responses sparsify and decorrelate, but surprisingly find no changes in the ability of an ideal observer to decode stimulus identity or timing.

These results are an important contribution to the understanding of how temporal sequences and expectations are encoded in the primary visual cortex. However, there are several methodological concerns with the study, and some of the authors' interpretations and conclusions are unsupported by data.

Major concerns:

(1) Experimental design using a block structure. The use of a block structure on test days (0 and 5) in which sequences were presented in 100 repetition blocks leads to several potential confounds. First, there is the potential for plasticity within blocks, which could alter the responses and induce learned expectations. The ability of the authors to clearly distinguish blocks 1 and 2 on Day 0 with a decoder suggests this change over time may be meaningful.

Repeating the experiments with fully interleaved sequences on test days would alleviate this concern. With the existing data, the authors should compare responses from the first trials in a block to the last trials in a block.

This block design likely also accounts for the ability of a decoder to readily distinguish stimulus A in ABCD from A in ABBD. As all ABCD sequences were run in a contiguous block separate from ABBD, the recent history of experience is different for A stimuli in ABCD versus ABBD. Running fully interleaved sequences would also address this point, and would also potentially mitigate the impact of drift over blocks (discussed below).

(2) The computation of prediction error differs significantly for omission as opposed to substitutions, in meaningful ways the authors do not address. For omission errors, PE compares the responses of B1 and B2 within ABBD blocks. These responses are measured from the same trial, within tens of milliseconds of each other. In contrast, substitution PE is computed by comparing C in ABCD to C in ACBD. As noted above, the block structure means that these C responses were recorded in different blocks, when the state of the brain could be different. This may account for the authors' detection of prediction error for omission but not substitution. To address this, the authors should calculate PE for omission using B responses from ABCD.

(3) The behavior of responses to B and C within the trained sequence ABCD differs considerably, yet is not addressed. Responses to B in ABCD potentiate from d0-> d5, yet responses to C in the same sequence go down. This suggests there may be some difference in either the representation of B vs C or position 2 vs 3 in the sequence that may also be contributing to the appearance of prediction errors in ABBD but not ACBD. The authors do not appear to consider this point, which could potentially impact their results. Presenting different stimuli for A,B,C,D across mice would help (in the current paper B is 75 deg and C is 165 deg in all cases). Additionally, other omissions or substitutions at different sequence positions should be tested (eg ABCC or ABDC).

(4) The authors' interpretation of their PCA results is flawed. The authors write "Experience simplifies activity in principal component space". This is untrue based on their data. The variance explained by the first set of PCs does not change with training, indicating that the data is not residing in a lower dimensional ("simpler") space. Instead, the authors show that the first 5 PCs better align with their a priori expectations of the stimulus structure, but that does not mean these PCs necessarily represent more information about the stimulus (and the fact that the authors fail to see an improvement in decoding performance argues against this case). Addressing such a question would be highly interesting, but is lacking in the current manuscript. Without such analysis, referring to the PCs after training as "highly discretized" and "untangled" are largely meaningless descriptions that lack analytical support.

(5) The authors report that activity sparsifies, yet provide only the fraction of stimulus-selective cells. Given that cell detection was automated in a manner that takes into account neural activity (using Suite2p), it is difficult to interpret these results as presented. If the authors wish to claim sparsification, they need to provide evidence that the total number of ROIs drawn on each day (the denominator for sparseness in their calculation) is unbiased. Including more (or less) ROIs can dramatically change the calculated sparseness.

The authors mention sparsification as contributing to coding efficiency but do not test this. Training a decoder on variously sized subsets of their data on days 0 and 5 would test whether redundant information is being eliminated in the network over training.

(6) The authors claim their results show representational drift, but this isn't supported in the data. Rather they show that there is some information in the structure of activity that allows a decoder to learn block ID. But this does not show whether the actual stimulus representations change, and could instead reflect an unrelated artifact that changes over time (responsivity, alertness, bleaching, etc). To actually assess representational drift, the authors should directly compare representations across blocks (one could train a decoder on block 1 and test on blocks 2-5). In the absence of this or other tests of representational drift over blocks, the authors should remove the statement that "These findings suggest that there is a measurable amount of representational drift".

(7) The authors allude to "temporal echoes" in a subheading. This term is never defined, or substantiated with analysis, and should be removed.

Reviewer #3 (Public Review):

Summary:

The study by Yao, Dai and colleagues successfully describes the design of a viral gene drive against herpes simplex virus 1. Gene drives are genetic modifications designed to spread efficiently in a population. Most applications have been developed in insects to eradicate diseases such as malaria, and the design of gene drives in viruses is an exciting recent development. A viral gene drive system was first described with human cytomegalovirus, another virus of the herpesvirus family (PMID: 32985507), and the authors followed similar methods to design a gene drive against HSV-1. While some key experiments lack rigorous controls, overall the authors convincingly showed that an HSV-1 gene drive could spread efficiently in the target population in cell culture experiments. Cytomegalovirus and HSV-1 have very different infection dynamics, and these new findings suggest that viral gene drives could be developed in a wide variety of herpesviruses. This significantly expands the potential of the technology and will be of interest to readers interested in gene drives, viral engineering, or biotechnology in general.

The most novel and interesting part of the study is the comparison of gene drives relying on spCas9 and Un1Cas12f1 nuclease. Most gene drives developed to date have relied on Cas9 or similar nucleases. Cleavage and repair of the target site by non-homologous end-joining (NHEJ) can lead to the formation of drive-resistant sequences, and, depending on the selective pressure on the wild-type, gene drive and drive-resistant alleles, prevent successful gene drive propagation. By contrast to most RNA-guided nucleases, Un1Cas12f1 cleaves outside of the RNA-recognition site. The authors hypothesized that it could prevent the appearance of drive-resistant sequences, since the target sequence would be preserved after NHEJ repair. Indeed, the study convincingly showed that Un1Cas12f1 induced fewer drive-resistant mutations, which led to almost complete penetrance of the drive. However, the claim in the abstract that an "Un1Cas12f1 gene drive yielded a greater conversion" rate than Cas9 appears unsupported. Together with its smaller size, this positions Un1Cas12f1 as an interesting alternative to Cas9 for gene drives in any organism. This development will be of great interest to researchers interested in gene drives.

Strengths:

Overall, this study is well done and the main conclusions are supported by the data. The authors used flow cytometry to follow gene drive propagation, detecting either fluorescent or cell surface proteins expressed by the different viral populations. This represents an indirect but adequate way of measuring the proportion of the different viral populations, assuming that each of the target BHK cells is infected with only one virus.<br /> In particular, the results in Fig 3 showing that Un1Cas12f1 induces fewer drive-resistant mutations than Cas9 are convincing.

Weaknesses:

The manuscript presents several conceptual and methodological weaknesses that could be discussed or addressed experimentally, which would improve the overall rigor of the study.

(1) In the abstract and the text, the author claims that "HSV1 emerges as a dependable and swift platform for gene drive assessment". It is unclear if the author believes that the main interest of their work with HSV-1 is to provide a platform for testing gene drive for other organisms, or whether a gene drive for HSV-1 could be useful by itself. While their findings with Un1Cas12f1 certainly warrant investigation in other systems, the dynamics of DNA cleavage, recombination, and selection of drive-resistant alleles will be very different between a viral infection where hundreds or thousands of genome copies co-exist in a cell nucleus, and during sexual reproduction where only one gene drive and wild-type allele are present in a fertilized egg. As such, it is unsure whether gene drive dynamics in HSV-1 will be informative for other organisms besides other herpesviruses. On the other hand, the authors provide little perspectives on the potential usage of an HSV-1 gene drive, beyond concluding that "Our study opens new possibilities for using the HSV1 gene drive for the prevention and treatment of diseases". The authors designed a drive against the important viral protein gE in an attempt to limit infectivity, but it is unclear from the data presented whether this was successful. An extended discussion on the potential use case of an HSV-1 gene drive would be informative.

(2) Unfortunately, the experiments presented lack rigorous controls to unambiguously show that gene drive propagation is mediated by CRISPR-directed recombination into the target genome. Gene drive-mediated recombination converts wild-type viruses into new recombinant viruses and the population of recombinants is expected to increase in frequency, as observed with the yellow population in Fig 2G and 3G. However, a rigorous experimental design would show that this population of recombinant viruses does not appear with a non-functional CRISPR system (for example if Cas9 is deleted in the gene drive virus) or if the target site is absent in the recipient virus. The comparison of Fig 2B and 2D does show that gene drive viruses do not increase in frequency when the target site is absent in the V19 virus, but these experiments could not distinguish between original and recombinant gene drive viruses. Thus, it is unknown if the increase in gene drive frequency in Fig 2B is because wild-type viruses have been converted to gene drive viruses, or because the WT and v23 viruses replicate with different dynamics (one could imagine for example that CRISPR cleavage of the WT genomes impaired the replication of the WT virus without inducing recombination, thus giving an advantage to v23). In Fig 2G and 3B, the authors do follow the population of recombinant viruses, in yellow, which increase in frequency as expected. However, in these experiments, either the donor or recipient viruses are mutated for gE, and the different viral populations might replicate with different dynamics, which confounds the interpretation of the results (see point 4. below). Overall, while the data presented suggests that CRISPR-mediated gene drive propagation is happening, it does not conclusively rule out other explanations, especially if viruses have different fitness.

(3) In Fig 2F-G-H, the authors designed a gene drive knocking out an important viral gene, gE, in an attempt to build a drive that reduces infectivity. gE knockout viruses V10 and V15 had smaller plaques but replicated with similar titers (Fig 1B, 1C). The gene drive against gE spread efficiently in Fig 2G. However, gE-KO viruses did not appear to have a meaningful disadvantage in the experimental system used, since the high MOI used in the co-infection experiments allowed to bypass the cell-to-cell defect of gE mutants. It would have been interesting to characterize the final population composed primarily of original and recombinant viruses (at P3 in Fig 2G), and in particular measure the plaque size of these viruses. Recombinant viruses should have smaller plaque sizes, and showing that the gene drive was able to propagate an attenuating phenotype would be a meaningful result that hints at potential therapeutic applications.

(4) Experiments presented in Fig 3 compared the dynamics of Cas9 and Un1Cas12f1 gene drives, but the experimental system used is a bit puzzling and makes the interpretation of the results challenging. In particular, the authors chose to use gE-knockout virus v10 as the recipient for the gene drive, which allowed them to use gE in their flow cytometry assay. Unfortunately, this added a confounding factor to the experiments, since gE- viruses might replicate with different dynamics than gE+ viruses (for example v10 titers are one log higher than WT at 12h in Fig 1C). In Fig 3B, gD+ gE- viruses (in blue) disappear and are replaced by gD+ GFP+ gE- recombinants (in yellow), which is suggestive of efficient gene drive recombination, as pointed out by the authors. However, the population of gD+ GFP+ virus (in green) representing the original gene drive virus also disappeared over time. At the end of the experiments in Fig 3B, the population of gE+ viruses is gone. This is unexpected and suggests that the gD+ GFP+ gE- (yellow) has a replicative advantage over gD+ GFP+ (green), and that the gE- mutation is actually positively selected in these viral competition assays. So in these experiments, both gene drive-mediated recombination and competition between viral genotypes appear to be happening at the same time, which makes interpretation of the results challenging. However, despite these limitations, the results presented convincingly suggested that Un1Cas12f1 gene drives achieved higher penetrance than Cas9's, which is one of the most important findings of the study.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, the authors have used biochemical approaches to provide compelling evidence for the cleavage of TRMT1 by SARS-CoV-2 Nsp5 protease.<br /> This work is of wide interest to biochemists, cell biologists, and structural biologists in the coronavirus (CoV) field. Furthermore, it substantially advances the understanding of how CoV's interact with host factors during infection and modify cellular metabolism.

Strengths:<br /> The authors provide multiple lines of biochemical evidence to report a TRMT1-Nsp5 interaction during SARS-CoV-2 infection. They show that the host enzyme TRMT1 is cleaved at a specific site, and that it generates fragments that are incapable of functioning properly. This is an important result because TRMT1 is a critical player in host protein synthesis. This also advances our understanding of virus-host interactions during SARS-CoV-2 infections. Furthermore, this revised submission attempts to address the mechanistic role of TRMT1-Nsp5 interaction.

Weaknesses:<br /> The discussion on the enhanced viral infectivity upon expression of the non-cleavable TRMT1 is unclear. As presented, this is a bit contradictory to the suggested function of the TRMT1-Nsp5 interaction in diverting the host tRNA pools towards viral propagation. If the authors' model were correct, then one would expect a non-cleavable TRMT1 to inhibit viral infectivity because the virus would be unable to divert the host tRNA pools towards its propagation. I think this section needs to be written more clearly. But other than this, I have no further questions/suggestions for the authors.

Reviewer #3 (Public Review):

Human complex traits including common diseases are highly polygenic (influenced by thousands of loci). This observation is in need of an explanation. The authors of this manuscript propose a model that a competition for a single global resource (such as RNA polymerase II) may lead to a highly polygenic architecture of traits. Following an analytical examination the authors reject their hypothesis. This work is of clear interest to the field. It remains to be seen if the model covers the variety of possible competition models.

Reviewer #3 (Public Review):

The message conveyed by figure 1b is now clearer, but could still be improved. The authors explained the meaning of this figure well in their response to the reviewers: "For example, the results for CRISPR were obtained from 15 focus studies (original research) and 18 subsequent studies (papers citing focus articles). Those 15 studies identified 9,268 genes where loss-of-function changed phenotypes but, in their titles and abstracts, mentioned only 18 of those 9,268 genes. While the 9,268 hit genes have received similar research attention to the entirety of protein-coding genes, the 18 hit genes mentioned in the title or abstract are significantly more well studied. The articles citing the focus articles also only mentioned in their titles and abstracts 19 highly studied hit genes".<br /> The new Figure S8 is good.

Reviewer #3 (Public Review):

Summary:

Li et al investigated the initial target of the herbicidal caprilic acid (CAP). Using a combination of proteomic and metabolomic approaches, they generated a list of candidate targets for CAP and identified a Serine hydroxymethyl transferase (SHMT) as the best candidate.

CAP application to Conyza canadensis induces an early and brief increase in SHMT1 protein and transcript. Studies with purified recombinant CcSHMT1 indicate that enzymatic activity is inhibited by CAP. The authors suggest a kinetic mechanism of CAP inhibition but more data should be collected to reach a firm conclusion on this point.

Transgenic Arabidopsis and rice plants expressing CcSHMT1 show increased tolerance to CAP, as measured by biomass reduction 7 days after treatment with CAP. Similar results were obtained with Arabidopsis and rice plants overexpressing AtSHMT2 and OsSHMT1, respectively. OsSHMT1 single and double mutant rice plants showed increased tolerance to CAP. These results strongly link CAP tolerance to the level of SHMT, which can be manipulated by transgenesis, and suggest that engineered SHMT can also lead to higher CAP tolerance.

Finally, structural analysis allowed the identification of three residues close to the active site involved in the binding of CAP. Arabidopsis plants containing AtSHMT2 modified in these three residues are more sensitive to CAP.

Strengths:

The work of Li et al. includes a large number of assays using different methodologies. The evidence suggests that SHMT inhibition by CAP is effective in inhibiting plant growth. In addition, new technologies that manipulate SHMT levels or activity may improve crop yield by controlling weeds. Structural analysis can be the starting point for the design of more complex molecules that exceed the herbicidal activity of CAP.

Weaknesses:

The methods are rather incomplete, lacking many details necessary to fully understand the author's reasoning. It is not possible to reproduce the experiments on the basis of the information provided.

Although the conclusions are generally well supported, the results are presented in an incorrect or confusing manner. In the comparison of wild-type and transgenic plants, the control condition is missing in some experiments (Figures 4A and 5A). In some plots, the scales are not logical, making them difficult to interpret and fit into an equation (Figures 4B, 4C, 4E, 5E, 6E, 6F).

A final concern is the finding that some point mutations in the SHMT1 gene lead to more tolerant plants (Figures 6D, 6E, 6F). The authors could then explain whether this means that resistance to CAP could be easily acquired by weeds.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Wang et al. investigates the effects of B. velezensis HBXN2020 in alleviating S. Typhimurium-induced mouse colitis. The results showed that B. velezensis HBXN2020 could alleviate bacterial colitis by enhancing intestinal homeostasis (decreasing harmful bacteria and enhancing the abundance of Lactobacillus and Akkermansia) and gut barrier integrity and reducing inflammation. Overall, the manuscript is of potential interest to readers.

Strengths:<br /> B. velezensis HBXN2020 is a novel species of Bacillus that can produce a great variety of secondary metabolites and exhibit high antibacterial activity against several pathogens. B. velezensis HBXN2020 is able to form endospores and has strong anti-stress capabilities. B. velezensis HBXN2020 has a synergistic effect with other beneficial microorganisms, which can improve intestinal homeostasis.

Weaknesses:<br /> There are few studies about the clinical application of Bacillus velezensis. Thus, more studies are still needed to explore the effectiveness of Bacillus velezensis before clinical application.

Reviewer #3 (Public Review):

Summary:

Grattan and colleagues were trying to establish the neural mechanism underlying lactational infertility, in particular trying to establish the relative importance of the neurogenic effects of the suckling stimulus versus prolactin per se. They have shown that in the mouse it is rather prolactin and more specifically its action on the hypothalamic arcuate kisspeptin neuronal system, which is the key neural construct underlying gonadotrophin-releasing hormone (GnRH) pulse generation and central to the neuroendocrine control of reproduction, that mediates lactational infertility. The authors have taken a measured tone to emphasise the data pertaining to the mouse without extravagant extrapolation to humans. Nevertheless, the key findings provide a substantial foundation to facilitate interpretation of studies in other species.

Strengths:

The major strength of this study is the use of a combination of cutting-edge technologies, which of course underlie the majority of scientific advances rather than intellectual prowess favoured by the majority of scientists. Their approach avoided the major confounding effects of using pharmacological strategies to suppress prolactin action that has complicated the vast majority of previous studies. The study also provides an elegant and comprehensive contiguous description of GnRH pulse generator frequency across the ovarian cycle, through pregnancy and lactation, and into weaning in individual animals.

Weaknesses:<br /> There are no significant weaknesses.

Reviewer #3 (Public Review):

In this manuscript, Nishi et al. propose a new model to explain the previously reported myeloid-biased hematopoiesis associated with aging. Traditionally, this phenotype has been explained by the expansion of myeloid-biased hematopoietic stem cell (HSC) clones during aging. Here, the authors question this idea and show how their Hoxb5 reporter model can discriminate long-term (LT) and short-term (ST) HSC and characterized their lineage output after transplant. From these analyses, the authors conclude that changes during aging in the LT/ST HSC proportion explain the myeloid bias observed.

Although the topic is appropriate and the new model provides a new way to think about lineage-biased output observed in multiple hematopoietic contexts, some of the experimental design choices, as well as some of the conclusions drawn from the results could be substantially improved. Also, they do not propose any potential mechanism to explain this process, which reduces the potential impact and novelty of the study. Specific concerns are outlined below.

Major

(1) As a general comment, there are experimental details that are either missing or not clear. The main one is related to transplantation assays. What is the irradiation dose? The Methods sections indicates "recipient mice were lethally irradiated with single doses of 8.7 or 9.1 Gy". The only experimental schematic indicating the irradiation dose is Figure 7A, which uses 8.7 Gy. Also, although there is not a "standard", 11 Gy split in two doses is typically considered lethal irradiation, while 9.5 Gy is considered sublethal. Is there any reason for these lower doses? Same question for giving a single dose and for performing irradiation a day before transplant.

(2) The manuscript would benefit from the inclusion of references to recent studies discussing hematopoietic biases and differentiation dynamics at a single-cell level (e.g., Yamamoto et. al 2018; Rodriguez-Fraticelli et al., 2020). Also, when discussing the discrepancy between studies claiming different biases within the HSC pool, the authors mentioned that Montecino-Rodriguez et al. 2019 showed preserved lymphoid potential with age. It would be good to acknowledge that this study used busulfan as the conditioning method instead of irradiation.

(3) When representing the contribution to PB from transplanted cells, the authors show the % of each lineage within the donor-derived cells (Figures 3B-C, 5B, 6B-D, 7C-E, and S3 B-C). To have a better picture of total donor contribution, total PB and BM chimerism should be included for each transplantation assay. Also, for Figures 2C-D and Figures S2A-B, do the graphs represent 100% of the PB cells? Are there any radioresistant cells?

(4) For BM progenitor frequencies, the authors present the data as the frequency of cKit+ cells. This normalization might be misleading as changes in the proportion of cKit+ between the different experimental conditions could mask differences in these BM subpopulations. Representing this data as the frequency of BM single cells or as absolute numbers (e.g., per femur) would be valuable.

(5) Regarding Figure 1B, the authors argue that if myeloid-biased HSC clones increase with age, they should see increased frequency of all components of the myeloid differentiation pathway (CMP, GMP, MEP). This would imply that their results (no changes or reduction in these myeloid subpopulations) suggest the absence of myeloid-biased HSC clones expansion with age. This reviewer believes that differentiation dynamics within the hematopoietic hierarchy can be more complex than a cascade of sequential and compartmentalized events (e.g., accelerated differentiation at the CMP level could cause exhaustion of this compartment and explain its reduction with age and why GMP and MEP are unchanged) and these conclusions should be considered more carefully.

(6) Within the few recipients showing good donor engraftment in Figure 2C, there is a big proportion of T cells that are "amplified" upon secondary transplantation (Figure 2D). Is this expected?

(7) Do the authors have any explanation for the high level of variability within the recipients of Hoxb5+ cells in Figure 2C?

(8) Can the results from Figure 2E be interpreted as Hoxb5+ cells having a myeloid bias? (differences are more obvious/significant in neutrophils and monocytes).

(9) Is Figure 2G considering all primary recipients or only the ones that were used for secondary transplants? The second option would be a fairer comparison.

(10) When discussing the transcriptional profile of young and aged HSCs, the authors claim that genes linked to myeloid differentiation remain unchanged in the LT-HSC fraction while there are significant changes in the ST-HSCs. However, 2 out of the 4 genes shown in Figure S4B show ratios higher than 1 in LT-HSCs.

(11) When determining the lymphoid bias in ST-HSCs, the authors focus on the T-cell subtype, not considering any other any other lymphoid population. Could the authors explain this?

(12) Based on the reduced frequency of donor cells in the spleen and thymus, the authors conclude "the process of lymphoid lineage differentiation was impaired in the spleens and thymi of aged mice compared to young mice". An alternative explanation could be that differentiated cells do not successfully migrate from the bone marrow to these secondary lymphoid organs. Please consider this possibility when discussing the data.

Reviewer #3 (Public Review):

Fister and colleagues use regeneration of the larval zebrafish caudal fin to compare the effects of two modes of tissue damage-transection and burn-on cutaneous sensory axon regeneration. The authors found that restoration of sensory axon density and function is delayed following burn injury compared to transection.

The authors hypothesized that thermal injury triggers signals within the wound microenvironment that impair sensory neuron regeneration. The authors identify differences in the responses of epithelial keratinocytes to the two modes of injury: keratinocytes migrate in response to burn but not transection. Inhibiting keratinocyte migration with the small-molecule inhibitor of Arp2/3 (CK666) resulted in decreased production of reactive oxygen species (ROS) at early, but not late, time points. Preventing keratinocyte migration by wounding in isotonic media resulted in increased sensory function 24 hours after burn.

Strengths of the study include the beautiful imaging and rigorous statistical approaches used by the authors. The ability to assess both axon density and axon function during regeneration is quite powerful. The touch assay adds a unique component to the paper and strengthens the argument that burns are more damaging to sensory structures and that different treatments help to ameliorate this.

A weakness of the study is the lack of genetic and cell-autonomous manipulations. Additional comparisons between transection and burns, in particular with manipulations that specifically modulate ROS generation or cell migration without potentially confounding effects on other cell types or processes would help to strengthen the manuscript. In terms of framing their results, the authors refer to "sensory neurons" and "sensory axons" throughout the text - it should be made clear what type of neuron(s)/axon(s) are being visualized/assayed. Along these lines, a broader discussion of how burn injuries affect sensory function in other systems - and how the authors' results might inform our understanding of these injury responses - would be beneficial to the reader.

In summary, the authors have established a tractable vertebrate system to investigate different sensory axon wound healing outcomes in vivo that may ultimately allow for the identification of improved treatment strategies for human burn patients. Although the study implicates differences in keratinocyte migration and associated ROS production in sensory axon wound healing outcomes, the links between these processes could be more rigorously established.

Reviewer #3 (Public Review):

Induced pluripotent stem cells, or iPSCs, are cells that scientists can push to become new, more mature cell types like neurons. iPSCs have a high potential to transform how scientists study disease by combining precision medicine gene editing with processes known as high-content imaging and drug screening. However, there are many challenges that must be overcome to realize this overall goal. The authors of this paper solve one of these challenges: predicting cell types that might result from potentially inefficient and unpredictable differentiation protocols. These predictions can then help optimize protocols.

The authors train advanced computational algorithms to predict single-cell types directly from microscopy images. The authors also test their approach in a variety of scenarios that one may encounter in the lab, including when cells divide quickly and crowd each other in a plate. Importantly, the authors suggest that providing their algorithms with just the right amount of information beyond the cells' nuclei is the best approach to overcome issues with cell crowding.

The work provides many well-controlled experiments to support the authors' conclusions. However, there are two primary concerns: (1) The model may be relying too heavily on the background and thus technical artifacts (instead of the cells) for making CNN-based predictions, and (2) the conclusion that their nucleocentric approach (including a small area beyond the nucleus) is not well supported, and may just be better by random chance. If the authors were to address these two concerns (through additional experimentation), then the work may influence how the field performs cell profiling in the future.

Additionally, the impact of this work will be limited, given the authors do not provide a specific link to the public source code that they used to process and analyze their data.

Reviewer #3 (Public Review):

Summary:

The mechanical properties of DNA wrapped in nucleosomes affect the stability of nucleosomes and may play a role in the regulation of DNA accessibility in eukaryotes. In this manuscript, Ngo and coworkers study how the stability of a nucleosome is affected by the introduction of a CC mismatched base pair, which has been reported to increase the flexibility of DNA. Previously, the group has used a sophisticated combination of single-molecule FRET and force spectroscopy with an optical trap to show that the more flexible half of a 601 DNA segment provides for more stable wrapping as compared to the other half. Here, it is confirmed with a single-molecule cyclization essay that the introduction of a CC mismatch increases the flexibility of a DNA fragment. Consistent with the previous interpretation, it also increased the unwrapping force for the half of the 601 segment in which the CC mismatch was introduced, as measured with single-molecule FRET and force spectroscopy. Enhanced stability was found up to 56 bp into the nucleosome. The intricate role of mechanical stability of nucleosomes was further investigated by comparing force-induced unwrapping profiles of yeast and Xenopus histones. Intriguingly, asymmetric unwrapping was more pronounced for yeast histones.

Strengths:

(1) High-quality single-molecule data.

(2) Novel mechanism, potentially explaining the increased prominence of mutations near the dyads of nucleosomes.

(3) A clear mechanistic explanation of how mismatches affect nucleosome stability.

Weaknesses:

(1) Disconnect between mismatches in nucleosomes and measurements comparing Xenopus and yeast nucleosome stability.

(2) Convoluted data in cyclization experiments concerning the phasing of mismatches and biotin site.

Reviewer #3 (Public Review):

Summary:

In this study, Wang et al. have demonstrated that TMC7, a testis-enriched multipass transmembrane protein, is essential for male reproduction in mice. Tmc7 KO male mice are sterile due to reduced sperm count and abnormal sperm morphology. TMC7 co-localizes with GM130, a cis-Golgi marker, in round spermatids. The absence of TMC7 results in reduced levels of Golgi proteins, elevated abundance of ER stress markers, as well as changes of Ca2+ and pH levels in the KO testis. However, further confirmation is required because the analyses were performed with whole testis samples in spite of the differences in the germ cell composition in WT and KO testis. In addition, the causal relationships between the reported anomalies await thorough interrogation.

Strengths:<br /> The microscopic images are of great quality, all figures are properly arranged, and the entire manuscript is very easy to follow.

Weaknesses:<br /> Tmc7 KO male mice show multiple anomalies in sperm production and morphogenesis, such as reduced sperm count, abnormal sperm head, and deformed midpiece. Thus, it is confusing that the authors focused solely on impaired acrosome biogenesis. Further investigations are warranted to determine whether the abnormalities reported in this manuscript (e.g., changes in protein, Ca2+, and pH levels) are directly associated with the molecular function of TMC7 or are the byproducts of partially arrested spermiogenesis. Please find additional comments in "Recommendations for the authors".

Reviewer #3 (Public Review):

Summary:

A known feature of cilia in vertebrates and many, if not all, invertebrates is the striking heterogeneity of their lengths among different cell types. The underlying mechanisms, however, remain largely elusive. In the manuscript, the authors addressed this question from the angle of intraflagellar transport (IFT), a cilia-specific bidirectional transportation machinery essential to biogenesis, homeostasis, and functions of cilia, by using zebrafish as a model organism. They conducted a series of experiments and proposed an interesting mechanism. Furthermore, they achieved in situ live imaging of IFT in zebrafish larvae, which is a technical advance in the field.

Strengths:

The authors initially demonstrated that ectopically expressed Ift88-GFP through a certain heat-shock induction protocol fully sustained the normal development of mutant zebrafish that would otherwise be dead by 7 dpf due to the lack of this critical component of IFT-B complex. Accordingly, cilia formations were also fully restored in the tissues examined. By imaging the IFT using Ift88-GFP in the mutant fish as a marker, they unexpectedly found that both anterograde and retrograde velocities of IFT trains varied among cilia of different cell types and appeared to be positively correlated with the length of the cilia.

For insights into the possible cause(s) of the heterogeneity in IFT velocities, the authors assessed the effects of IFT kinesin Kif3b and Kif17, BBSome, and glycylation or glutamylation of axonemal tubulin on IFT and excluded their contributions. They also used a cilia-localized ATP reporter to exclude the possibility of different ciliary ATP concentrations. When they compared the size of Ift88-GFP puncta in crista cilia, which are long, and spinal cord cilia, which are relatively short, by imaging with a cutting-edge super-resolution microscope, they noticed a positive correlation between the puncta size, which presumably reflected the size of IFT trains, and the length of the cilia.

Finally, they investigated whether it is the size of IFT trains that dictates the ciliary length. They injected a low dose (0.5 ng/embryo) of ift88 MO and showed that, although such a dosage did not induce the body curvature of the zebrafish larvae, crista cilia were shorter and contained less Ift88-GFP puncta. The particle size was also reduced. These data collectively suggested mildly downregulated expression levels of Ift88-GFP. Surprisingly, they observed significant reductions in both retrograde and anterograde IFT velocities. Therefore, they proposed that longer IFT trains would facilitate faster IFT and result in longer cilia.

Weaknesses:

The current manuscript, however, contains serious flaws that markedly limit the credibility of major results and findings. Firstly, important experimental information is frequently missing, including (but not limited to) developmental stages of zebrafish larvae assayed (Figures 1, 3, and 5), how the embryos or larvae were treated to express Ift88-GFP (Figures 3-5), and descriptions on sample sizes and the number of independent experiments or larvae examined in statistical results (Figures 3-5, S3, S6). For instance, although Figure 1B appears to be the standard experimental scheme, the authors provided results from 30-hpf larvae (Figure 3) that, according to Figure 1B, are supposed to neither express Ift88-GFP nor be genotyped because both the first round of heat shock treatment and the genotyping were arranged at 48 hpf. Similarly, the results that ovl larvae containing Tg(hsp70l:ift88 GFP) (again, because the genotype is not disclosed in the manuscript, one can only deduce) display normal body curvature at 2 dpf after the injection of 0.5 ng of ift88 MO (Fig 5D) is quite confusing because the larvae should also have been negative for Ift88-GFP and thus displayed body curvature. Secondly, some inferences are more or less logically flawed. The authors tend to use negative results on specific assays to exclude all possibilities. For instance, the negative results in Figures 4A-B are not sufficient to "suggest that the variability in IFT speeds among different cilia cannot be attributed to the use of different motor proteins" because the authors have not checked dynein-2 and other IFT kinesins. In fact, in their previous publication (Zhao et al., 2012), the authors actually demonstrated that different IFT kinesins have different effects on ciliogenesis and ciliary length in different tissues. Furthermore, instead of also examining cilia affected by Kif3b or Kif17 mutation, they only examined crista cilia, which are not sensitive to the mutations. Similarly, their results in Figures 4C-G only excluded the importance of tubulin glycylation or glutamylation in IFT. Thirdly, the conclusive model is based on certain assumptions, e.g., constant IFT velocities in a given cell type. The authors, however, do not discuss other possibilities.

Reviewer #3 (Public Review):

Summary:

The authors studied the function of Hsp110 co-chaperones (e.g. yeast Sse1) in Hsp70-dependent protein disaggregation reactions. The study builds on former work by the authors (Wyszkowski et al., 2021, PNAS), analyzing the binding of Hsp70 and J-domain protein (JDP) cochaperones to protein aggregates using bio-layer interferometry (BLI). It was shown before by other groups that Hsp110 enhances Hsp70 disaggregation activity. The mechanism of Hsp110-stimulated disaggregation activity, however, remained poorly defined. Here, the authors show that yeast Hsp110 increases Hsp70 recruitment to the surface of protein aggregates. The effect is largely dependent on J-domain protein (JDP) identity and is particularly pronounced for class B JDPs (e.g. yeast Sis1), which are also more effective in disaggregation reactions. The authors also confirm former results, showing inhibition by increased Hsp110 levels, and provide novel evidence that the inhibitory effect is caused by competition between Hsp110 and JDPs for Hsp70 binding.

Strengths:

The work represents a very thoroughly executed study, which provides novel insights into the mechanism of Hsp70-mediated protein disaggregation. Key findings established for yeast chaperones are also documented for human counterparts. The observation that Hsp110 might displace JDPs from Hsp70 during the disaggregation reaction is very appealing. It will now become important to validate this initial finding and dissect how it propels the disaggregation reaction.

Weaknesses:

How exactly the interplay between JDPs and Hsp110 orchestrates protein disaggregation remains largely speculative and further analysis is required for a deeper mechanistic understanding. Enhanced recruitment of Hsp70 in the presence of Hsp110 was shown for amyloid fibrils before (Beton et al., EMBO J 2022) and should be acknowledged. The assay reporting on the refolding activity of Hsp70 seems problematic due to the high spontaneous refolding of the substrate Luciferase and should be modified.

Reviewer #3 (Public Review):

Overall:

ExoIII has been described and commercialized as a dsDNA-specific nuclease. Several lines of evidence, albeit incomplete, have indicated this may not be entirely true. Therefore, Wang et al comprehensively characterize the endonuclease and exonuclease enzymatic activities of ExoIII on ssDNA. A strength of the manuscript is the testing of popular kits that utilize ExoIII and coming up with and testing practical solutions (e.g. the addition of SSB proteins ExoIII variants such as K121A and varied assay conditions).

Comments:

(1) The footprint of ExoIII on DNA is expected to be quite a bit larger than 5-nt, see structure in manuscript reference #5. Therefore, the substrate design in Figure 1A seems inappropriate for studying the enzymatic activity and it seems likely that ExoIII would be interacting with the FAM and/or BHQ1 ends as well as the DNA. Could this cause quenching? Would this represent real ssDNA activity? Is this figure/data necessary for the manuscript?

(2) Based on the descriptions in the text, it seems there is activity with some of the other nucleases in 1C, 1F, and 1I other than ExoIII and Cas12a. Can this be plotted on a scale that allows the reader to see them relative to one other?

(3) The sequence alignment in Figure 2N and the corresponding text indicates a region of ExoIII lacking in APE1 that may be responsible for their differences in substrate specificity in regards to ssDNA. Does the mutational analysis support this hypothesis?

Reviewer #3 (Public Review):

This study aimed to explore the computational mechanisms of view invariance, driven by the observation that in some regions of monkey visual cortex, neurons show comparable responses to (1) a given face and (2) to the same face but horizontally flipped. Here they study this known phenomenon using AlexNet and other shallow neural networks, using an index for mirror symmetric viewpoint tuning based on representational similarity analyses. They find that this tuning is enhanced at fully connected- or global pooling layers (layers which combine spatial information), and that the invariance is prominent for horizontal- but not vertical- or rotational transformations. The study shows that mirror tuning can be learned when a given set of images are flipped horizontally and given the same label, but *not* if they are flipped and given different labels. They also show that networks learn this tuning by focusing on local features, not global configurations.

I found the study to be a mixed read. Some analyses were fascinating: for example, it was satisfying to see the use of well-controlled datasets to increase or decrease the rate of mirror-symmetry tuning. The insertion- and deletion¬ experiments were elegant tests to probe the mechanisms of mirror symmetry, asking if symmetry could arise from (1) global feature configurations (in a holistic sense) vs. (2) local features, with stronger evidence for the latter. These two sets of results were successful and interpretable. They stand in contrast with the first analysis, which relies on observations that do not seem justified. Specifically, Figure 2D shows mirror-symmetry tuning across 11 stages of image processing, from pixels space to fully connected layers. It shows that images from different object categories evoke considerably different tuning index values. The explanation for this result is that some categories, such as "tools," have "bilaterally symmetric structure," but this is not explicitly measured anywhere. "Boats" are described as having "front-back symmetry," more so than flowers. One imagines flowers being extremely symmetric, but perhaps that depends on the metric. What is the metric? At first I thought it was the mirror-symmetric viewpoint tuning index in the image (pixel) space, but this cannot be, as the index for faces and flowers is negative, cars have no symmetry, and boats are positive. To support these descriptions, one must have an independent variable (for object class symmetry) that can be related to the dependent variable (the mirror-symmetric viewpoint tuning index). If it exists, it is not a part of the Results section. This omission undermines other parts of the Results section: "some car models have an approximate front-back symmetry...however, a flower typically does not..." "Some," "typically:" how many in the dataset exactly, and how often? The description of CIFAR-10 as having bilaterally symmetric categories - are all these categories equally symmetric? If not, would such variability matter in terms of these results? These assessments of object category symmetry values are made before experiments are presented, so they are not interpretations of the results, and it would be circular to write it otherwise.

Overall, my bigger concern is that the framing is misleading or at best incomplete. The manuscript successfully showed that if one introduces left-right symmetry to a dataset, the network will develop population-level representations that are also bilaterally symmetric. But the study does not explain that the model's architecture and random weight distribution are sufficient for symmetry tuning to emerge, without training, just to a much more limited degree. Baek et al. showed in 2021 that viewpoint-invariant face-selective units and mirror-symmetric units emerge in untrained networks ("Face detection in untrained deep neural networks"; this current manuscript cites this paper but does not mention that mirror symmetry is a feature of the 2021 study). This current study also used untrained networks as controls (Fig. 3), and while they were useful in showing that learning boosts symmetry tuning, the results also clearly show that horizontal-reflection invariance is far from zero. So, the simple learning-driven explanation for the mirror-symmetric viewpoint tuning for faces is wrong: while (1) network training and (2) pooling are mechanisms that charge the development of mirror-symmetric tuning, the lottery ticket hypothesis is enough for its emergence. Faces and numbers are simple patterns, so the overparameterization of networks is enough to randomly create units that are tuned to these shapes and to wire many of them together. How learning shapes this process is an interesting direction, especially now that this current study has outlined its importance.

Finally, it would help to cite other previous demonstrations of equivariance and mirror symmetry in neural networks. Chris Olah, Nick Cammarata, Chelsea Voss, Ludwig Schubert, and Gabriel Goh of OpenAI wrote of this phenomenon in 2020 (Distill journal).

Some other observations that might help:

- I am enthusiastic about the experiments using different datasets to increase or decrease the rate of mirror-symmetry tuning (sets including CIFAR10, SVHN, symSVHN, asymSVHN); it is worth noting, however, that the lack of a ground truth metric for category symmetry is a problem here too. In the asymSVHN dataset, images are flipped and given different labels. If some categories are naturally symmetric after horizontal flips, such as images containing "0" or "8", then changing the label is likely to disturb training. This would explain why the training loss is larger for this condition (Figure S4D).

- It is puzzling why greyscale 3D rendered images are used. By using greyscale 3D render (at least as shown in the figures) the study proceeds as if the units are invariant under color transformations. Unfortunately, this is not true and using greyscale images impact the activations of different layers of Alexnet in a way that is not fully defined. Moreover, many units in shallow networks focus on color and exactly these units could be invariant to other transformation like the mirror symmetry, but grey scaling the images makes them inactive.

Reviewer #3 (Public Review):

It has recently been demonstrated that bacteria in biofilms show changes in membrane potential in response to changes in their environment, and that these can propagate signals through the biofilm to coordinate bacterial behavior. Akabuogu et al. contribute to this exciting research area with a study of blue light-induced membrane potential dynamics in E. coli biofilms. They demonstrate that Thioflavin-T (ThT) intensity (a proxy for membrane potential) displays multiphasic dynamics in response to blue light treatment. They additionally use genetic manipulations to implicate the potassium channel Kch in the latter part of these dynamics. Mechanosensitive ion channels may also be involved, although these channels seem to have blue light-independent effects on membrane potential as well. In addition, there are challenges to the quantitative interpretation of ThT microscopy data which require consideration. The authors then explore whether these dynamics are involved in signaling at the community level. The authors suggest that cell firing is both more coordinated when cells are clustered and happens in waves in larger, 3D biofilms; however, in both cases evidence for these claims is incomplete. The authors present two simulations to describe the ThT data. The first of these simulations, a Hodgkin-Huxley model, indicates that the data are consistent with the activity of two ion channels with different kinetics; the Kch channel mutant, which ablates a specific portion of the response curve, is consistent with this. The second model is a fire-diffuse-fire model to describe wavefront propagation of membrane potential changes in a 3D biofilm; because the wavefront data are not presented clearly, the results of this model are difficult to interpret. Finally, the authors discuss whether these membrane potential changes could be involved in generating a protective response to blue light exposure; increased death in a Kch ion channel mutant upon blue light exposure suggests that this may be the case, but a no-light control is needed to clarify this.

In a few instances, the paper is missing key control experiments that are important to the interpretation of the data. This makes it difficult to judge the meaning of some of the presented experiments.

(1) An additional control for the effects of autofluorescence is very important. The authors conduct an experiment where they treat cells with CCCP and see that Thioflavin-T (ThT) dynamics do not change over the course of the experiment. They suggest that this demonstrates that autofluorescence does not impact their measurements. However, cellular autofluorescence depends on the physiological state of the cell, which is impacted by CCCP treatment. A much simpler and more direct experiment would be to repeat the measurement in the absence of ThT or any other stain. This experiment should be performed both in the wild-type strain and in the ∆kch mutant.

(2) The effects of photobleaching should be considered. Of course, the intensity varies a lot over the course of the experiment in a way that photobleaching alone cannot explain. However, photobleaching can still contribute to the kinetics observed. Photobleaching can be assessed by changing the intensity, duration, or frequency of exposure to excitation light during the experiment. Considerations about photobleaching become particularly important when considering the effect of catalase on ThT intensity. The authors find that the decrease in ThT signal after the initial "spike" is attenuated by the addition of catalase; this is what would be predicted by catalase protecting ThT from photobleaching (indeed, catalase can be used to reduce photobleaching in time lapse imaging).

(3) It would be helpful to have a baseline of membrane potential fluctuations in the absence of the proposed stimulus (in this case, blue light). Including traces of membrane potential recorded without light present would help support the claim that these changes in membrane potential represent a blue light-specific stress response, as the authors suggest. Of course, ThT is blue, so if the excitation light for ThT is problematic for this experiment the alternative dye tetramethylrhodamine methyl ester perchlorate (TMRM) can be used instead.

(4) The effects of ThT in combination with blue light should be more carefully considered. In mitochondria, a combination of high concentrations of blue light and ThT leads to disruption of the PMF (Skates et al. 2021 BioRXiv), and similarly, ThT treatment enhances the photodynamic effects of blue light in E. coli (Bondia et al. 2021 Chemical Communications). If present in this experiment, this effect could confound the interpretation of the PMF dynamics reported in the paper.

(5) Figures 4D - E indicate that a ∆kch mutant has increased propidium iodide (PI) staining in the presence of blue light; this is interpreted to mean that Kch-mediated membrane potential dynamics help protect cells from blue light. However, Live/Dead staining results in these strains in the absence of blue light are not reported. This means that the possibility that the ∆kch mutant has a general decrease in survival (independent of any effects of blue light) cannot be ruled out.

(6) Additionally in Figures 4D - E, the interpretation of this experiment can be confounded by the fact that PI uptake can sometimes be seen in bacterial cells with high membrane potential (Kirchhoff & Cypionka 2017 J Microbial Methods); the interpretation is that high membrane potential can lead to increased PI permeability. Because the membrane potential is largely higher throughout blue light treatment in the ∆kch mutant (Fig. 3AB), this complicates the interpretation of this experiment.

Throughout the paper, many ThT intensity traces are compared, and described as "similar" or "dissimilar", without detailed discussion or a clear standard for comparison. For example, the two membrane potential curves in Fig. S1C are described as "similar" although they have very different shapes, whereas the curves in Fig. 1B and 1D are discussed in terms of their differences although they are evidently much more similar to one another. Without metrics or statistics to compare these curves, it is hard to interpret these claims. These comparative interpretations are additionally challenging because many of the figures in which average trace data are presented do not indicate standard deviation.

The differences between the TMRM and ThT curves that the authors show in Fig. S1C warrant further consideration. Some of the key features of the response in the ThT curve (on which much of the modeling work in the paper relies) are not very apparent in the TMRM data. It is not obvious to me which of these traces will be more representative of the actual underlying membrane potential dynamics.

A key claim in this paper (that dynamics of firing differ depending on whether cells are alone or in a colony) is underpinned by "time-to-first peak" analysis, but there are some challenges in interpreting these results. The authors report an average time-to-first peak of 7.34 min for the data in Figure 1B, but the average curve in Figure 1B peaks earlier than this. In Figure 1E, it appears that there are a handful of outliers in the "sparse cell" condition that likely explain this discrepancy. Either an outlier analysis should be done and the mean recomputed accordingly, or a more outlier-robust method like the median should be used instead. Then, a statistical comparison of these results will indicate whether there is a significant difference between them.

In two different 3D biofilm experiments, the authors report the propagation of wavefronts of membrane potential; I am unable to discern these wavefronts in the imaging data, and they are not clearly demonstrated by analysis.

The first data set is presented in Figures 2A, 2B, and Video S3. The images and video are very difficult to interpret because of how the images have been scaled: the center of the biofilm is highly saturated, and the zero value has also been set too high to consistently observe the single cells surrounding the biofilm. With the images scaled this way, it is very difficult to assess dynamics. The time stamps in Video S3 and on the panels in Figure 2A also do not correspond to one another although the same biofilm is shown (and the time course in 2B is also different from what is indicated in 2B). In either case, it appears that the center of the biofilm is consistently brighter than the edges, and the intensity of all cells in the biofilm increases in tandem; by eye, propagating wavefronts (either directed toward the edge or the center) are not evident to me. Increased brightness at the center of the biofilm could be explained by increased cell thickness there (as is typical in this type of biofilm). From the image legend, it is not clear whether the image presented is a single confocal slice or a projection. Even if this is a single confocal slice, in both Video S3 and Figure 2A there are regions of "haze" from out-of-focus light evident, suggesting that light from other focal planes is nonetheless present. This seems to me to be a simpler explanation for the fluorescence dynamics observed in this experiment: cells are all following the same trajectory that corresponds to that seen for single cells, and the center is brighter because of increased biofilm thickness.

The second data set is presented in Video S6B; I am similarly unable to see any wave propagation in this video. I observe only a consistent decrease in fluorescence intensity throughout the experiment that is spatially uniform (except for the bright, dynamic cells near the top; these presumably represent cells that are floating in the microfluidic and have newly arrived to the imaging region).

3D imaging data can be difficult to interpret by eye, so it would perhaps be more helpful to demonstrate these propagating wavefronts by analysis; however, such analysis is not presented in a clear way. The legend in Figure 2B mentions a "wavefront trace", but there is no position information included - this trace instead seems to represent the average intensity trace of all cells. To demonstrate the propagation of a wavefront, this analysis should be shown for different subpopulations of cells at different positions from the center of the biofilm. Data is shown in Figure 8 that reflects the velocity of the wavefront as a function of biofilm position; however, because the wavefronts themselves are not evident in the data, it is difficult to interpret this analysis. The methods section additionally does not contain sufficient information about what these velocities represent and how they are calculated. Because of this, it is difficult for me to evaluate the section of the paper pertaining to wave propagation and the predicted biofilm critical size.

There are some instances in the paper where claims are made that do not have data shown or are not evident in the cited data:

(1) In the first results section, "When CCCP was added, we observed a fast efflux of ions in all cells"- the data figure pertaining to this experiment is in Fig. S1E, which does not show any ion efflux. The methods section does not mention how ion efflux was measured during CCCP treatment.

(2) In the discussion of voltage-gated calcium channels, the authors refer to "spiking events", but these are not obvious in Figure S3E. Although the fluorescence intensity changes over time, it's hard to distinguish these fluctuations from measurement noise; a no-light control could help clarify this.

(3) The authors state that the membrane potential dynamics simulated in Figure 7B are similar to those observed in 3D biofilms in Fig. S4B; however, the second peak is not clearly evident in Fig. S4B and it looks very different for the mature biofilm data reported in Fig. 2. I have some additional confusion about this data specifically: in the intensity trace shown in Fig. S4B, the intensity in the second frame is much higher than the first; this is not evident in Video S6B, in which the highest intensity is in the first frame at time 0. Similarly, the graph indicates that the intensity at 60 minutes is higher than the intensity at 4 minutes, but this is not the case in Fig. S4A or Video S6B.

Reviewer #3 (Public Review):

The authors present in this study the characterization of two mutant lines of the filasterean Capsaspora owczarzaki, a unicellular holozoan with a key phylogenetic position to understand multicellular development in animals. The present study is built on a previous work from the same research group, on the mutant of the orthologue of the Yorki gene in C. owczarzaki. By knocking out the two upstream kinases of the same pathway, coHpo-/- and coWts-/-, in single cell and aggregates of C. owkzarzaki, they now have mutated the entire pathway and in different cellular contexts.

The authors obtain results in the same direction as the previous work, demonstrating that the Hippo pathway of the unicellular holozoan C. owczarzaki, is not involved in the control of cell proliferation but is related with cytoskeletal dynamics through the actin-myosin mechanism.

In this revised version of the study, the authors have addressed my concerns by providing additional experiments, references and discussing further the points of controversy.

I think the authors have done a great job improving the robustness of the paper proving further some of the claims raised in the previous version of the manuscript.

Reviewer #3 (Public Review):

Summary:

The manuscript examined the role of large versus small prediction errors (PEs) in creating a state-based memory distinction between acquisition and extinction. The premise of the paper is based on theoretical claims and empirical findings that gradual changes between acquisition and extinction would lead to the potential overwriting of the acquisition memory with extinction, resulting in a more durable reduction in conditioned responding (i.e. more durable extinction effect). The paper tests the hypotheses in a series of elegant experiments in which the shock intensity is decreased across extinction sessions before non-reinforced CS presentations are given. Additional manipulations include context change, shock devaluation, and controlling for lower shock intensity exposure. The critical comparison was standard non-reinforced extinction training. The critical tests were done in spontaneous recovery and reinstatement.

Strengths:<br /> The findings are of tremendous importance in understanding how memories can be updated and reveal a well-defined role of PE in this process. It is well-established that PE is critical for learning, so delineating how PE is critical for generating memory states and the role it serves in keeping memories dissociable (or not) is exciting and clever. As such the paper addresses a fundamental question in the field.

The studies test clear and defined predictions derived from simulations of the state-belief model of Cochran & Cisler (2019). The designs are excellent: well-controlled and address the question.

The authors have done an excellent job of explaining the value of the latent state models.

The authors have studied both sexes in the study presented, providing generality across the sexes in their findings. However, depicting the individual data points in the bar graphs and noting which data represent males and which represent females would be of great value.

Weaknesses:

(1) While it seems obvious that delivering a lower intensity shock will generate a smaller PE than say no shock, it would have been nice to see data from say a compound testing procedure that confirms this.

(2) The devaluation experiment is quite clever, but it also would be strengthened if there was evidence in the paper that this procedure does indeed lead to shock devaluation.

(3) It would have been very exciting to see even more parametric examinations of this idea, like maintaining shock intensity but gradually reducing shock duration, which would have increased the impact of the paper.

(4) Individual data points should be represented in the test figures (see above also).

Reviewer #3 (Public Review):

Summary:<br /> This manuscript describes the symptoms of patients harboring KCNQ2 mutation G256W, functional changes of the mutant channel in exogenous expression, and phenotypes of G256W/+ mice. The patients presented seizures, the mutation reduced currents of the channel, and the G256W/+ mice showed seizures, increased firing frequency in neurons, reduced KCNQ2 expression,<br /> and altered subcellular distribution.

Strengths:

This is a large amount of work and all results corroborated the pathogenicity of the mutation in KCNQ2, providing an interesting example of KCNQ2-associated neurological disorder's impact on functions at all levels including molecular, cellular, tissue, animal model, and patients.

Weaknesses:

The manuscript described observations of changes in association with the mutation at molecular cellular functions and animal phenotype, but the results in some aspects are not as strong as in others. Nevertheless, the manuscript made overarching conclusions even when the evidence was not sufficiently strong.

yeah. but the actual problem is pacifism and overpopulation.<br /> all other problems, including world wars, are only symptoms of pacifism and overpopulation.<br /> this is just another intelligence test, and again, most people fail, most people are idiots

Reviewer #3 (Public Review):

Stout et al investigate the link between prefrontal-hippocampal (PFC-HPC) theta-band coherence and accurate decisions in spatial decision making tasks. Previous studies show that PFC-HPC theta coherence positively correlates with task learning and correct decisions but the nature of this relation relies on correlations that cannot show whether coherence leads, coincides or is a consequence of decision making. To investigate more precisely this link, the authors devise a novel paradigm. In this paradigm the rat is blocked during a delay period preceding its choice and they control on a trial-by-trial basis the level of PFC-HPC theta coherence prior to the decision by allowing the rat to access the choice point only at a time when coherence reaches above or below a threshold. The design of the paradigm is very well controlled in many ways. First, using the PFC-HPC theta coherence during the delay period to gate when the rat accesses the choice zone clearly separates this coherence from the behavioural decision itself. Moreover, the behaviour of the animal is similar during high and low coherence periods. Finally, control trials are matched trial-by-trial to the time spent waiting by the rat when gated on theta coherence, which is crucial given that working memory performance depends on delay duration. All these features bolster the specificity of the author's main finding which is that PFC-HPC theta coherence prior to choice making is strongly predictive of accuracy in two tasks : one that requires working memory and another in which behaviour is cue-guided. Although this paradigm does not provide direct causal evidence, it convincingly supports the idea that PFC-HPC theta coherence prior to the behavioural decision is related to correct decision making and is not simply temporally coincidental or a consequence of the decision output.

The authors also investigate the mechanisms behind the increase in PFC-HPC coherence during the task and show that it likely involves the recruitment of a small population of PFC neurons, via interactions with the Ventral Midline Thalamus that could mediate prefrontal/hippocampal dialogue.

A key point of interest is the unexpected result showing a link between theta coherence even in the cue-driven version of the task. As the authors point out, muscimol inhibition of neither PFC nor HPC, nor the ventral midline thalamus impacts performance in this task. This raises the question of why coherence between two areas is predictive of choice accuracy when neither area appears to be causally involved. The authors discuss various options and explanations for this discrepancy which clearly adds to the current debate. Moreover their novel paradigm provides new tools to interrogate when inter-area synchrony is associated with information transfer and when this information is then used to drive behavioural decisions.

"we have been winning battles... but losing the war

big environmentalist shifting toward anti-capitalism

Reviewer #3 (Public Review):

Summary and strengths:

In the manuscript, Abd El Hay et al investigate the role of thermally sensitive ion channels TRPM2 and TRPV1 in warm preference and their dynamic response features to thermal stimulation. They develop a novel thermal preference task, where both the floor and air temperature are controlled, and conclude that mice likely integrate floor with air temperature to form a thermal preference. They go on to use knockout mice and show that TRPM2-/- mice play a role in the avoidance of warmer temperatures. Using a new approach for culturing DRG neurons they show the involvement of both channels in warm responsiveness and dynamics. This is an interesting study with novel methods that generate important new information on the different roles of TRPV1 and TRPM2 on thermal behavior.

Open questions and weaknesses:

(1) Differences in the response features of cells expressing TRPM2 and TRPV1 are central and interesting findings but need further validation (Figures 3 and 4). To show differences in the dynamics and the amplitude of responses across different lines and stimulus amplitudes more clearly, the authors should show the grand average population calcium response from all responsive neurons with error bars for all 3 groups for the different amplitudes of stimuli (as has been presented for the thermal stimuli traces). The authors should also provide a population analysis of the amplitude of the responses in all groups to all stimulus amplitudes. Prior work suggests that thermal detection is supported by an enhancement or suppression of the ongoing activity of sensory fibers innervating the skin. The authors should present any data on cells with ongoing activity.

(2) The authors should better place their findings in context with the literature and highlight the novelty of their findings. The introduction builds a story of a 'disconnect' or 'contradictory' findings about the role of TRPV1 and TRPM2 in warm detection. While there are some disparate findings in the literature, Tan and McNaughton (2016) show a role for TRPM2 in the avoidance of warmth in a similar task, Paricio et al. (2020) show a significant reduction in warm perception in TRPM2 and TRPV1 knock out lines and Yarmolinksy et al. (2016) show a reduction in warm perception with TRPV1 inactivation. All these papers are therefore in agreement with the authors finding of a role for these channels in warm behavior. The authors should change their introduction and discussion to more correctly discuss the findings of these studies and to better pinpoint the novelty of their own work.

(3) The responses of 60 randomly selected cells are shown in Figure 2B. But, looking at the TRPM2-/- data, warm responses appear more obvious than in WTs and the weaker responders of the WT group appear weaker than the equivalent group in the TRPV1-/- and TRPM2-/- data. This does not necessarily invalidate the results, but it may suggest a problem in the data selection. Because the correct classification of warm-sensitive neurons is central to this part of the study more validation of the classifier should be presented. For example, the authors could state if they trained the classifier using equal amounts of cells, show some randomly selected cells that are warm-insensitive for all genotypes, and show the population average responses of warm-insensitive neurons.

(4) The interpretation of the main behavioral results and justification of the last figure is presented as the result of changes in sensing but differences in this behavior could be due to many factors and this needs clarification and discussion. (i) The authors mention that 'crucially temperature perception is not static' and suggest that there are fluctuating changes in perception over time and conclude that their modelling approach helps show changes in temperature detection. They imply that temperature perceptual threshold changes over time, but the mouse could just as easily have had exactly the same threshold throughout the task but their motivation (or some other cognitive variable) might vary causing them to change chamber. The authors should correct this. (ii) Likewise, from their fascinating and high-profile prior work the authors suggest a model of internal temperature sensing whereby TRPM2 expression in the hypothalamus acts as an internal sensory of body temperature. Given this, and the slow time course of the behavior in chambers with different ambient temperatures, couldn't the reason for the behavioral differences be due to central changes in hypothalamic processing rather than detection by skin temperature? If TRPM2-/- were selectively ablated from the skin or the hypothalamus (these experiments are not necessary for this paper) it might be possible to conclude whether sensation or body temperature is more likely the root cause of these effects but, without further experiments it is tough to conclude either way. (iii) Because the ambient temperature is controlled in this behavior, another hypothesis is that warm avoidance could be due to negative valence associated with breathing warm air, i.e. a result of sensation within the body in internal pathways, rather than sensing from the external skin. Overall, the authors should tone down conclusions about sensation and present a more detailed discussion of these points.

(5) It is an excellent idea to present a more in-depth analysis of the behavioral data collected during the preference task, beyond 'the mouse is on one side or the other'. However, the drift-diffusion approach is complex to interpret from the text in the results and the figures. The results text is not completely clear on which behavioral parameters are analyzed and terms like drift, noise, estimate, and evidence are not clearly defined. Currently, this section of the paper slightly confuses and takes the paper away from the central findings about dynamics and behavioral differences. It seems like they could come to similar conclusions with simpler analysis and simpler figures.

(6) In Figure 2D the % of warm-sensitive neurons are shown for each genotype. Each data point is a field of view, however, reading the figure legend there appear to be more FOVs than data points (eg 10 data points for the TRPV1-/- but 17 FOVs). The authors should check this.

(7) Can the authors comment on why animals with over-expression of TRPV1 spend more time in the warmest chamber to start with at 38C and not at 34C?