Reviewer #3 (Public review):

Summary:

In this manuscript, the investigators identified LMOD1 as one of a subset of cytoskeletal proteins that levels increase in early stages of myogenic differentiation. Lmod1 is understudied in striated muscle and in particular in myogenic differentiation. Thus, this is an important study. It is also a very thorough study, with perhaps even too much data presented. Importantly, the investigators observed that LMOD1 appears to be important for skeletal regeneration, myogenic differentiation and that it interacts with SIRT1. Both primary myoblast differentiation and skeletal muscle regeneration were studied. Rescue experiments confirmed these observations: SIRT1 can rescue perturbations of myogenic differentiation as a result of LMOD1 knockdown.

Strengths:

Particular strengths include: an important topic, the use of primary skeletal cultures, the use of both cell culture and in vivo approaches, careful biomarker analysis of primary mouse myoblast differentiation, the use of two methods to probe the function of the Lmod1/SIRT1 pathway via using depletion approaches and inhibitors, and the generation of six independent myoblast cultures. Results support their conclusions.

Weaknesses:

(1) Figure 1. Images of cells in Figure 1A are too small to be meaningful (especially in comparison to the other data presented in this figure). Perhaps make graphs smaller?

(2) Line 148 "We found LMOD2 to be the most abundant Lmod in whole skeletal muscle." This is confusing since most, if not all, prior studies have shown that Lmod3 is the predominant isoform in skeletal muscle. The two papers that are cited are incorrectly cited. Clarification to resolve this discrepancy is needed.

(3) Figure 2. Immunofluorescence (IF) panels are too small to be meaningful. Perhaps the graphs could be made smaller and more space allocated for the IF panels? This issue is apparent for just about all IF panels - they are simply too small to be meaningful. Additionally, in many of the immunofluorescence figures, the colors that were used make it difficult to discern the stained cellular structures. For example, in Figure S1, orange and purple are used - they do not stand out as well as other colors that are more commonly used.

(4) There is huge variability in many experiments presented - as such, more samples appear to be required to allow for meaningful data to be obtained. For example, Figure S2. Many experimental groups, only have 3 samples - this is highly problematic - I would estimate that 5-6 would be the minimum.

(5) Ponceau S staining is often used as a loading control in this manuscript for western blots. The area/molecular weight range actually used should be specified. Not clear why in some experiments GAPDH staining is used, in other experiments Ponceau S staining is used, and in some, both are used. In some experiments the variability of total protein loaded from lane-to-lane is disconcerting. For example, in Figure S4C there appears to be more than normal variability. Can the protein assay be redone and the samples run again?

(6) Figure S3 - Lmod3 is included in the figure but no mention of it occurs in the title of the figure and/or legend.

(7) Abstract, line 25. "overexpression accelerates and improves the formation of myotubes". This is a confusing sentence. How is it improving the formation? A little more information about how they are different than developing myotubes in normal/healthy muscle would be helpful

(8) Impossible from IF figures presented to determine where Lmod1 localizes in the myocytes. Information on its subcellular localization is important. Does it localize with Lmod2 and Lmod3 at thin filament pointed ends?

Comments on revisions:

Many comments have been adequately addressed. However, some concerns remain.

Former Concern #2. The issue with the lack of detection of LMOD3 in their muscle samples is troublesome and has not been adequately resolved in the revised manuscript. It is a fact that most, if not all, studies on Lmod3 report that it is the most abundant isoform in skeletal muscle. This issue should be discussed in the manuscript. It is recognized that a different assay was utilized in this paper. The papers that are cited continue to remain incorrect. Specifically:

Tsukada et al., reports abundance of LMOD2 in cardiac muscle, not in skeletal muscle.

Nworu et al., 2015 reports on LMOD3 in skeletal muscle.

Kiss et al.,2020. While this paper reveals an important function for Lmod2 in thin filament length regulation, it is clearly shows many examples of high expression of Lmod3 in various skeletal muscles isolated from mice.

Former Concern #3. With respect to small sample numbers. Hopefully a statistical editor is available to comment. While this reviewer is happy that other assays were used to verify their data, the problem still remains that many experimental groups only have 3 samples (with high variability).

Former Concern #3. Many immunofluorescence panels are hard to evaluate because of their small size.

Reviewer #4 (Public review):

Summary and background:

This report entitled "The insulin/IGF axis is critically important (for) controlling gene transcription in the podocyte" from Hurcombe et al is based on a mouse double knockdown of the IR and IGF1R and a parallel cultured mouse podocyte model. Insulin/IGF signaling system in mammals evolved as three gene reduplicated peptides (insulin, IGF-1, and IGF-2) and their two receptors IR and IGF1R that cross-react to variable extents with the peptides, are ubiquitously expressed, and signal through parallel pathways. The major downstream effect of insulin is to regulate glucose uptake and metabolism, while that of the IGF pathways is to regulate growth and cell cycling in part through mTORC1. The GH-IGF-1-IGF1R pathway regulates post-natal growth. IGF-2 signaling is thought to play a major role in regulating intrauterine growth and development, although IGF-2 is also present at high levels in post-natal life. Thus, one would anticipate that reducing IR/IGF1R signaling in any cell would slow growth and cell cycling by reducing growth factor and metabolic mTORC1-mediated and other processes including the splicing of RNA for protein synthesis.

Mouse IR/IGF1R double knockdown model:

A double knockdown mouse model was generated by interbreeding mice with different genetic backgrounds carrying floxed sites for IR and IGF-1R to produce mixed background offspring with both floxed IR and IGF-1R genes. These mice were crossed so that the podocin promoter driven-Cre (that comes on at about embryonic day 12 bas podocytes are developing) would delete IR and IGF-1R genes. Since podocin is believed to be an absolutely podocyte-specific protein, this podocin promoter this is predicted to specifically knock down the IR and IGF1R genes only in podocytes. The weight and growth of double KO offspring was not different from controls, but some proportion of the double knockdown mice subsequently developed proteinuria by 6 months and 20% died, although no specific data is provided to identify the cause of the deaths since eGFR was not decreased. Surviving mice were evaluated at 6 months of age. The efficacy of knockdown was not demonstrated in the mouse model itself, although a temperature-sensitive cell line developed from these double knockdown mice showed that expression of IR and IGF-1R proteins in the Cre-treated cell line were both reduced by about 50% (no statistical analysis of this result provided). In the knockout mice, proteinuria was significantly increased by 6 months, but not at earlier time points. Histologic analysis showed proteinaceous casts, glomerulosclerosis and interstitial fibrosis. Podocyte number was stated to be reduced by about 30% in double knockdown mice, although the method by which this was evaluated seems to have been by counting WT1 positive nuclei in glomerular cross-sections, an approach that is well-known not to be a reliable way of assessing true podocyte number. No information is provided about podocyte size, density or glomerular volume.

Comment: If IR/IGF1R deletion plays a significant role in normal podocyte function sufficient to cause proteinuria and glomerulosclerosis then the effect of reduced IR and IGF1R protein expression on podocyte function would have been expected to produce a phenotype before 6 months. A more likely scenario to explain the overall result is that deleting the IR and IGF1R genes at about embryonic day12 impacted podocyte development to a variable extent such that some mice developed fewer podocytes per glomerulus than other mice. As mice grow and their glomeruli and glomerular capillary area increases, those mice with fewer podocytes would not be able to completely cover the filtration surface with foot processes and would develop proteinuria and glomerulosclerosis. If reduced podocyte number per glomerulus is the proximate cause of the observed proteinuria, then modulation of the body and kidney growth rate by calorie restriction to slow growth (lower circulating IGF-1 levels) would be expected to be protective, while a high protein high calorie diet (higher circulating IGF-1 levels) or uni-nephrectomy to increase kidney growth rate would be expected to enhance proteinuria and glomerulosclerosis.

The model as used may be more representative of a variable degree of podocyte depletion than an effect of impaired IR/IGF1R signaling. Therefore, although the phenotype may be ultimately attributable to the IR/IGF1R gene deletions the proteinuria and glomerulosclerotic phenotype itself was probably a consequence of defective podocyte development. Examining podocyte number, size, density and glomerular volume at earlier time points (4 weeks) would help to answer this question. Therefore, a more appropriate title would be "The insulin/IGF axis is critically important (for) normal podocyte development and deployment". In this context the effect of the knockdowns on splicing would make more sense.

Cell culture studies. A cell line was generated using a temperature sensitive SV40 system that has been previously reported from this laboratory. A detailed analysis is provided to show that double knockout cells exhibited abnormal spliceosome activity. This forms the basis for the conclusion that "The insulin/IGF axis is critically important (for) controlling gene transcription in the podocyte". There are several concerns that weaken this conclusion.

(1) In the double knockdown cell culture system about 30% of cells were "lost" by 3 days and about 70% of cells were "lost" by 5days. The studies were done at the 3 day time point. It is not clear whether "lost" cells were in the process of dying, stress-induced detachment, or just growing more slowly than control due to reduced IR and IGF-1R signaling. These processes could have impacted splicing in a non-specific way independent of IR/IGF1R signaling itself.

(2) Can a single cell line derived from the double floxed mice be relied on to provide an unbiased picture of the effect of deleting IR and IGF-1R? Presumably, the transfection and selection process will select for cells that survive thereby including unknown biases, possibly related to spliceosome function. Is a single cell line adequate? These investigators have extensive experience with this type of analysis, but this question is not addressed in the discussion.

(3) To determine whether the effect is specific to reduced IR/IGFR signaling the deletion of IR and IGF-1R could be corrected by transfecting full length IR and IGF-1R cDNAs into the cells to restore normal IR/IGF1R signaling. If transfected cells with intact IR and IGF-1R expression and activity returns spliceosome activity to normal this would be evidence that receptors themselves play some role in spliceosome activity, as opposed to the downstream effect on growth limitation/stress on the cells.

(4) Other ways of testing whether the splicing effect is specifically due to reduced IR/IGF-1R signaling would be to (a) block IR and IGF1R receptors using available inhibitors, (b) remove or reduce insulin, IGF-1 and IGF-2 levels in the culture medium, (c) use low glucose and amino acid culture medium to slow growth rate independent of receptor function, (d) or block intra-cellular signaling via the IR and IGF-1R receptors through mTORC1 inhibition using rapamycin or other signaling targets.

(5) It would be useful to determine whether the cultured cells stressed in other ways (e.g. ischemia, toxins, etc.) also results in the same splicing abnormalities.

Reviewer #3 (Public review):

Summary:

Shen et al. investigate the contribution of the type VI secretion system of Bacteroidales in the gut microbiome assembly and targeting of closely related species. They demonstrate that B. acidifaciens relies on T6SS-mediated antagonism to prevent displacement by co-resident Bacteroidales and other members of the microbiome, allowing B. acidifaciens to persist in the gut.

Strengths:

Using a gnotobiotic model colonized with a wild-mouse microbiome is a significant strength of this study. This approach allows tracking of microbiome changes over time and directly examining targeting by Bacteroidales carrying T6SS in a more natural setting. The development of ICE-seq for mapping the distribution of the T6SS in the microbiome is remarkable, enabling the study of how this bacterial weapon is transferred between microbiome members without requiring long-read metagenomics methods.

Weaknesses:

Some conclusions are based on only four mice per condition. The author should consider increasing the sample size.

Overall, the authors successfully achieved their objectives, and their experimental design and results support their findings. As mentioned in the discussion, it would be important to investigate the role of the T6SS in resilience to disturbances in the microbiome, such as antibiotics, diet, or pathogen invasion. This work represents a step forward in understanding how contact-dependent competition influences the gut microbiome in relevant ecological contexts.

Reviewer #3 (Public review):

Summary:

It is well established that poly(A) tails at the 3' end of mRNA are critical for mRNA stability, providing another layer of gene regulation. TENT5A is one of the non-canonical poly(A) polymerases that add an extra poly(A) tail. This manuscript demonstrates that the Tent5A mutation leads to mineralization abnormalities in the tooth, shorter poly(A) tails in amelogenin mRNA and some other selected mRNAs, and provides a list of TENT5A interacting proteins.

Strengths:

(1) The authors show in vivo genetic evidence that Tent5a is critical for normal tooth mineralization.

(2) The authors show that the length of the poly(A) tail in amelogenin (AmelX) is 13 bases shorter in Tent5a mutants but not in other mRNAs, such as ameloblastin (Ambn).

(3) Differentially expressed genes (DEGs) in Tent5A mutant tissues (cervical loop) are identified, and some of them show different lengths of poly(A) tails.

(4) TENT5A interacting proteins are identified. Together with the DEGs, these datasets will provide valuable research tools to the community.

Weaknesses:

(1) There is no direct evidence to support the main conclusion; the length of the poly(A) tail is critical for normal tooth mineralization.

(2) The RNAseq data to identify TENT5A substrate is based on the assumption that shorter poly(A) tailed RNA is less stable. However, there are multiple reasons for the differential expression of RNA in Tent5A mutant tissues.

(3) Several TENT5A-interacting proteins have been identified, but, beyond their colocalization with a target mRNA, no mechanistic studies have been conducted.

DOI: 10.1016/j.celrep.2026.116971

Resource: (ZFIN Cat# ZDB-ALT-120723-3,RRID:ZFIN_ZDB-ALT-120723-3)

Curator: @areedewitt04

SciCrunch record: RRID:ZFIN_ZDB-ALT-120723-3

What is this?

Reviewer #3 (Public review):

Summary:

Fibroblast growth factor receptor 2 (FGFR2) is a receptor tyrosine kinase that can be amplified in gastric cancer and serves as a potential therapeutic target for this patient population. However, targeting FGFR2 has shown limited efficacy. Thus, this study seeks to identify additional molecules that can be effectively targeted in FGFR2 amplified gastric cancer, with a focus on Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2). The authors first demonstrate that 6% of gastric cancer patients in a cohort of human patient samples exhibit FGFR2 amplification. Furthermore, they demonstrate that FGFR2 mRNA expression is positively correlated with PTPN11 gene expression (which is the gene that encodes the SHP2 protein). Using human gastric cancer cell lines with amplified FGFR2, the authors then test the effects of combining the FGFR inhibitor AZD4547 with the SHP2 inhibitor SHP099 on tumor cell death and signaling molecules. They demonstrate that combining the two inhibitors is more effective at tumor cell killing and reducing activation of downstream signaling pathways than either inhibitor alone. In further studies, the authors obtained gastric cancer cells with FGFR2 amplification from a patient that was treated with FGFR2 inhibitor. While this patient initially showed a partial response, the patient ultimately progressed, demonstrating resistance to FGFR2 inhibition. Following isolation of tumor cells from the patient's ascites, the authors demonstrate that these cells are sensitive to the combination treatment of AZD4547 and SHP099. Further studies were performed using a xenograft model using athymic nude mice in which the combination of SHP099 and AZD4547 were found to reduce tumor growth more significantly than either treatment alone. Finally, the authors demonstrate using an in vitro culture model that this combination treatment enhances T cell mediated cytotoxicity. The authors conclude that targeting FGFR2 and SHP2 represents a potential combination strategy in gastric patients with FGFR2 amplification.

Strengths:

The authors demonstrate that FGFR2 amplification positively correlates with PTPN11 in human gastric cancer samples, providing a rationale for combination therapies. Furthermore, convincing data are provided demonstrating that targeting both FGFR and SHP2 is more effective than targeting either pathway alone using in vitro and in vivo models. The use of cells derived from a gastric cancer patient that progressed following treatment with an FGFR inhibitor is also a strength. The findings from this study support the conclusion that SHP2 inhibitors enhance the efficacy of FGFR-targeted therapies in cancer patients. This study also suggests that targeting SHP2 may also be an effective strategy for targeting cancers that are resistant to FGFR-targeted therapies.

Weaknesses:

The main caveat with these studies is the lack of an immune competent model with which to test the finding that this combination therapy enhances T cell cytotoxicity in vivo.

Reviewer #3 (Public review):

Summary:

In this manuscript titled "Mycobacterial Metallophosphatase MmpE Acts as a Nucleomodulin to Regulate Host Gene Expression and Promote Intracellular Survival", Chen et al describe biochemical characterisation, localisation and potential functions of the gene using a genetic approach in M. bovis BCG and perform macrophage and mice infections to understand the roles of this potentially secreted protein in the host cell nucleus. The findings demonstrate the role of a secreted phosphatase of M. bovis BCG in shaping the transcriptional profile of infected macrophages, potentially through nuclear localisation and direct binding to transcriptional start sites, thereby regulating the inflammatory response to infection.

Strengths:

The authors demonstrate using a transient transfection method that MmpE when expressed as a GFP-tagged protein in HEK293T cells, exhibits nuclear localisation. The authors identify two NLS motifs that together are required for nuclear localisation of the protein. A deletion of the gene in M. bovis BCG results in poorer survival compared to the wild type parent strain, which is also killed by macrophages. Relative to the WT strain infected macrophages, macrophages infected with the mmpE strain exhibited differential gene expression. Overexpression of the gene in HEK293T led to occupancy of the transcription start site of several genes, including the Vitamin D Receptor. Expression of VDR in THP1 macrophages was lower in case of mmpE infection compared to WT infection. This data supports the utility of the overexpression system in identifying potential target loci of MmpE using the HEK293T transfection model. The authors also demonstrate that the protein is a phosphatase and the phosphatase activity of the protein is partially required for bacterial survival but not for regulation of the VDR gene expression.

Weaknesses:

There are significant differences in lysosomal retention between M. tuberculosis and M. bovis BCG. This study uses BCG and MMPE overexpression to draw conclusions about the impact of the MMPE gene on host gene expression and the bacteria's lysosomal localisation. While the authors have convincingly supported their claims with this model system, the relevance of this mechanism in M. tuberculosis infection remains unaddressed.

Reviewer #3 (Public review):

Summary:

Bhattacharya et al. describe significant differences in prey capture behaviour in PSD-95 KO (Knockout) and wild-type (WT) mice. This work develops logically from their previous findings that KO of PSD-95 inhibits the maturation in the primary visual cortex. However, their previous work revealed that the visual deficits in the KO mice were relatively modest. Here, by employing an ethologically-relevant behavioural task, they show that several aspects of prey capture are impaired in the KO. Importantly, the deficits in predatory behavior in the KO mouse improved with monocular deprivation, consistent with deficits in binocular vision.

Strengths:

Overall, the data presented are convincing and valuable, and support the idea that PSD-95 expression is important for the maturation of visual responses.

Weaknesses:

The manuscript could be strengthened by consideration of the following points:

(1) The deficits in predatory behavior are interpreted to reveal several possible visual defects, including the absence of binocularity, binocular summation, or binocular mismatch in V1 neurons. Yet this is done with insufficient detail about each possible mechanism and without direct neuronal evidence.

(2) The observation that binocular visual field bias is intact in the PSD-95 KO mice is interesting but appears to contradict other data suggesting the absence of binocularity in the KO visual system, and this is not discussed in sufficient detail.

(3) No consideration of previous work using constitutive PSD-95 KOs that documented a learning deficit.

(4) Throughout the manuscript, including the first paragraph of the discussion, the authors state that "This study explored whether the maturation of CP closure, inhibited by PSD-95 influences binocular visual behaviour". However, if this were the case, the current experiments would have compared cricket capture behavior at two ages across the two genotypes: pre- and post-CP closure in WTs and at matching chronological ages in KOs.

(5) Freeman and others have shown that the influence of binocular summation on orientation discrimination is highest at low stimulus contrast and short duration stimuli. How does this impact the interpretation of predatory behavior and discrimination in the VWT?

Reviewer #3 (Public review):

Summary:

The LC3 family of proteins, which includes LC3B, are ubiquitin-like proteins that are covalently linked to phosphatidylethanolamine in the expanding autophagosomal membrane during autophagy. LC3 family members bind to short sequences of amino acids that reside within dynamic regions in a wide variety of proteins. These sequences, termed LC3 Interacting Regions (LIRs), were initially thought to function primarily to link LIR-containing autophagy cargo receptors to LC3 family members to help facilitate their capture during autophagy. However, the functional importance of LIRs in autophagy has broadened to include more general functions in autophagy as well. While a general consensus for LIR sequences has been described as [FWY]0-X1-X2-[LVI]3, recent work has suggested that additional sequences outside of the canonical LIR sequence can bind LC3 family members and play important roles in autophagy. In this manuscript by Kosmatka et al, the authors perform a high-throughput screen using bacterial surface display coupled with fluorescence-associated cell sorting to identify which human sequences can bind to LC3B. They identify a variety of peptides capable of binding LC3B, including peptides from proteins that have not previously been described as LC3B-binding proteins. The results from the bacterial surface display were then used to guide sequence analysis, mutational analysis, and structural studies to further characterize the range of LIR sequences that are capable of binding LC3B. Taken together, this work adds to the growing knowledge of how LIR sequences interact with LC3 family members and demonstrates which amino acids both inside and outside of the LIR sequence aid in binding. This work also identifies new potential LC3 binding proteins, which may play unknown roles in autophagy regulation. Lastly, this work reinforces the importance of alternative LIR sequences such as the [WFY]0-X1-X2-[WFY]3 sequence, which the authors have dubbed the LIR+ sequence.

Strengths:

The manuscript uses a robust approach to identify and characterize different peptide sequences that can interact with LC3B. They validate a large number of sequences using biolayer interferometry (BLI) and attempt to correlate different amino acids with their binding affinity for LC3B. The large number of LC3B binding sequences and their dissociation constants adds significant new information to the field that will help others understand what sequences can bind to LC3B. The authors are also very careful to accurately report on their data and not overly interpret their findings.

Weaknesses:

After the authors identify proteins from their bacterial display assay, the remainder of the manuscript is focused on characterizing the different types of sequences that are identified in addition to validating the LC3B-LIR interactions using biochemical approaches, including BLI and X-ray crystallography. However, it's not entirely clear if the screen identified novel LC3B binders that interact with LC3B in cells. While I acknowledge that the focus of the manuscript is on the characterization of LIR sequences that can bind LC3B, it seems like a missed opportunity not to validate a few of the novel LC3B binders in vivo. This may result in the demonstration of novel binders of LC3B in cells and may further demonstrate the strength of this approach for identifying LC3 family member binding partners. Therefore, it would be helpful to look at a few proteins identified in the HC set that have not previously been identified as LC3B binders in cells to determine if they CO-IP with LC3B or interact with LC3B using a different approach.

Reviewer #3 (Public review):

In this manuscript, the authors examine the role of Syt7 in the plasticity of synchronous and asynchronous release in cultured neurons. The experimental approach is the imaging of SF-iGluSnFR.A184V expressed in cultured neurons while delivering stimulation through whole-cell patch clamping of single neurons in the culture. In this manner, they could examine the optical signature of glutamate release in single presynaptic terminals, while separating the release events into synchronous (<10ms) and asynchronous (>10ms) while delivering both paired pulses or trains of stimuli (at 20 Hz, 50 ms between stimuli).

This manuscript employs techniques previously reported by the research group in their Mendoca et al., Nat Comms 2022 paper. This paper uses this approach to specifically examine the role of Syt7. The use of iGluSnFR in this manner provides significant rigor to the paper. The most significant weakness is that some of the events the authors discuss in this manuscript are rare, and the strength of the conclusions regarding those is somewhat unclear.

The main novel contribution of this manuscript is that single-bouton high-frequency imaging allowed them to examine paired-pulse plasticity in boutons that had not released neurotransmitter during the first pulse (failure-based analysis), thus separating between the effects of vesicle depletion and facilitation of the release machinery. This approach also allowed them to segregate their observations according to bouton-specific release efficacy. Both examinations are unavailable when performing cell-level analysis of neurotransmitter release, as is reported by most electrophysiological approaches.

The authors conclude that Syt7 contributes specifically to facilitation of synchronous release, not asynchronous release, while reducing the magnitude of the asynchronous component. Finally, the authors suggest segregation of synchronous and asynchronous release, either by differential use of calcium sensors or spatial segregation of the vesicles contributing to both modes of release.

This report contributes significantly to the discussion of the control of synaptic plasticity by different molecular players. It is not the first to examine Syt7, but its contribution to the examination of this protein is significant.

I find this report to be well executed and reasoned. In my opinion, the authors could improve the manuscript by clarifying the description of several methodological and experimental sections. Furthermore, in my opinion, some of the conclusions are overstated.

The authors state: "Because boutons along a single axon originate from the same presynaptic neuron, they are expected to share broadly similar molecular components of the vesicular release machinery and experience comparable presynaptic action potential waveforms." The authors should address the idea that presynaptic terminals from the same neuron on different postsynaptic targets can differ in the molecular components, as well as in the presynaptic side. There is ample evidence for differences between synapses onto glutamatergic and GABAergic neurons of the same neuron.

The authors used 4ms-long frames, but the stimuli are delivered at 20Hz (50ms apart). Therefore, in paired pulse stimulation, isn't there going to be a difference between the first and second stimuli regarding the timing of the frames relative to the stimulus? Isn't the deconvolution sensitive to such an offset?

A 10ms threshold for defining synchronous vs. asynchronous release full in-between frames. Doesn't this increase the chance of assigning borderline events to the wrong category?

On page 11 of the conclusion, the authors state that "Our data indicate that in our conditions during paired-pulse protocol Syt7 primarily enhances release probability rather than increasing the RRP size." While I understand the reasoning behind this statement, it should be toned down. The authors did not directly address the RRP size.

In failure-based analysis, the number of failure events in high-efficiency boutons is expected to be low. How does this affect the conclusions of the authors concerning the effects of Syt7 deletion on facilitation in high-efficiency boutons?<br /> SourceData.xlsx was not available to me, as far as I could tell.

How can the conclusions of the authors on the differential molecular composition of vesicles contributing to synchronous and asynchronous release be related to the reported effect of strontium on the nature of release? (see 10.1523/JNEUROSCI.20-12-04414.2000)

Is this the first use of failure-based analysis? If not, the authors should cite precedents. In 10.1016/s0896-6273(00)80338-4, failure of release during the 1st AP was presented, with facilitation during the 2nd, although no formal analysis was performed.

Reviewer #3 (Public review):

Summary:

Protein complexes, like the GID/CTLH-type E3 ligase, adopt a complex three-dimensional structure, which is of functional importance. Several domains are known to be involved in shaping the complexes. Structural information based on cryo-EM is available, but its resolution does not always provide detailed information on protein-protein interactions. The work by van gen Hassend and Schindelin provides additional structural data based on crystal structures.

Strengths:

The work is solid and very carefully performed. It provides high-resolution insights into the domain architecture, which helps to understand the protein-protein interactions on a detailed molecular level. They also include mutant data and can thereby draw conclusions on the specificity of the domain interactions. These data are probably very helpful for others who work on a functional level with protein complexes containing these domains.

Weaknesses:

The manuscript contains a lot of useful, very detailed information. This information is likely very helpful to investigate functional and regulatory aspects of the protein complexes, whose assembly relies on the LisH-CTLH-CRA modules. However, this goes beyond the scope of this manuscript.

Reviewer #3 (Public review):

Summary:

The manuscript of Spokaite et al. focuses on the Vps34 complex involved in PI3P production. This complex exists in two variants, one (class I) specific for autophagy, and a second one (class II) specific for the endocytic system. Both differ only in one subunit. The authors previously showed that the Vps34 complexes interact with Rab GTPases, Rab1 or Rab5 (for class II), and the identified site was found at Vps34. Now, the authors identify a conserved and overlooked Rab5 binding site in Vps15, which is required for the function of the Class II complex. In support of this, they show cryo-EM data with a second Rab5 bound to Vps15, identify the corresponding residues, and show by mutant analysis that impaired Rab5 binding also results in defects using yeast as a model system.

Overall, this is a most complete study with little to criticize. The paper shows convincingly that the two Rab5 binding sites are required for Vps34 complex II function, with the Vps15 binding site being critical for endosomal localization. The structural data is very much complete. What I am missing are a few controls that show that the mutations in Vps15 do not affect autophagy. I also found the last paragraph of the results section a bit out of place, even though this is a nice observation that the N-terminal part of BECLIN has these domains. However, what does it add to the story?

Reviewer #3 (Public review):

Summary:

In this study, Tisdale et al. studied the sleep/wake patterns in the biological mouse model of Alzheimer's disease. The results in this study, together with the established literature on the relationship of sleep and Alzheimer's disease progression, guided the authors to propose this mouse model for the mechanistic understanding of sleep states that translates to Alzheimer's disease patients. However, the manuscript currently suffers from a disconnect between the physiological data and the mechanistic interpretations. Specifically, the claim of "impaired transitions" is logically at odds with the observed increase in wake-state stability or possible hyperactivity. Additionally, the description of the methods, the quantification, and the figure presentation could be substantially improved. I detail some of my concerns below.

Strengths:

The selection of the knock-in model is a notable strength as it avoids the artifacts associated with APP overexpression and more closely mimics human pathology. The study utilizes continuous 14-day EEG recordings, providing a unique dataset for assessing chronic changes in arousal states. The assessment of sex as a biological variable identifies a more severe "insomniac-like" phenotype in females, which aligns with the higher prevalence and severity of Alzheimer's disease in women.

Weaknesses:

The study seems to lack a clear hypothesis-driven approach and relies mostly on explorative investigations. Moreover, lack of quantitative analytical methods as well as shaky logical conclusions, possibly not supported by data in its current form, leaves room for major improvement.

Since this paper studied sleep states, the "Methods" section is quite unclear on what specific criteria were used to classify sleep states. There is no quantitative description of classifying sleep based on clear, reproducible procedures. There are many reasonably well-characterized sleep scoring systems used in rat electrophysiological literature, which could be useful here. The authors are generally expected to describe movement speed and/or EMG and/or EEG (theta/delta/gamma) criteria used to classify these epochs. The subjective (manual) nature of this procedure provides no verifiable validation of the accuracy and interpretability of the results.

One of the bigger claims is that "state transition mechanism(s)" are impaired. However, Figure 7 shows that model mice exhibit significantly more long wake bouts (>260s) and fewer short wake bouts (<60s). Logically, an "impaired switch" (the flip-flop model, Saper et al., 2010) results in state fragmentation. The data here show the opposite: the wake state has become too stable. This suggests the primary defect is not in the transition mechanism itself, but possibly in a pathological increase in arousal drive (hyper-arousal), likely linked to the dark-phase hyperactivity shown in Figures 4 and 5. Also, a point to note is that this finding is not new.

Figure 3 heatmaps lack color bars and units. Spectral power must be quantitatively defined and methods well-explained in the Methods section. Without these, the reader cannot discern if the "reduced power" in females is a global suppression of signal or a frequency-specific shift. Additionally, the representative example used to claim shorter sleep bouts lacks the statistical weight required for a major physiological conclusion. How does a cooler color (not clear what range and what the interpretation is) mean shorter sleep bout in female mice? The authors should clearly mark the frequency ranges that support their claims. In this figure, there is a question mark following the theta/delta range. The authors should avoid speculation and state their claims based on facts. They should also add the theta and delta ranges in the plot, such that readers can draw their own conclusions.

Figure 8 and the MSLT results show that model mice are "no sleepier than WT mice" and have a functional homeostatic rebound. This presents a logical flaw in the "insomnia" narrative. True insomnia in AD patients typically involves a failure of the homeostatic process or a debilitating accumulation of sleep debt. If these mice do not show increased sleepiness (shorter latency) despite ~19% less sleep, the authors might be describing a "reduced need" for sleep or a "hyper-aroused" state, possibly not a clinical insomnia phenotype.

In Figure 9, LFP power shown and compared in percentages is problematic, as LFP power distribution is known to be skewed (follows power law). This is particularly problematic here because all the frequencies above ~20 Hz seem to be totally flattened or nonexistent, which makes this comparison of power severely limited and biased towards the relative frequency in the highly skewed portion of the LFP power spectrum, i.e., very low frequency ranges like delta, theta, and possibly beta. This ignores low, mid, and high gamma as well as ripple band frequencies. NREM sleep is known to have relatively greater ripple band (100-250 Hz) power bursts in hippocampal regions, and REM sleep is known to have synchronous theta-gamma relationships.

Reviewer #3 (Public review):

Summary:

The authors develop a tool for marking presynaptic active zones in Drosophila brains, dependent on the GAL4 construct used to express a fragment of GFP, which will incorporate with a genome-engineered partial GFP attached to the active zone protein bruchpilot - signal will be specific to the GAL4 expressing neuronal compartment. They then use various GAL4s to examine innervation onto the mushroom bodies to dissect compartment specific differences in the size and intensity of active zones. After a description of these differences, they induce learning in flies with classic odour/electric shock pairing and observe changes after conditioning that are specific to the paired conditioning/learning paradigm.

Strengths:

The imaging and analysis appears strong. The tool is novel and exciting.

Weaknesses:

I feel that the tool could do with a little more characterisation. It is assumed that the puncta observed are AZs with no further definition or characterisation. It is not resolved if the AZs visualised here simply tagged, or are the constructs incorporated to be an active functional part of the AZ.

Comments on revisions:

Apologies, I should have thought of this in the first round of review. An experiment I would suggest (and it is not a difficult one) to address the functionality of the marker: It is mentioned that the genetically tagged half of the construct is homozygous lethal. Can this be placed in trans to a brp null, with a neuronal UAS-expression of the other half of Brp-GFP - Are the animals then 1) alive, and 2) able to fly (brp mutants can't fly, hence the name 'crashpilot') - a rescue would suggest (and that is all that would be needed here) that the reconstituted brp-GFP has function.

On another note, the paper keeps switching between different DAN-GAL4 lines. In 1H, 2Band 4A, there are informative cartoons showing the extension of the neurons for PPL1, APL and DPM neurons - could these be incorporated into figures 5, 6 and 7, and the supplementary figures to help orient the reader. Ideally they would refer to a figure (in Fig 1?) -to refer to the groups of DANs in the adult brain that are known to innervate the MBs (e.g. Fig1 in Mao and Davis, Front in Neural Circuits 2009). I suggest this because I feel that this tool will be widely used, and if non-MB aficionados can follow what's being done here I feel it will be more widely accepted.

Reviewer #3 (Public review):

Summary

This is an exciting and timely study addressing the role of descending noradrenergic systems in nocifensive responses. While it is well-established that spinally released noradrenaline (aka norepinephrine) generally acts as an inhibitory factor in spinal sensory processing, this system is highly complex. Descending projections from the A6 (locus coeruleus, LC) and the A5 regions typically modulate spinal sensory processing and reduce pain behaviours, but certain subpopulations of LC neurons have been shown to mediate pronociceptive effects, such as those projecting to the prefrontal cortex (Hirshberg et al., PMID: 29027903).

The study proposes that descending cerulean noradrenergic neurons potentiate touch sensation via alpha-1 adrenoceptors on Hes5+ spinal astrocytes, contributing to mechanical hyperalgesia. This finding is consistent with prior work from the same group (dd et al., PMID:). However, caution is needed when generalising about LC projections, as the locus coeruleus is functionally diverse, with differences in targets, neurotransmitter co-release, and behavioural effects. Specifying the subpopulations of LC neurons involved would significantly enhance the impact and interpretability of the findings.

Strengths

The study employs state-of-the-art molecular, genetic, and neurophysiological methods, including precise CRISPR and optogenetic targeting, to investigate the role of Hes5+ astrocytes. This approach is elegant and highlights the often-overlooked contribution of astrocytes in spinal sensory gating. The data convincingly support the role of Hes5+ astrocytes as regulators of touch sensation, coordinated by brain-derived noradrenaline in the spinal dorsal horn, opening new avenues for research into pain and touch modulation.

Furthermore, the data support a model in which superficial dorsal horn (SDH) Hes5+ astrocytes act as non-neuronal gating cells for brain-derived noradrenergic (NA) signalling through their interaction with substantia gelatinosa inhibitory interneurons. Locally released adenosine from NA-stimulated Hes5+ astrocytes, following acute restraint stress, may suppress the function of SDH-Vgat+ inhibitory interneurons, resulting in mechanical pain hypersensitivity. However, the spatially restricted neuron-astrocyte communication underlying this mechanism requires further investigation in future studies.

Comments on revisions:

One important point remains insufficiently resolved. In Figure S4C, two of the three visible neurons in the A5 example appear to show a white "halo" at the cell border, suggesting a merge of eGFP (green) and TH (magenta) and therefore possible transgene positivity. To draw a confident conclusion about the specificity of the approach for the A6 (LC) population, the authors are kindly asked to provide high-resolution images of several representative A5 sections, presented both as merged and as separate colour channels. Ideally, quantification across multiple rostrocaudal sections of A5, A6 and A7 should be provided. This is essential for determining whether any transgene expression occurs within the A5 nucleus, particularly given its several-millimetre rostrocaudal extent. As the behavioural phenotype arises from manipulation of only a small subset of A6 neurons, ruling out any contribution from A5 (or A7) is critical for validating pathway specificity, especially in light of prior reports showing that similar approaches can label A5 fibres.

Reviewer #3 (Public review):

Disclaimer:

My expertise is in live single-molecule imaging of RNA and transcription, as well as associated data analysis and modeling. While this aligns well with the technical aspects of the manuscript, my background in translation is more limited, and I am not best positioned to assess the novelty of the biological conclusions.

Summary:

This study combines live-cell imaging of nascent proteins on single mRNAs with time-series analysis to investigate the kinetics of mRNA translation.<br /> The authors (i) used a calibration method for estimating absolute ribosome counts, and (ii) developed a new Bayesian approach to infer ribosome counts over time from run-off experiments, enabling estimation of elongation rates and ribosome density across conditions.

They report (i) translational bursting at the single-mRNA level, (ii) low ribosome density (~10% occupancy {plus minus} a few percents), (iii) that ribosome density is minimally affected by perturbations of elongation (using a drug and/or different coding sequences in the reporter), suggesting a homeostatic mechanism potentially involving a feedback of elongation onto initiation, although (iv) this coupling breaks down upon knockout of elongation factor eIF5A.

Strengths:

(1) The manuscript is well written and the conclusions are in general appropriately cautious (besides the few improvements I suggest below).

(2) The time-series inference method is interesting and promising for broader application.

(3) Simulations provide convincing support for the modeling (though some improvements are possible).

(4) The reported homeostatic effect on ribosome density is surprising and carefully validated with multiple perturbations.

(5) Imaging quality and corrections (e.g., flat-fielding, laser power measurements) are robust.

(6) Mathematical modeling is clearly described and precise; a few clarifications could improve it further.

Weaknesses:

(1) The absolute quantification of ribosome numbers (via the measurement of $i_{MP}$​) should be improved. This only affects the finding that ribosome density is low, not that it appears to be under homeostatic control. However, if $i_{MP}$​ turns out to be substantially overestimated (hence ribosome density underestimated), then "ribosomes queuing up to the initiation site and physically blocking initiation" could become a relevant hypothesis. In my first review of this work, I made recommendations, which the authors did not follow. In my view, the robustness of this particular aspect of this study remains moderate.

(2) The proposed initiation-elongation coupling is plausible, but alternative explanations such as changes in abortive elongation frequency should be considered. In their response to my previous comments, the authors indicate that this is "beyond the scope of the present work".

(3) More an opportunity for improvement than a weakness: It is unclear what the single-mRNA nature of the inference method is bringing since it is only used here to report _average_ ribosome elongation rate and density (averaged across mRNAs and across time during the run-off experiments -although the method, in principle, has the power to resolve these two aspects). In response to my previous comment, the authors note that such analyses could be incorporated in future work.

Reviewer #3 (Public review):

Summary:

The manuscript by Chaya and Syed focuses on understanding the link between cell cycle and temporal patterning in central brain type II neural stem cells (NSCs). To investigate this, the authors perturb the progression of the cell cycle by delaying the entry into M phase and preventing cytokinesis. Their results convincingly show that temporal factor expression requires progression of the cell cycle in both Type 1 and Type 2 NSCs in the Drosophila central brain. Overall, this study establishes an important link between the two timing mechanisms of neurogenesis.

Strengths:

The authors provide solid experimental evidence for the coupling of cell cycle and temporal factor progression in Type 2 NSCs. The quantified phenotype shows an all-or-none effect of cell cycle block on the emergence of subsequent temporal factors in the NSCs, strongly suggesting that both nuclear division and cytokinesis are required for temporal progression. The authors also extend this phenotype to Type 1 NSCs in the central brain, providing a generalizable characterization of the relationship between cell cycle and temporal patterning.

Weaknesses:

One major weakness of the study is that the authors do not explore the mechanistic relationship between cell cycle and temporal factor expression. Although their results are quite convincing, they do not provide an explanation as to why Cdk1 depletion affects Syp and EcR expression but not the onset of svp. This result suggests that at least a part of the temporal cascade in NSCs is cell-cycle independent which isn't addressed or sufficiently discussed.

Reviewer #3 (Public review):

Summary:

This study investigates the computational role of top-down feedback in artificial neural networks (ANNs), a feature that is prevalent in the brain but largely absent in standard ANN architectures. The authors construct hierarchical recurrent ANN models that incorporate key properties of top-down feedback in the neocortex. Using these models in an audiovisual integration task, they find that hierarchical structures introduce a mild visual bias, akin to that observed in human perception, not always compromising task performance.

Strengths:

The study investigates a relevant and current topic of considering top-down feedback in deep neural networks. In designing their brain-like model, they use neurophysiological data, such as externopyramidisation and hierarchical connectivity. Their brain-like model exhibits a visual bias that qualitatively matches human perception.

Weaknesses:

While the model is brain-inspired, it has limited bioplausibility. The model assumes a simplified and fixed hierarchy. The authors acknowledge this limitation in the discussion.

While the brain-like model showed an advantage in ignoring distracting auditory inputs, it struggled when visual information had to be ignored. This suggests that its rigid bias toward visual processing could make it less adaptive in tasks requiring flexible multimodal integration. It hence does not necessarily constitute an improvement over existing ANNs. The study does not evaluate whether the top-down feedback architecture scales well to more complex problems or larger datasets. A valuable future contribution would be to evaluate how the network's behaviour fits to human data.

Reviewer #3 (Public review):

Summary:

The study aims to investigate sensorimotor plasticity mechanisms by exposing a cohort of 20 subjects to manipulation activities while using wearable finger extensions. With a series of experiments involving localization and motor tasks, the authors provide evidence that the finger extensions are integrated into the body representation of the subjects.

Strengths:

The study deserves attention, and the psychophysical protocols are carefully designed, and the statistical analyses are solid.

Weaknesses:

However, the current version of the manuscript, in my opinion, makes an exaggerated use of the term plasticity, and this should be amended. This is because the authors support the plasticity claims with psychophysical experiments, without providing evidence of neural-plasticity mechanisms (e.g., neuroimaging methods are not used).

The authors are recommended to revise the wording of the manuscript and possibly perform additional experiments with brain imaging methods (e.g., EEG or fMRI).

Reviewer #3 (Public review):

Summary:

Combining electrophysiological recording, circuit tracing, single cell RNAseq, and optogenetic and chemogenetic manipulation, Howe and colleagues have identified a graded division between anterior and posterior plCoA and determined the molecular characteristics that distinguish the neurons in this part of the amygdala. They demonstrate that the expression of slc17a6 is mostly restricted to the anterior plCoA whereas slc17a7 is more broadly expressed. Through both anterograde and retrograde tracing experiments, they demonstrate that the anterior plCoA neurons preferentially projected to the MEA whereas those in the posterior plCoA preferentially innervated the nucleus accumbens. Interestingly, optogenetic activation of the aplCoA drives avoidance in a spatial preference assay whereas activating the pplCoA leads to preference. The data support a model that spatially segregated and molecularly defined populations of neurons and their projection targets carry valence specific information for the odors. Moreover, the intermingling of neurons in the plCoA is consistent with prior observations. The presence of a gradient rather than a distinct separation of the cells fits the model being proposed. The discoveries represent a conceptual advance in understanding plCoA function and innate valence coding in the olfactory system.

Strengths:

The strongest evidence supporting the model comes from single-cell RNASeq, genetically facilitated anterograde and retrograde circuit tracing, and optogenetic stimulation. The evidence clear demonstrates two molecularly defined cell populations with differential projection targets. Stimulating the two populations produced opposite behavioral responses.

Weaknesses:

The weaknesses noted in primary review have all been addressed adequately.

Reviewer #3 (Public review):

Summary:

The authors present an in vitro evaluation of drug-drug interactions between artemisinins and quinoline antimalarials, as an important aspect for screening the current artemisinin-based combination therapies for Plasmodium falciparum. Using a revised pulsing assay, they report antagonism between dihydroartemisinin (DHA) and several quinolines, including chloroquine, piperaquine (PPQ), and amodiaquine. This antagonism is increased in CQ-resistant strains in isobologram analyses. Moreover, CQ co-treatment was found to induce artemisinin resistance even in parasites lacking K13 mutations during the ring-stage survival assay. This implies that drug-drug interactions, not just genetic mutations, can influence resistance phenotypes. By using a chemical probe for reactive heme, the authors demonstrate that quinolines inhibit artemisinin activation by rendering cytosolic heme chemically inert, thereby impairing the cytotoxic effects of DHA. The study also observed negative interactions in triple-drug regimens (e.g., DHA-PPQ-Mefloquine) and in combinations involving OZ439, a next-generation peroxide antimalarial. Taken together, these findings raise significant concerns regarding the compatibility of artemisinin and quinoline combinations, which may promote resistance or reduce efficacy.

With the additive profile as the comparison and a lack of synergistic effect in any of the comparisons, it is hard to contextualize the observed antagonism. Including a known synergistic pair (e.g., artemisinin + lumefantrine) would have provided a useful benchmark to assess the relative impact of the drug interactions described.

Strengths:

This study demonstrates the following strengths:

• The use of a pulsed in vitro assay that is more physiologically relevant over the traditional 48h or 72h assays

• Small molecule probes, H-FluNox, and Ac-H-FluNox to detect reactive cytosolic heme, demonstrating that quinolines render heme inert and thereby block DHA activation.

• Evaluates not only traditional combinations but also triple-drug combinations and next-generation artemisinins like OZ439. This broad scope increases the study's relevance to current treatment strategies and future drug development.

• By using the K13 wild-type parasites, the study suggests that resistance phenotypes can emerge from drug-drug interactions alone, without requiring genetic resistance markers.

Weaknesses:

• The study would benefit from a future characterization of the molecular basis for the observed heme inactivation by quinolines to support this hypothesis - while the probe experiments are valuable, they do not fully elucidate how quinolines specifically alter heme chemistry at the molecular level.

• Suggestion of alternative combinations that show synergy could have improved the significance of the work. The invitro study did not include pharmacokinetic/pharmacodynamic modeling, hence it leaves questions about how the observed antagonism would manifest under real-world dosing conditions, necessitating furture work based on these findings.

Reviewer #3 (Public review):

The authors identified EOLA1 in a CRISPR/Cas9 screen for essential mitochondrial genes in a mouse B16-F10 cell line; however, no information on the library used for this screen or the list of all identified essential genes is provided. What was the p-value for EOLA1 in Figure 1b?

The authors show that EOLA1 is indeed a mitochondrial protein (using both mouse and human cell lines). It is valuable that the authors use different cell lines to investigate the function of this protein; however, this also presents a challenge, as four different cell lines (two mouse and two human) are used across individual experiments, with no consistency between them. Knock-out (KO) experiments were performed in mouse cell lines only, and human cell lines were used in overexpression experiments, in which EOLA1 was tagged with FLAG-HA. It would be beneficial if a knock-out were also generated in a human cell line to confirm the effect on the expression of mitochondria-encoded proteins, along with a rescue experiment in which the EOLA1 protein is reintroduced into KO cells.

Functional analysis of EOLA1: The authors performed affinity immunoprecipitation of FLAG-HA-tagged EOLA1 from stably overexpressing cells, and identified 202 co-immunoprecipitating proteins, of which 71 were known mitochondrial proteins; however, no list of these proteins is provided. Why did the authors choose TUFM? Were any mitochondrial ribosomal proteins co-immunoprecipitated, if EOLA1 is suggested to regulate translation? Were levels of TUFM affected in EOLA1-KO cells?

The authors continued to analyze mitochondrial ribosomes using sucrose gradient fractionation and in-vitro mitochondrial translation. However, there are several technical problems with the presented data: It has been established that mitochondrial ribosomes do not form polysomes in mammalian cells but rather perform translation as monosomes. The authors indirectly confirm this: almost no 12S or 16S rRNA (Fig. 3f) or MRP proteins (Extended data 3c) are present in "polysome" fractions. Although indeed 12S and 16S rRNAs are decreased in monosome fractions, the levels of mRNAs are not different between KO and WT cells, and neither is the migration of mitochondrial ribosomal proteins. As there is no loading control provided for the sucrose gradients blots (such as SDHA, VDAC), it is not possible to assess the overall levels of mitochondrial ribosomes. The gel presented for mitochondrial translation is of poor quality, as it is impossible to identify any of the expected 13 polypeptides. Although the intensity of the signal is weaker for KO, so is the intensity in the portion of Coomassie stained gel. A better-quality gel and quantification need to be provided to support the claims.

What is the difference between endogenous and exogenous RIP-qPCR? EOLA1 pulled down 12S rRNA without cross-linking (Figure 3d) or with UV-crosslinking (Figure 3e), however, both 12S and 16S rRNAs were enriched in UV-crosslinked cells (Figure 3c) and by UV-RIP-seq (Extended data 3b; although no control is provided here). Is no discussion offered for this observation? Is it possible that EOLA1 plays a role in the maturation of the mito-ribosome, rather than translation? Does EOLA1 co-migrate with the mito-ribosome on sucrose gradients?

Altogether, there is insufficient evidence to support the conclusion that EOLA1 plays a role in mitochondrial translation.

To investigate EOLA1 biological function, the authors created a whole-body EOLA1-/- mouse that exhibited no overall developmental abnormalities; however presented with an abnormal cardiac function. This is an ideal model to confirm prior observations in cellular models; however, apart from one western-blot for three mitochondrial encoded subunits, no other experiments were provided (such as measurements of the levels of 12S, or 16S rRNA, TUFM levels, ribosomes profile, mitochondrial translation, OXPHOS assembly, respirometry).

In Figure 2 g-i: TEM images are presented, but the method is not described, nor is any information on the cells used provided, nor is it clear how the circularity was determined. KO cells certainly look abnormal; however, are the authors sure that the indicated structures are mitochondria? They rather resemble autophagosomes/lysosomes with lamellar inclusions.

Reviewer #3 (Public review):

This work by Du et al. addresses a critical problem in cryo-electron microscopy. To date, there are few ways of generating phase contrast during cryo-EM imaging while remaining in focus. Cryo-EM practitioners today must generate contrast by collecting out-of-focus exposures, a process that introduces aberrations in the resulting image data. Recent work has shown that standing wave lasers are capable of using the ponderomotive effect to shift the phase of electrons in transmission electron microscopy to generate in-focus phase contrast imaging for cryo-EM. A limitation of this 'laser phase plate' is the high laser power required, which can damage optical mirrors and necessitate high laser safety. Thus, alternative approaches are needed for phase contrast imaging in cryo-EM.

In this manuscript, Du et al. exploit their expertise in ultrafast electron microscopy to explore the ability to shift the phase of electrons using pulsed electrons and lasers. The motivation for exploring pulsed laser phase plates stems from the fact that femtosecond pulses from 9W lasers can generate extremely high power (as much as the standing-wave laser phase plate, > 1 gigawatt) at the back focal plane. If successful, this type of instrument will likely be much more affordable and easier to deploy worldwide.

The work outlined here shows a proof of principle, highlighting that an ultrafast scanning electron microscopy beam at 30 kV can have the electron packets phase shift by 430 radians (24637 degrees), which is much greater than the required 1.5 radians (90 degrees) needed for phase contrast imaging. The data presented do not use any biological samples; instead, they measure the spread of the electron beam on a test sample to assess the ability to target pulsed lasers onto electron packets and the amount of electron spread (which relates to the phase shift). They were also able to take their system a step further to measure how changes to the system in terms of laser power affect performance, and show that the system can be stable for 10+ hours.

The only weaknesses relate to the broad readability of the text. Improved textual clarity will help ensure a wider readership.

Overall, this work is an important step toward developing lower-cost alternatives to the standing-wave laser phase plate.

Reviewer #3 (Public review):

Summary:

The authors of this report wish to show that distinct populations of meningeal macrophages respond to cortical spreading depolarization (CSD) via unique calcium activity patterns depending on their location in the meningeal sub-compartments. Perivascular macrophages display calcium signaling properties that are sometimes in opposition to non-perivascular macrophages. Many of the meningeal macrophages also displayed synchronous activity at variable distances from one another. Other macrophages were found to display calcium signals in response to dural vasomotion. CSD could induce variable calcium responses in both perivascular and non-perivascular macrophages in the meninges, in part due to RAMP1-dependent effects. Results will inform future research on the calcium responses displayed by macrophages in the meninges under both normal and pathological conditions.

Strengths:

Sophisticated in vivo imaging of meningeal immune cells is employed in the study, which has not been performed previously. A detailed analysis of the distinct calcium dynamics in various subtypes of meningeal macrophages is provided. Functional relevance of the responses is also noted in relation to CSD events.

Weaknesses:

The specificity of the methods used to target both meningeal macrophages and RAMP1 is limited. Additional discussion points on the functional relevance of the two subtypes of meningeal macrophages and their calcium responses are warranted. A section on potential pitfalls should be included.

Reviewer #3 (Public review):

Summary:

In the current manuscript, Sun et al aimed to determine the metabolic function of hepatocyte DHHC7, one of the key enzymes in protein palmitoylation. They generated inducible liver-specific Dhhc7 knockout mice and discovered that Dhhc7-LKO mice are more prone to gain weight and develop adipose expansion and obesity. Via unbiased proteomic analysis, they identified PRG4 as one of the top secreted factors in the liver of Dhhc7-LKO mice. Hepatic overexpression of PRG4 recapitulates the obesity phenotype observed in Dhh7-LKO mice. At the mechanistic level, PRG4, once secreted from the liver, can bind to GPR146 on adipocytes and inhibit PKA-HSL signaling and lipolysis. Taken together, their findings suggest a novel pathway by which the liver communicates with adipose tissue and impacts systemic metabolism.

Strengths:

(1) The systemic metabolic homeostasis depends on coordination among metabolically active tissues. Thus, active communication between the liver and adipose tissue when facing nutritional challenges (such as high-fat diet feeding) is crucial for achieving metabolic health. The concept that the liver can communicate with adipose tissue and impact the lipolysis process via secreted hepatokines is quite significant but remains poorly understood.

(2) Hepatocyte Dhhc7 knockout mice developed a significant obesity phenotype, which is associated with adipose expansion.

(3) Unbiased proteomic analysis identified PRG4 as one of the top secreted factors in the liver of Dhh7-LKO mice. Hepatic overexpression of PRG4 recapitulates the obesity phenotype observed in Dhh7-LKO mice.

(4) In vitro cell-based assay showed that PRG4 can bind to adipocyte GPR146, inhibit PKA-mediated HSL phosphorylation, and subsequently, the lipolysis process.

Weaknesses:

(1) Lack of a causal-effect study to generate evidence directly linking hepatocyte DHH7 and PRG4 in driving adipose expansion and obesity upon HFD feeding.

(2) Lack of direct evidence to support that PRG4 inhibits adipocyte lipolysis via GPR146. A functional assay demonstrating adipocyte lipolysis is required.

(3) The conclusion is largely based on the correlation evidence.

Reviewer #3 (Public review):

Summary:

In this study, the authors describe modeling of neuronopathic Gaucher disease (nGD) using midbrain-like organoids (MLOs) derived from hiPSCs carrying GBA1 L444P/P415R or L444P/RecNciI variants. These MLOs recapitulate several disease features, including GCase deficiency, reduced enzymatic activity, lipid substrate accumulation, and impaired dopaminergic neuron differentiation. Correction of the GBA1 L444P variant restored GCase activity, normalized lipid metabolism, and rescued dopaminergic neuronal defects, confirming its pathogenic role in the MLO model. The authors further leveraged this system to evaluate therapeutic strategies, including: (i) SapC-DOPS nanovesicles for GCase delivery, (ii) AAV9-mediated GBA1 gene therapy, and (iii) GZ452, a glucosylceramide synthase inhibitor. These treatments reduced lipid accumulation and ameliorated autophagic, lysosomal, and neurodevelopmental abnormalities.

Strengths:

This manuscript demonstrates that nGD patient-derived MLOs can serve as an additional platform for investigating nGD mechanisms and advancing therapeutic development.

Comments:

(1) It is interesting that GBA1 L444P/P415R MLOs show defects in midbrain patterning and dopaminergic neuron differentiation (Figure 3). One might wonder whether these abnormalities are specific to the combination of L444P and P415R variants or represent a general consequence of GBA1 loss. Do GBA1 L444P/RecNciI (GD2-10-257) MLOs also exhibit similar defects?

(2) In Supplementary Figure 3, the authors examined GCase localization in SapC-DOPS-fGCase-treated nGD MLOs. These data indicate that GCase is delivered to TH⁺ neurons, GFAP⁺ glia, and various other unidentified cell types. In fruit flies, the GBA1 ortholog, Gba1b, is only expressed in glia (PMID: 35857503; 35961319). Neuronally produced GluCer is transferred to glia for GBA1-mediated degradation. These findings raise an important question: in wild-type MLOs, which cell type(s) normally express GBA1? Are they dopaminergic neurons, astrocytes, or other cell types?

(3) The authors may consider switching Figures 2 and 3 so that the differentiation defects observed in nGD MLOs (Figure 3) are presented before the analysis of other phenotypic abnormalities, including the various transcriptional changes (Figure 2).

Reviewer #3 (Public review):

The hypothesis is that Trehalose metabolism regulates transcriptional control of muscle development in lepidopteran insects.

The manuscript investigates the role of Trehalose metabolism in muscle development. Through sequencing and subsequent bioinformatics analysis of insects with perturbed trehalose metabolism (knockdown of TPS/TPP), the authors have identified transcription factor E2F, which was validated through RT-PCR. Their hypothesis is that trehalose metabolism regulates E2F, which then controls the myogenic genes. Counterintuitive to this hypothesis, the investigators perform EMSAs with the E2F protein and promoter of the TPP gene and show binding. Their knockdown experiments with Dp, the binding partner of E2F, show direct effect on several trehalose metabolism genes. Similar results are demonstrated in the trehalose feeding experiment, where feeding trehalose leads to partial rescue of the phenotype observed as a result of Dp knockdown. This seems contradictory to their hypothesis. Even more intriguing is a similar observation between paramyosin, a structural muscle protein, and E2F/Dp - they show that paramyosin regulates E2F/Dp and E2F/Dp regulated paramyosin. The only plausible way to explain the results is the existence of a feed-forward loop between TPP-E2F/Dp and paramyosin-E2F/Dp. But the authors have mentioned nothing in this line. Additionally, I think trehalose metabolism impacts amino acid content in insects, and that will have a direct bearing on muscle development. The sequencing analysis and follow-up GSEA studies have demonstrated enrichment of several amino acid biosynthetic genes. Yet authors make no efforts to measure amino acid levels or correlate them with muscle development. Any study aiming to link trehalose metabolism and muscle development and not considering the above points will be incomplete.

The result section of the manuscript is quite concise, to my understanding (especially the initial few sections), which misses out on mentioning details that would help readers understand the paper better. While technical details of the methods should be in the Materials and Methods section, the overall experimental strategy for the experiments performed should be explained in adequate detail in the results section itself or in figure legends. I would request authors to include more details in the results section. As an extension of the comment above, many times, abbreviations have been used without introducing them. A thorough check of the manuscript is required regarding this.

The Spodoptera experiments appear ad hoc and are insufficient to support conservation beyond Helicoverpa. To substantiate this claim, please add a coherent, minimal set of Spodoptera experiments and present them in a dedicated subsection. Alternatively, consider removing these data and limiting the conclusions (and title) to H. armigera.

In order to check the effects of E2F/Dp, a dsRNA-mediated knockdown of Dp was performed. Why was the E2F protein, a primary target of the study, not chosen as a candidate? The authors should either provide justification for this or perform the suggested experiments to come to a conclusion. I would like to point out that such experiments were performed in Drosophila.

Silencing of HaDp resulted in a significant decrease in HaE2F expression. I find this observation intriguing. DP is the cofactor of E2F, and they both heterodimerise and sit on the promoter of target genes to regulate them. I would request authors to revisit this result, as it contradicts the general understanding of how E2F/Dp functions in other organisms. If Dp indeed controls E2F expression, then further experiments should be conducted to come to a conclusion convincingly. Additionally, these results would need thorough discussion with citations of similar results observed for other transcription factor-cofactor complexes.

I consider the overall bioinformatics analysis to remain very poorly described. What is specifically lacking is clear statements about why a particular dry lab experiments were conducted.

In my judgement, the EMSA analysis presented is technically poor in quality. It lacks positive and negative controls, does not show mutation analysis or super shifts. Also, it lacks any competition assays that are important to prove the binding beyond doubt. I am not sure why protein is not detected at all in lower concentrations. Overall, the EMSA assays need to be redone; I find the current results to be unacceptable.

GSEA studies clearly indicate enrichment of the amino acid synthesis gene in TPP knockdown samples. This supports the plausible theory that a lack of Trehalose means a lack of enough nutrients, therefore less of that is converted to amino acids, and therefore muscle development is compromised. Yet the authors make no effort to measure amino acid levels. While nutrients can be sensed through signalling pathways leading to shut shutdown of myogenic genes, a simple and direct correlation between less raw material and deformed muscle might also be possible.

The authors are encouraged to stick to one color palette while demonstrating sequencing results. Choosing a different color palette for representing results from the same sequencing analysis confuses readers.

Expression of genes, as understood from sequencing analysis in Figure 1D, Figure 2F, and Figure 3D, appears to be binary in nature. This result is extremely surprising given that the qRT-PCR of these genes have revealed a checker and graded expression.

In several graphs, non-significant results have been interpreted as significant in the results section. In a few other cases, the reported changes are minimal, and the statistical support is unclear; please recheck the analyses and include exact statistics. In the results section, fold changes observed should be discussed, as well as the statistical significance of the observed change.

Finally, I would add that trehaolse metabolism regulates cell cycle genes, and muscle development genes establish correlation and causation. The authors should ensure that any comments they make are backed by evidence.

Reviewer #3 (Public review):

Summary:

The study assesses whether CRISPR-generated founder (F0) "crispant" mice can be reliably used for initial phenotypic assessment and screening. By targeting seven genes with known visible recessive phenotypes, the authors show that, despite genetic mosaicism, the expected null phenotypes were observed in all targeted genes. These findings demonstrate that the phenotyping and screening of F0 "crispant" mice is a valid (and efficient) approach to selecting candidate alleles for follow-up studies, thereby streamlining mouse breeding and animal husbandry-related costs.

Strengths:

The study is comprehensive, carefully executed, and provides deep insight into the utility of F0 "crispant" mice for phenotypic screening. The authors evaluated the CRISPR/Cas9 editing outcomes across seven genes using multiple sequencing modalities, providing a robust framework for determining and interpreting complex founder genotypes. Importantly, the study examines/highlights the biological insight gained from compound heterozygous founders and naturally arising allelic series, enabling genotype-phenotype associations and functional inferences about protein domains.

More broadly, the authors' thorough evaluation of the CRISPR/Cas9-based gene editing events in the founders can serve as a benchmark for others in the field, engineering their own mouse "crispants."

Weaknesses:

The relationship between the sgRNA/Cas9 concentrations delivered to the zygotes and the resulting editing efficiencies are not explicitly investigated.

Reviewer #3 (Public review):

Summary:

Kumar et al. examine the H3K115 epigenetic mark located on the lateral surface of the histone core domain and present evidence that it may serve as a marker enriched at transcription start sites (TSSs) of active CpG island promoters and at polycomb-repressed promoters. They also note enrichment of the H3K115ac mark is found on fragile nucleosomes within nucleosome-depleted regions, on active enhancers and CTCF bound sites. They propose that these observations suggest that H3K115ac contributes to nucleosome destabilization and so may servers a marker of functionally important regulatory elements in mammalian genomes.

Strengths:

The authors present novel observations suggesting that acetylation of a histone residue in a core (versus on a histone tail) domain may serve a functional role in promoting transcription in CPG islands and polycomb-repressed promoters. They present a solid amount of confirmatory in silico data using appropriate methodology that supports the idea that H3K115ac mark may function to destabilize nucleosomes and contribute to regulating ESC differentiation. These findings are quite novel.

Weaknesses:

Additional experiments to confirm specificity of the antibodies used have been done, improving confidence in the study.

Reviewer #3 (Public Review):

Primary neutrophils are difficult to modify genetically, whereas the generation of knockout mice to study the role of specific proteins is time-consuming and expensive. CRISPR-Cas 9 genetic modification of neutrophil progenitors in vitro offers a platform to study neutrophil biology. Hoxb8 cells are immortalized neutrophil progenitors that differentiate into neutrophils when cultured in the presence of G-CSF, and have been shown to recapitulate the stages of murine neutrophil differentiation. They have also been shown to be amendable to CRISPR-Cas 9 genetic editing with successful deletion of key transcriptional regulators of neutrophil maturation and function. The authors of this manuscript offer an extension to this technique, by generating Hoxb8 cells that constitutively express Cas9. This may reduce the variation between the generated knock-out cells by avoiding the introduction of Cas9 in a plasmid every time together with a guide RNA.

The first part of the manuscript is dedicated to the characterisation of Cas9+HoxB8 cells throughout their differentiation. Considering the existing body of literature on HoxB8 progenitors and their differentiation into neutrophils ex vivo, it does not significantly further our understanding of these cells, but rather provides a good validation to their Cas9+ modified version of them. Gene editing using Cas9+ Hoxb8 progenitors seems to be highly efficient, which is an important technical point, however, it is hard to assess the degree of improvement in efficiency compared to the published protocols with Cas9 delivery in a plasmid.

As a test, the authors use Cas9+HoxB8 progenitor to generate a knockout of CEBPE, known for its important role in neutrophil development. They convincingly demonstrate its impact on HoxB8 cell differentiation, with in vivo reconstitution of wild-type and CEBPE-deficient HoxB8 progenitors into irradiated mice being especially elegant. However, the transfer into different recipient mice assumed no differences in the recipient environment, while immunophenotyping for mature neutrophils within the HoxB8 progenitor-derived cells did not account for possible differences in numbers of wt and CEBPE KO surviving cells, limiting the conclusions.

Finally, the authors put the system to the test by screening a library of Brie gRNA library of ~80K mouse sgRNAs, targeting almost 20K genes with 4 gRNA per gene coverage, to identify genes that are needed for the differentiation of Cas9+ERHoxb8 progenitors into mature neutrophils. They identify a number of hits, amongst which the WASH complex and CEBPE are highlighted. A comparison of cell numbers prior to differentiation and at 4 days post differentiation indicates that they are indeed required for neutrophil survival. To validate the role of these hits in neutrophil maturation itself, as they stated in the aims, i.e. "to identify genes that modulate the differentiation of Cas9+ERHoxb8 progenitors into mature neutrophils", phenotypic, functional, and morphological characterization of these cell lines could have been appropriate.

Overall, this study has the potential to improve on the established lentiviral CRISPR-Cas9 editing of Hoxb8 cells and be valuable for library-screening approaches for neutrophil modulators, which will benefit the community.

Reviewer #3 (Public review):

Summary:

In this study, Wang et al., investigate how herbivorous insects overcome plant receptor-mediated immunity by targeting plant receptor-like proteins. The authors identify two independently evolved salivary effectors, BtRDP in whiteflies and NlSP694 in brown planthoppers, that promote the degradation of plant RLP4 through the ubiquitin-dependent proteasome pathway. NtRLP4 from tobacco and OsRLP4 from rice are shown to confer resistance against herbivores by activating defense signaling, while BtRDP and NlSP694 suppress these defenses by destabilizing RLP4 proteins.

Strengths:

This work highlights a convergent evolutionary strategy in distinct insect lineages and advances our understanding of insect-plant coevolution at the molecular level.

Two minor comments:

In line 140, yeast two-hybrid (Y2H) was used to screen for interacting proteins in plants. However, it is generally difficult to identify membrane receptors using Y2H. Please provide more methodological details to justify this approach, or alternatively, include a discussion explaining this.

In Figure S12C, the interaction between the two proteins appears to be present in the nucleus as well. Please provide a possible explanation for this observation.

Reviewer #3 (Public review):

Summary:

This study investigates the molecular underpinnings of immune responses in the leptomeninges in neonatal bacterial meningitis. Bacterial meningitis is a major disease burden, particularly for neonates, and it has previously been noted that the meningeal immune environment in infants is permissive to opportunistic infection (Kim et al., Sci Immunol, 2023). There is less known about the contribution of the stromal compartment to meningeal immune responses. Seegren et al. interrogate the role of leptomeningeal endothelium in host defence in E. coli infected neonatal mice using mouse genetic tools to delete the LPS receptor Tlr4 from either endothelial cells (using Cdh5-CreER) or macrophages (using LysM-Cre). The authors use snRNAseq, cleared cortical mounts, and in vitro work to define the impact of E. coli infection on leptomeningeal endothelial cells. This study uses a range of innovative techniques to probe the role of the stromal compartment in meningitis.

Strengths:

This study makes excellent use of cleared cortical mounts to examine the biology of the leptomeninges, in particular, changes to the endothelium, with unprecedented detail. In combination with high-quality sequencing data provide new insights into the impact of meningitis on the leptomeninges. The data presented by the authors is of very high quality.

Weaknesses:

The weaknesses of the study were in terms of interpretation and perhaps study design.

(1) Most importantly, the authors need to provide additional validation of their conditional knockout models. The authors need to confirm that the Cdh5-CreER does not impact leptomeningeal fibroblasts and to confirm gene deletion in macrophages.

(2) The authors could also strengthen the paper by providing data on the impact of these conditional knockout models on the course of meningitis and bacterial burden.

(3) Finally, it is perhaps not surprising that Tlr4 is required for meningitis responses with E. coli. However, it is unclear if these findings can be generalised to other, more common, meningitis infections (streptococcal/pneumococcal).

(4) There are additional minor issues; for instance, the arachnoid fibroblast 2 population appears to closely resemble dural border cells.

(5) The cell line model (bEnd.3) is a relatively low-fidelity model of BBB endothelial cells, and this should be acknowledged.

With these caveats, it is difficult to be certain that the endothelium alone is the driver of meningeal immune responses in meningitis, and what the impact of these is.

https://www.facebook.com/groups/721704878218903/posts/2997989990590369/

Reviewer #3 (Public review):

Summary:

The purpose of this study was to test the hypothesis that the inverted pendulum mechanism contributes to the gait transition from quadrupedal to bipedal gait in Japanese macaques. The author uses a neuromusculoskeletal model to generate different motor tasks by varying motor command parameters during forward dynamics simulations. After simulations were done, the authors used dynamical system analysis of the inverted pendulum model to reveal the underlying principles of gait transition control. The authors showed that successful gait transition from quadrupedal to bipedal gait mostly depends on increased step length of a hindlimb.

Strengths:

This study is important not only for understanding gait transition, but also to understand stability control of bipedal gaits. Another advantage of this study is that it allows us to estimate the effect of one control mechanism and find its effect and limits. In animal studies, we also have a combination of compensatory stability control mechanisms.

Weaknesses:

Any simulation is not perfect, so discrepancies from experimental data are expected. A 2D model is used, but the advantage of using a 3D model is not clear, and it is much more complicated.

Reviewer #3 (Public review):

Summary:

The authors examine firing of dysgranular retrosplenial cortex (dRSC) neurons in relation to head orientation and location for rats exploring open-field environments. One environment utilized was a square arena with high walls that is split into two rectangular spaces connected by a doorway. Another environment is a square arena split into quadrants connected by doors near the center. For each, the different sub-spaces of the environments are either identical in terms of visual and tactile cues or different. For head direction neurons, the authors present one population where each neuron maintains a single tuning direction for the two or four sub-compartments of the two environments. A second population exhibits what is termed multi-directional firing, wherein neurons exhibit (overall) two or four head direction peaks in firing. For such neurons, firing in each of the sub-compartments is associated with only a single preferred direction, but the directions across compartments are shown to be at 180-degree (two-compartment environment) or 90-degree offsets. The offsets evidence tuning to the "same" orientation for the sub-compartments that are, in the global reference frame, oriented at 180 or 90 degree offsets. The results are similar whether or not the sub-compartments have the same or different tactile and visual cues. Thus, the first population is said to be global in its head direction tuning, while the second relates to each local environment in a way that is systematic across sub-compartments. Spatially-specific activity of another population of non-direction-tuned RSC neurons is examined, and comparisons of sub-compartment spatial firing maps suggest that spatial tuning in RSC also repeats across compartments when the firing maps for the compartments are rotated to match each other (as in physical space). Finally, a population of hippocampal "place" cells exhibited different location mapping across sub-compartments. The findings are interpreted to indicate that RSC can simultaneously map orientation in both local and global reference frames, possibly forming a mechanism whereby the sub-compartments' shared geometry (given by the boundary shapes and the door locations) can be related to each other and to the global space they share.

Strengths:

This paper addresses an interesting problem and expands how the field will think about directional tuning.

Weaknesses:

It is not clear that the experimental design allows for a clear interpretation of the data. Rates for preferred turning are low, as are ratemap correlations for spatially-tuned neurons.

(1) It is concerning that the neurons with head direction tuning have fairly low peak firing rates (mean close to 5 Hz), where prior studies examining head direction tuning in dRSC found head direction-tuned neurons with peak rates more than an order of magnitude higher (100 Hz or more). Under circumstances where neurons are tuned well to variables other than head direction (for example, angular velocity of movement), weak head direction tuning may be observed if those other variables are not sampled equally across head directions. The manuscript contains no rigorous control for this possibility. One place to start to address this issue would be to map out variables such as angular velocity by head orientation, and to test whether such relationships also carry 90 and 180 degree offsets.

(2) There is some question as to whether dRSC neurons (spatial or directional) following the sub-compartment "geometry" is appropriate in terms of interpreting the data. In the condition with sub-compartments carrying different tactile and visual cues, it seems that such cues pertain only to the floor of the environments. The distal visual space of the boundaries appears to be identical. One is left to wonder whether distinguishing environments according to boundary wall visual cues would lead to different results. The CA1 data does not help to rule this possibility out. A second reason to doubt the "shared geometry" interpretation is that there is no condition where sub-compartment geometry is varied. It is also the case that the sub-compartment doorways may stand as the only salient distal visual cue linking the environments. Local sensory cues and geometry seem not so disentangled in this study, but this is a major claim in the abstract.

(3) There is some concern with the interpretation that the spatial tuning of some dRSC neurons repeats in rotated form across sub-compartments. The firing rate map correlations are very low on average (~0.2), and far lower than the population of CA1 having repeating fields across the same vs different visual/tactile cue conditions. The authors should define the chance level of ratemap correlation by shuffling neuron identities. Apologies if this is indeed the current approach, but it seems not to be (I was left a bit lost by the description in the methods). For any population of hippocampal place cells, the cross-neuron correlations of firing rate maps are typically not zero, and correlations at 0.2 would normally be evidence for remapping.

(4) A somewhat picky point here that is not meant to claim that multi-compartment studies are not useful - the introduction states that real-world environments typically consist of multi-compartment rooms. This is certainly not true for rodents and is only sometimes true in humans.

(5) The discussion lacks a consideration of how such dRSC output might impact the target structures of dRSC.

(6) The discussion speaks to the idea that multi-directional neurons may aid in transitioning between contexts (sub-compartments). But it is notable that none of the multidirectional neurons have multi-directional tuning in all sub-compartments, but such firing was seen in the 2017 Nature Neuroscience study by Jacob/Jeffery. The discussion should address this difference and perhaps posit a means by which the firing of global and local head direction neurons can be related to each other to yield navigation that depends on both scales.

(7) The authors should provide the size of the smoothing function for spatial firing rate maps.

(8) The authors should devise a measure to define directional tuning in 4 directions (with 90-degree offsets).

(9) Figures 2D and 2H - The offsets in preferred tuning across sub-compartments are rather variable.

Reviewer #3 (Public review):

Summary:

The manuscript presents an elegant and cost-effective approach for generating a tunable Bessel beam on a conventional two-photon microscope. The authors assemble a compact optical module comprising three axicons and a series of lenses that permits rapid adjustment of both lateral resolution and axial extent without modifying the focal plane. This flexibility enables the system to be readily adapted to a variety of biological preparations. As a proof of concept, the authors employ the device to record blood flow velocities in cortical microcapillaries, arterioles, and venules, thereby directly visualizing vasodilatation and vasoconstriction dynamics and permitting quantitative analysis of neurovascular coupling across cortical layers in awake mice.

The authors demonstrate that the tunability of the Bessel beam can be exploited to match the numerical aperture to the vessel type: a high NA configuration, albeit slower scan, is optimal for resolving flow in capillaries, whereas a low NA setting provides faster acquisition suitable for arterioles and venules. By implementing a one-dimensional line scan with the Bessel beam, they achieve an imaging speed that is twentyfold faster than conventional frame-by-frame scanning, which proves sufficient to capture hemodynamic transients before and after an induced ischemic stroke.

In addition to pure observation, the authors integrate a co-propagating Gaussian line to the system, allowing simultaneous imaging and photostimulation within the same focal plane. This capability addresses a common limitation of other Bessel beam implementations, in which the observation and perturbation planes often become misaligned when the Bessel beam is altered. The manuscript also emphasizes the advantage of Bessel beam excitation for calcium imaging after a perturbation, because it captures neuronal activity in planes both above and below the nominal focal plane, signals that would be missed with a standard Gaussian focus. Finally, the authors apply the technique to investigate the neuroimmune response following targeted microglial ablation; they report that adjacent microglia extend processes toward the injury site while retracting processes in the opposite direction.

Overall, the work offers a technically straightforward yet powerful extension to existing two-photon platforms, providing high-speed, volumetric imaging and stimulation capabilities that are well-suited to a broad range of neurovascular and neuroimmune studies. The experimental validation is quite thorough, and the presented data convincingly illustrates the benefits of the approach.

Strengths:

The authors present a truly clever and inexpensive optical module that can be integrated into almost any two-photon microscope, providing a tunable Bessel beam with a minimal modification of the existing system. The experimental data and accompanying quantitative analysis convincingly demonstrate that the system can reveal physiological events, such as capillary flow, calcium transients across multiple axial planes, and microglial process dynamics, that are difficult or impossible to capture with a conventional Gaussian beam. The breadth of experiments chosen for the manuscript illustrates the practical utility of the device and supports the authors' conclusions that it extends the functional repertoire of standard two-photon microscopy.

Weaknesses:

The manuscript would benefit from a more detailed contextualisation of the claimed speed advantage. Although the authors mention other techniques in the introduction, they do not provide any direct comparison with other state-of-the-art high-speed two-photon approaches such as light beads microscopy (Demas et al., Nat. Methods 2021), temporal multiplexing schemes (Weisenburger et al., Cell 2019), or random access microscopy (Villette et al., Cell 2019). A brief comparison of imaging speed, spatial resolution, and instrumental complexity would enable readers to assess the relative merits of the present method.

A second limitation that warrants discussion is the inherent trade off between volumetric coverage and image specificity. Because the Bessel beam excites fluorescence throughout an extended axial range, the detector inevitably integrates signal from a three dimensional volume into a two dimensional image. In densely labelled tissue, this can lead to significant signal crosstalk, reducing contrast and complicating quantitative interpretation. A brief analysis of how labeling density affects the fidelity of flow or calcium measurements, or suggestions for mitigating crosstalk (e.g., computational deconvolution, adaptive excitation shaping, or combinatorial sparse labeling), would broaden the applicability of the technique.

Reviewer #3 (Public review):

Summary:

While our knowledge regarding visual opsins is largely very good, a lot more uncertainty exists around the role of non-visual opsins. Using the power of the Drosophila melanogaster model system, Kirsh et al. investigate the role of the non-visual opsin Rhodopsin7 (Rh7). Expression analysis, based on Rh7-Gal4>UAS-GFP and HRC in situ staining, reveals strong expression in the optic lobes and somewhat weaker, but nevertheless extensive expression in the brain. An investigation of motor activity reveals that loss of function leads to an altered day and night rhythm, specifically decreasing activity during the dark phase. These flies were also less sensitive, but still responsive to a light-induced startle response and showed deficiencies in the optomotor response. To further investigate how Rh7 may modulate these responses, inspired by the Dark line of flies (which were kept in the dark for ~1400 generations) and which has accumulated C-terminal related losses, the authors conducted rescues with an intact and a C-terminal-deficient Rh7 and were able to pinpoint that region as an important driver of related behavioral shifts. These findings are particularly intriguing as Rh7 represents an ancient opsin with phylogenetic and mechanistic parallels to mammalian melanopsin.

Strengths:

The paper is well-written and contains high-quality data with appropriate sample sizes, and the conclusions are well supported.

Weaknesses:

No weaknesses were identified by this reviewer, but the following recommendations are made:

(1) The authors should clarify exactly what tissues were taken for the comparative qPCR. This is particularly interesting in terms of the retina. Since Rh7 appears not to be expressed within the photoreceptor cells of the retina, this raises the important question as to which cells it is expressed in. To address this important question, it would also be helpful to include an expression analysis of the retina itself (by extending the RH7-GFP expression patterns and/or adding HCR in situ of the ommatidia array). The cell types of the retina are very well classified, and some evidence already exists for Rh7 expression in support cells (e.g., Charlton-Perkins et al., (2017); PMID: 28562601). This study has a unique opportunity to investigate this further by adding these critical data for a more complete picture of Rh7.

(2) Mammalian opsins should be included in the phylogenetic analysis illustrated in Figure 2A and indicate their position on the tree. This will allow readers to better put the authors' statements regarding the intermediate position of Rh7 into perspective. In addition, note that the distinction between red and deep red is easy to miss regarding the Rh7 cluster. Perhaps the authors could use a more distinct colour scheme, for example, orange and deep red.

(3) More details should be provided on the optomotor response experiments. Specifically, specifications of the frequencies used for the optomotor response are needed. Results show a relatively large level of variation, which may be due to different angular perspectives that flies may have had while viewing the stimulus. If possible, provide videos as examples, as they will make it clearer to viewers how much flies could move around in the setup (from the methods, it seems they could move within the 2.2 of the 3 cm diameter of the arena, which would lead to substantial differences in the visual angle of the viewed grating.

Reviewer #3 (Public review):

Summary

This study aimed to investigate whether the differences observed in the organization of visual brain networks between blind and sighted adults result from a reorganization of an early functional architecture due to blindness, or whether the early architecture is immature at birth and requires visual experience to develop functional connections. This question was investigated through the comparison of 3 groups of subjects with resting-state functional MRI (rs-fMRI). Based on convincing analyses, the study suggests that: 1) secondary visual cortices showed higher connectivity to prefrontal cortical regions (PFC) than to non-visual sensory areas (S1/M1 and A1) in infants like in blind adults, in contrast to sighted adults; 2) the V1 connectivity pattern of infants lies between that of sighted adults (showing stronger functional connectivity with non-visual sensory areas than with PFC) and that of blind adults (showing stronger functional connectivity with PFC than with non-visual sensory areas); 3) the laterality of the connectivity patterns of infants resembled those of sighted adults more than those of blind adults, but infants showed a less differentiated fronto-occipital connectivity pattern than adults.

Strengths

- The question investigated in this article is important for understanding the mechanisms of plasticity during typical and impaired development, and the approach considered, which compares different groups of subjects including, neonates/infants and blind adults, is highly original.

- Overall, the presented analyses are solid and well-detailed, and the results and discussion are convincing.

Weaknesses

- While it is informative to compare the "initial" state (close to birth) and the "final" states in blind and sighted adults to study the impact of post-natal and visual experience, this study does not analyze the chronology of this development and when the specialization of functional connections is completed. This would require investigating the evolution of functional connectivity of the visual system as a function of visual experience and thus as a function of age, at least during toddlerhood given the early and intense maturation of the visual system after birth. This could be achieved by analyzing different developmental periods using open databases such as the Baby Connectome Project.

- The rationale for grouping full-term neonates and preterm infants (scanned at term-equivalent age) is not understandable when seeking to perform comparisons with adults. Even if the study results do not show differences between full-terms and preterms in terms of functional connectivity differences between regions and of connectivity patterns, preterms group had different neurodevelopment and post-natal (including visual) experiences (even a few weeks might have an impact). And actually they show reduced connectivity strength systematically for all regions compared with full-terms (Sup Fig 7). Considering a more homogeneous group of neonates would have strengthened the study design.

- The rationale for presenting results on the connectivity of secondary visual cortices before the one of primary cortices (V1) could be clarified.

- The authors acknowledge the methodological difficulties for defining regions of interest (ROIs) in infants in a similar way as adults. Since the brain development is not homogeneous and synchronous across brain regions (in particular with the frontal and parietal lobes showing a delayed growth), this poses major problems for registration. This raises the question of whether the study findings could be biased by differences in ROI positioning across groups.

Comments on revisions:

The authors have addressed my specific recommendations, but some weaknesses in the study remain, particularly the inclusion of preterm infants alongside full-term neonates.

Reviewer #3 (Public review):

Summary:

The authors aim to identify the peripheral end organ origin in the fly's wing of all sensory neurons in the Anterior Dorsal Mesothoracic nerve. They reconstruct the neurons and their downstream partners in an electron microscopy volume of a female ventral nerve cord, analyse the resulting connectome and identify their origin with review of the literature and imaging of genetic driver lines. While some of the neurons were already known through previous work, the authors expand on the identification and create a near complete map of the wing mechanosensory neurons at synapse resolution.

Strengths:

The authors elegantly combine electron microscopy neuron morphology, connectomics and light microscopy methods to bridge the gap between fly wing sensory neuron anatomy and ventral nerve cord morphology. Further, they use EM ultrastructural observations to make predictions on the signaling modality of some of the sensory neurons and thus their function in flight.

The work is as comprehensive as state of the art methods allow to create a near complete map of the wing mechanosensory neurons. This work will be of importance to the field of fly connectomics and modelling of fly behavior as well as a useful resource to the Drosophila research community.

Through this comprehensive mapping of neurons to the connectome the authors create a lot of hypotheses on neuronal function partially already confirmed with the literature and partially to be tested in the future. The authors achieved their aim of mapping the periphery of the fly's wing to axonal projections in the ventral nerve cord, beautifully laying out their results to support their mapping.

The authors identify the neurons in a previously published connectome of a male fly ventral nerve cord to enable cross-individual analysis of connections and find no indication of sexual dimorphism at the sensory neuron level. Further, together with their companion paper Dhawan et al., 2025 describing the haltere sensory neurons in the same EM dataset, they cover the entire mechanosensory space involved in Drosophila flight.

Reviewer #3 (Public review):

Summary:

In this study, the authors investigate how the structural state of the microtubule lattice influences the accessibility of the α-tubulin C-terminal tail (CTT). By developing and applying new biosensors, they reveal that the tyrosinated CTT is largely inaccessible under normal conditions but becomes more accessible upon changes to the tubulin conformational state induced by taxol treatment, MAP expression, or GTP-hydrolysis-deficient tubulin. The combination of live imaging, biochemical assays, and simulations suggests that the lattice conformation regulates the exposure of the CTT, providing a potential mechanism for modulating interactions with microtubule-associated proteins. The work addresses a highly topical question in the microtubule field and proposes a new conceptual link between lattice spacing and tail accessibility for tubulin post-translational modification. Future work is required to distinguish CTT exposure in the microtubule lattice is sensitive to additional factors present in vivo but not in vitro.

Strengths:

(1) The study targets a highly relevant and emerging topic-the structural plasticity of the microtubule lattice and its regulatory implications.

(2) The biosensor design represents a methodological advance, enabling direct visualization of CTT accessibility in living cells.

(3) Integration of imaging, biochemical assays, and simulations provides a multi-scale perspective on lattice regulation.

(4) The conceptual framework proposed lattice conformation as a determinant of post-translational modification accessibility is novel and potentially impactful for understanding microtubule regulation.

[Editors' note: the authors have responded to the reviewers and this version was assessed by the editors.]

Reviewer #3 (Public review):

This manuscript presents a study combining molecular dynamics simulations and Hedgehog (Hh) pathway assays to investigate cholesterol translocation pathways to Smoothened (SMO), a G protein-coupled receptor central to Hedgehog signal transduction. The authors identify and characterize two putative cholesterol access routes to the transmembrane domain (TMD) of SMO and propose a model whereby cholesterol traverses through the TMD to the cysteine-rich domain (CRD), which is presented as the primary site of SMO activation.

The MD simulations and biochemical experiments are carefully executed and provide useful data.

Comments on revisions:

I appreciate the authors' detailed response and the substantial revisions made to the manuscript. The changes addressing Comments 3.1-3.5 have significantly improved the balance and framing of the work, and my primary concerns regarding overstatement and selective interpretation have been satisfactorily addressed.

The authors' rebuttal to my initial review includes extended argumentation regarding specific interpretations of prior studies and broader models of SMO regulation. These issues represent longstanding differences in interpretation that have already been discussed extensively in the literature and are not essential to evaluating the quality or conclusions of the present study.

For readers seeking a comprehensive and balanced overview of cholesterol-dependent SMO activation that integrates both CRD- and TMD-centered models, I would point to recent review articles (e.g., Zhang and Beachy, Nat Rev Mol Cell Biol2023). I do not feel it is productive to rehash these debates further in the context of this review, and I have no additional substantive concerns with the revised manuscript.

Reviewer #3 (Public review):

Reinwald et al. present the NoSeMaze, a semi-natural behavioral system designed to track social behaviors alongside reinforcement-learning in large groups of mice. Accumulating more than 4,000 days of behavioral monitoring, the authors demonstrate that social rank (determined by tube competitions) is a stable trait across shuffled cohorts and correlated with active chasing behaviors. The system also provides a solid platform for long-term measurements of reinforcement learning, including flexibility, response adaptation, and impulsiveness. Yet, the authors show that social ranking and chasing are mostly independent of these cognitive traits, and both seem mostly independent of oxytocin signaling in the AON.

Strengths:

(1) The neuroethological approach for automated tracking of several mice under semi-natural conditions is still rare in social behavioral research and should be encouraged.

(2) The assessment of dominance by two independent measures, i.e., spontaneous tube competitions and proactive chasing, is innovative and valuable.

(3) The integration of a long-term reinforcement-learning module into the semi-natural system provides novel opportunities to combine cognitive traits into social personality assessments.

(4) The open-source system provides a valuable resource for the scientific community.

Limitations:

(1) Apparent ambiguity and inconsistency in age structure and cohort participation across rounds, raising concerns about uncontrolled confounds.

(2) Chasing behavior appears more stable than tube-test competitions (Figure 4D vs. Figure 3D), which challenges the authors' decision to treat tube competitions as the primary basis for hierarchy determination.

Major concerns:

(1) Unclear and inconsistent handling of age groups and repeated sampling. The manuscript repeatedly refers to "younger" and "older" adults, but it is unclear whether age was ever controlled for or included in models. Some mice completed only one round, others 2-5 rounds, without explanation of the criteria or balancing.

(2) Stability of chasing appears stronger than the stability of tube competitions. Figure 4D shows highly consistent chasing behavior across weeks, while Figure 3D shows weaker and more variable correlations for tube-based David scores. This is also evident from Figure 5A-B,D. Thus, it appears that chasing, which serves to quantify dominance in similar semi-natural setups, may be a more reliable and behaviorally meaningful measure of dominance than the incidental tube competitions.

(3) Unbalanced participation across rounds compromises stability analyses. Stability analyses (e.g., ICCs, round-to-round correlations) assume comparable sampling across individuals. However, some mice contribute 1 round, others 2, 3, 4, and even 5 rounds. This imbalance may inflate stability estimates or confound group reshuffling effects, and the rationale for variable participation is not explained.

Reviewer #3 (Public review):

Summary:

The manuscript introduces a visual paradigm aimed at studying tran-saccadic memory.

The authors observe how memory of object location is selectively impaired across eye movements, whereas object colour memory is relatively immune to intervening eye movements.<br /> Results are reported for young and elderly healthy controls, as well as PD and AD participants.

A computational model is introduced to account for these results, indicating how early differences in memory encoding and decay (but not tran-saccadic updating per se) can account for the observed differences between healthy controls and clinical groups.

In the revised manuscript, the authors have addressed most of my initial concerns. The dataset is generally compelling, as it includes healthy younger and older adults as well as clinical populations. In addition, the authors propose an interesting modelling approach designed to isolate and characterize the key components underlying the observed patterns of results.

It is important to acknowledge potential limitations of the modelling approach, particularly the differences in the number of parameters across the tested models. As models with more parameters typically achieve better fit, this issue warrants careful consideration. The authors have substantially addressed this point in their rebuttal.

Concerns regarding the specificity of the findings were also raised and have been adequately discussed in the authors' response. Specifically, they clarified the selective impact of saccade-related costs on spatial working memory updating across eye movements-without affecting feature‑based memory (e.g., color) -as well as the specificity of the updating effects observed with the Rey-Osterrieth Complex Figure.

Reviewer #3 (Public review):

Genetically encoded calcium indicators (GECIs) are essential tools in neurobiology and physiology. Technological constraints in targeting and kinetics of previous versions of GECIs have limited their application at the subcellular level. Chen et al. present a set of novel tools that overcome many of these limitations. Through systematic testing in the Drosophila NMJ, they demonstrate improved targeting of GCaMP variants to synaptic compartments and report enhanced brightness and temporal fidelity using members of the GCaMP8 series. These advancements are likely to facilitate more precise investigation of synaptic physiology. This manuscript could be improved by further testing the GECIs across physiologically relevant ranges of activity, including at high frequency and over long imaging sessions. Moreover, while the authors provide a custom software package (CaFire) for Ca2+ imaging analysis, comparisons to existing tools and more guidance for broader usability are needed.

In this revised version, Chen et al. answered most of our concerns. The tools developed here will be useful for the community.

Reviewer #3 (Public review):

Summary:

To explore the diverse nature of somatosensation, Parkes et al. established and characterized a system for precise cutaneous stimulation of mice as they walk and run in naturalistic settings. This paper provides a framework for real-time body part tracking and targeted optical stimuli with high precision, ensuring reliable and consistent cutaneous stimulation. It can be adapted in somatosensation labs as a general technique to explore somatosensory stimulation and its impact on behavior, enabling rigorous investigation of behaviors that were previously difficult or impossible to study.

Strengths:

The authors characterized the closed-loop system to ensure that it is optically precise and can precisely target moving mice. The integration of accurate and consistent optogenetic stimulation of the cutaneous afferents allows systematic investigation of somatosensory subtypes during a variety of naturalistic behaviors. Although this study focused on nociceptors innervating the skin (Trpv1::ChR2 animals), this setup can be extended to other cutaneous sensory neuron subtypes, such as low-threshold mechanoreceptors and pruriceptors. This system can also be adapted for studying more complex behaviors, such as the maze assay and goal-directed movements.

Weaknesses:

Although the paper has strengths, its weakness is that some behavioral outputs could be analyzed in more detail to reveal different types of responses to painful cutaneous stimuli. For example, paw withdrawals were detected after optogenetically stimulating the paw (Figures 3E and 3F). Animals exhibit different types of responses to painful stimuli on the hindpaw in standard pain assays, such as paw lifting, biting, and flicking, each indicating a different level of pain. The output of this system is body part keypoints, which are the standard input to many existing tools. Analyzing these detailed keypoints would greatly strengthen this system by providing deeper biological insights into the role of somatosensation in naturalistic behaviors. Additionally, if the laser spot size could be reduced to a diameter of 2 mm², it would allow the activation of a smaller number of cutaneous afferents, or even a single one, across different skin types in the paw, such as glabrous or hairy skin.

Comments on revisions:

The authors successfully addressed all of my questions and concerns.

Reviewer #3 (Public review):

Summary:

The manuscript is a comprehensive molecular and cell biological characterisation of the effects of P604 hydroxylation by PHD1 on RepoMan, a regulatory subunit of the PPIgamma complex. Conclusions are generally supported by results. Overall, a timely study that demonstrates the interplay between hydroxylase signalling and the cell cycle. The study extends the scope of prolyl hydroxylase signalling beyond canonical hypoxia pathways, providing a concrete example of hydroxylation regulating PP1 holoenzyme composition and function during mitosis.

The work would benefit from additional biochemical validation of direct targeting to characterise the specificity and mode of recognition, but this is beyond the scope of the study.

Strengths:

Compelling data, characterisation of how P604 hydroxylation induces the interaction between RepoMan and a phosphatase complex, resulting in loading of RepoMan on Chromatin. Knockdown of PHD1 mimics the disruption of the complex and loss of the regulation of the hydroxylation site by PHD1, resulting in mitotic defects.

Reviewer #3 (Public review):

Summary:

This study attempts to mimic in the laboratory changing seasonal phase relationships between light and temperature and determine their effects on Drosophila circadian locomotor behavior and on the underlying splicing patterns of a canonical clock gene, timeless. The results are then extended to strains that have been selected over many years for early or late circadian phase phenotypes.

Strengths:

A lot of work, and some results showing that the phasing of behavioral and molecular phenotypes is slightly altered in the predicted directions in the selected strains.

Weaknesses:

The experimental conditions are extremely artificial, with immediate light and temperature transitions compared to the gradual changes observed in nature. Studies in the wild have shown how the laboratory reveals artifacts that are not observed in nature. The behavioral and molecular effects are very small, and some of the graphs and second-order analyses of the main effects appear contradictory. Consequently, the Discussion is very speculative as it is based on such small laboratory effects

Reviewer #3 (Public review):

Summary:

The authors present a new method for detecting and identifying proline hydroxylation sites within the proteome. This tool utilizes traditional LC-MS technology with optimized parameters, combined with HILIC-based separation techniques. The authors show that they pick up known hydroxy-proline sites and also validate a new site discovered through their pipeline.

Strengths:

The manuscript utilizes state-of-the-art mass spectrometric techniques with optimized collision parameters to ensure proper detection of the immonium ions, which is an advance compared to other similar approaches before. The use of synthetic control peptides on the HILIC separation step clearly demonstrates the ability of the method to reliably distinguish hydroxy-proline from oxidized methionine - containing peptides. Using this method, they identify a site on CDCA2, which they go on to validate in vitro and also study its role in regulation of mitotic progression in an associated manuscript.

Weaknesses:

Despite the authors claim about the specificity of this method in picking up the intended peptides, there is a good amount of potential false positives that also happen to get picked (owing to the limitations of MS-based readout), and the authors' criteria for downstream filtering of such peptides requires further clarification. In the same vein, greater and more diverse cell-based validation approach will be helpful to substantiate the claims regarding enrichment of peptides in the described pathway analyses. Experiments must show reproducibility and contain appropriate controls wherever necessary.

Comments on revisions:

I thank the authors for their clarifications and opinions on my questions and suggestions. Based on the response, the following points are important while considering the significance of this manuscript:

- The manuscript provides a novel method to detect and identify proline hydroxylation residues in the proteome. While this provides several advances over previous methods, the probability of false positives, loss of true positives and incomplete removal of the interference of methionine oxidation in this strategy need to be addressed clearly in the discussion section of the manuscript, so that the strengths and limitations of this method are made aware to the reader.

- Going by the standards of publication in eLife, reproducibility is very important in the experiments done. Hence, I strongly recommend that the authors perform the experiments in triplicate with error bars to confirm reproducibility. Graphs with single data points do not convey that, and this is very important for eLife.

- As for Figure 9C, the authors have rejected the request for a control lane in the figure. It may sound trivial to the authors, but for completeness of the experiment, all applicable controls must be performed and shown alongside the main data. It is essential to show the PHD1 only control to rule out the possibility of the contribution of any non-specific signal in the dot blot by PHD1.

Reviewer #3 (Public review):

Summary:

This work aims to understand how cells repair damage to the plasma membrane (PM). This is important as failure to do so will result in cell lysis and death. Therefore, this is an important fundamental question with broad implications for all eukaryotic cells. Despite this importance, there are relatively few proteins known to contribute to this repair process. This study expands the number of experimentally validated PM from 8 to 80. Further, they use precise laser-induced damage of the PM/cell wall and use live-cell imaging to track the recruitment of repair proteins to these damage sites. They focus on repair proteins that are involved in either exocytosis or clathrin-mediated endocytosis (CME) to understand how these membrane remodeling processes contribute to PM repair. Through these experiments, they find that while exocytosis and CME both occur at the sites of PM damage, exocytosis predominates the early stages of repairs, while CME predominates in the later stages of repairs. Lastly, they propose that CME is responsible for diverting repair proteins localized to the growing bud cell to the site of PM damage.

Strengths:

The manuscript is very well written and the experiments presented flow logically. The use of laser-induced damage and live-cell imaging to validate the proteome-wide screen using SDS induced damage strengthen the role of the identified candidates in PM/cell wall repair.

Comments on revisions:

The authors have very nicely addressed my previous comments and I have no further concerns.

Reviewer #3 (Public review):

Summary:

The paper by Raghavan et. al. describes pathways that suppress the formation of meiotic DNA double-strand breaks (DSBs) for interhomolog recombination at the end of chromosomes. Previously, the authors' group showed that meiotic DSB formation is suppressed in a ~20kb region of the telomeres in S. cerevisiae by suppressing the binding of meiosis-specific axis proteins such as Red1 and Hop1. In this study, by precise genome-wide analysis of binding sites of axis proteins, the authors showed that the binding of Red1 and Hop1 to sub-telomeric regions with X and Y' elements is dependent on Rec8 (cohesin) and/or Hop1's chromatin-binding region (CBR). Furthermore, Dot1 functions in a histone H3K79 trimethylation-independent manner, and the silencing proteins Sir2/3 also regulate the binding of Red1 and Hop1 and also the distribution of DSBs in sub-telomeres.

Strengths:

The experiments were conducted with high quality and included nice bioinformatic analyses, and the results were mostly convincing. The text is easy to read.

Weaknesses:

The paper did not provide any new mechanistic insights into how DSB formation is suppressed at sub-telomeres.

Reviewer #3 (Public review):

Programmed DNA elimination (PDE) is a process that removes a substantial amount of genomic DNA during development. While it contradicts the genome constancy rule, an increasing number of organisms have been found to undergo PDE, indicating its potential biological function. Single-cell ciliates have been used as a prominent model system for studying PDE, providing important mechanistic insights into this process. Many of those studies have focused on the excision of internally eliminated sequences (IES) and the subsequent repair using non-homologous end joining (NHEJ). These studies have led to the identification of small RNAs that mark retained or eliminated regions and the transposons that generate double-strand breaks.

In this manuscript, Nagao and Mochizuki examined the other type of breaks in ciliates that were healed with telomere addition. They specifically focused on the sequences at the ends of the germline (MIC) chromosomes, which have received relatively less attention due to the technical challenges associated with the highly repetitive nature of the sequences. The authors used the Tetrahymena model and developed a set of new tools. They used a novel FISH strategy that enables the distinction between germline and somatic telomeres, as well as the retained and eliminated DNA near the chromosome ends. This allows them to track these sequences at the cellular level throughout the development process, where PDE occurs. They also analyzed the more comprehensive germline and somatic genomes and determined at the sequence level the loss of subtelomeric and telomere sequences at all chromosome ends. Their result is reminiscent of the PDE observed in nematodes, where all germline chromosome ends are removed and remodeled. Thus, the finding connects two independent PDE systems, a protozoan and a metazoan, and suggests the convergent evolution of chromosome end removal and remodeling in PDE.

The majority of sites (8/10) at the junctions of retained and eliminated DNA at the chromosome ends contain a chromosome breakage sequence (CBS). The authors created a set of mutants that modify the CBS at the ends of chromosome 4R. CBS regions are challenging for CRISPR due to their AT-rich sequences, making the creation of the 4R-CBS mutants a significant breakthrough. They used the FISH assay to determine if PDE still occurs in these mutant strains with compromised CBS. Surprisingly, they found that instead of blocking PDE, its adjacent retained DNA is now eliminated, suggesting a co-elimination event when the breakage is impaired. Furthermore, in biparental mutant crosses, no PDE occurred, and no viable progeny were produced, indicating that the removal of chromosome ends is crucial for proper PDE and sexual progeny development. Overall, the work demonstrates a critical role for 4R-CBS in separating retained and eliminated DNA.

Reviewer #3 (Public review):

Summary:

The work by Azur and colleagues makes use of the TEMPO (Temporal Encoding and Manipulation in a Predefined Order) methodology to trace cortical neurogenesis in combination with overexpression of Imp1. Imp1 is a mammalian homologue of the Drosophila Imp, which has been shown to control temporal identity in a stem cell context. In their work, they show that overexpression of Imp1 in radial glia, which generate neurons and macroglia in a sequential manner during cortical development, leads to a disruption of faithful neuron/glia generation. They show that continuous overexpression leads to a distinct phenotypic outcome when compared to paradigms where Imp1 was specifically overexpressed in defined temporal windows, enabled by the unique TEMPO approach. Interestingly, the observed phenotype with 'ectopic' generation of mainly lower cortical layer neurons appears not to be due to migration deficits. Strikingly, the overexpression of Imp1 specifically at later stages also leads to ectopic glia-like foci throughout the developing cortical plate. Altogether, the new data provide new insights regarding the role of the post-transcriptional Imp1 regulator in controlling temporal fate in radial glia for the faithful generation of neurons and glia during cortical development.

Strengths:

The TEMPO approach provides excellent experimental access to probe Imp1 gene function at defined temporal windows. The data is very robust and convincing. The overexpression paradigm and its associated phenotypes match very well the expected outcome based on Imp1 loss-of-function. Overall, the study contributes significantly to our understanding of the molecular cues that are associated with the temporal progression of radial glia fate potential during cortical development.

Weaknesses:

The authors provide some experimental evidence, including live imaging, that deficits related to Imp1 overexpression and subsequent overabundance of lower-layer neurons, or accumulation at the subplate, appear to evolve independently of neuronal migration deficits. However, the analysis at the population level might not suffice to make the claim robust. To analyze neuronal migration in more depth, the authors could trace individual neurons and establish speed and directional parameters for comparison.

In their analysis, the authors mainly rely on temporal parameters/criteria to associate the generation of certain neuron fates. While two markers were used to identify the neuronal fate, the variance seems quite high. The authors could consider utilizing an antibody against Satb2, which would provide additional data points that could help to establish statistical significance in some of the analyses.

The analysis of glia was done at postnatal day 10, although gliogenesis and, in particular, astrocyte maturation last at least until postnatal day 28. The authors could consider extending their analysis to capture the full spectrum of their astrocyte phenotype.

Reviewer #3 (Public review):

Summary:

In this study, the authors injected a contrast agent into patients and followed the induced signal change with MRI. Doing so, they observed cerebrospinal fluid (CSF) drainage whose magnitude and dynamics varied by anatomical location and scaled with a range of cognitive and socio-demographic metrics, including sleep scores and sex.

Strengths:

I would first like to stress that I am not a specialist in the topic of that paper; so my comments should be taken with a grain of salt, and feedback from the other reviewers should also be carefully considered.

I found the text concise and the figures straightforward to understand. Although they are manually defined, the authors compared drainage across different anatomical locations, which is a positive feature. Albeit purely correlative, the attempt to connect these otherwise 'peripheral' measures to cognitive variables is quite interesting. I also particularly liked the last paragraph of the discussion, which listed the main limitations of the study.

Weaknesses:

In the paragraph starting at line 446, the authors interpret poor sleep quality as being a cause and a consequence of impaired CSF clearance, but their approach is purely correlational. In other words, a third variable could be driving both of these parameters (correct?), thereby explaining their correlation. Later, they also proposed that therapeutically altering CSF clearance could improve cognitive symptoms, but, again, if there's a hidden cause of the correlation, that does not seem like a valid possibility. I believe there were other instances of this sort of inferential problem in the Discussion. It seems essential, particularly in clinical research, to precisely identify what the available evidence supports (correlation) and what is speculation (causation).

Assuming I did not miss it, the approach for testing and reporting correlations is not specified. In particular, the authors report correlation with CSF drainage and a variety of other metrics. But how many tests did the authors perform? They solely mention that they used the Benjamini-Hochberg method to correct for multiple comparisons. How were the decisions to test for this or that effect determined? Or did they test all the metrics they had? Also, that particular correction method is limited when statistics are negatively correlated. It would be helpful to validate findings with another approach.

I assume many of the metrics the authors use are also correlated with one another. Is it possible that a single principal component is driving the different correlations they see? Performing dimensionality reduction across available metrics and relating the resulting principal components to CSF drainage would help clarify the forces at play here.

In their interpretations, the authors claim that the CSF drainage they observe occurs through the bone marrow of the skull. How confident can we be in that claim? Is it that there are no other likely possibilities? It might be an unnecessary question, but given there seems to be no causal intervention (which is fine), and no consideration of alternatives, I am wondering whether this is because other possibilities are improbable or whether they were not adequately considered.

Reviewer #3 (Public review):

Summary:

Qiu et al. present a hierarchical framework that combines AI and molecular dynamics simulation to design an α-helical protein with enhanced thermal, chemical, and mechanical stability. Strategically, chemical modification by incorporating additional α-helix, site-specific salt bridges, and metal coordination further enhanced the stability. The experimental validation using single-molecule force spectroscopy and CD melting measurements provides fundamental physical chemical insights into the stabilization of α-helices. Together with the group's prior work on super-stable β strands (https://www.nature.com/articles/s41557-025-01998-3), this research provides a comprehensive toolkit for protein stabilization. This framework has broad implications for designing stable proteins capable of functioning under extreme conditions.

Strengths:

The study represents a complete framework for stabilizing the fundamental protein elements, α-helices. A key strength of this work is the integration of AI tools with chemical knowledge of protein stability.<br /> The experimental validation in this study is exceptional. The single-molecule AFM analysis provided a high-resolution look at the energy landscape of these designed scaffolds. This approach allows for the direct observation of mechanical unfolding forces (exceeding 200 pN) and the precise contribution of individual chemical modifications to global stability. These measurements offer new, fundamental insights into the physicochemical principles that govern α-helix stabilization.

Weaknesses:

(1) The authors report that appending an additional helix increases the overcall stability of the α-helical protein. Could the author provide a more detailed structural explanation for this? Why does the mechanical stability increase as the number of helixes increase? Is there a reported correlation between the number of helices (or the extent of the hydrophobic core) and the stability?

(2) The author analyzed both thermal stability and mechanical stability. It would be helpful for the author to discuss the relationship between these two parameters in the context of their design. Since thermal melting probes equilibrium stability (ΔG), while mechanical stability probes the unfolding energy barriers along the pulling coordinate.

(3) While the current study demonstrates a dramatic increase in global stability, the analysis focuses almost exclusively on the unfolding (melting) process. However, thermodynamic stability is a function of both folding (kf) and unfolding (ku) rates. It remains unclear whether the observed ultrastability is primarily driven by a drastic decrease in the unfolding rate (ku) or if the design also maintains or improves the folding rate (kf)?

(4) The authors chose the spectrin repeat R15 as the starting scaffold for their design. R15 is a well-established model known for its "ultra-fast" folding kinetics, with folding rates (kf ~105s), near three orders of magnitude faster than its homologues like R17 (Scott et.al., Journal of molecular biology 344.1 (2004): 195-205). Does the newly designed protein, with its additional fourth helix and site-specific chemical modifications, retain the exceptionally high folding rate of the parent R15?

Reviewer #3 (Public review):

In this study, Chen L et al. systematically analyzed the mycobacterial nucleomodulins and identified MgdE as a key nucleomodulin in pathogenesis. They found that MgdE enters into host cell nucleus through two nuclear localization signals, KRIR108-111 and RLRRPR300-305, and then interacts with COMPASS complex subunits ASH2L and WDR5 to suppress H3K4 methylation-mediated transcription of pro-inflammatory cytokines, thereby promoting mycobacterial survival.

Comments on revisions:

The authors have adequately addressed previous concerns through additional experimentation. The revised data robustly support the main conclusions, demonstrating that MgdE engages the host COMPASS complex to suppress H3K4 methylation, thereby repressing pro-inflammatory gene expression and promoting mycobacterial survival. This work represents a significant conceptual advance.

Reviewer #3 (Public review):

Summary:

AutoMorphoTrack is a Python package for quantitatively evaluating organelle shape, movement, and colocalization in high-resolution live cell imaging experiments. It is designed to be a beginning-to-end workflow from segmentation through metric graphing, which is easy to implement. The paper shows example results from their images of mitochondria and lysosomes within cultured neurons, demonstrating how it can be used to understand organelle processing.

Strengths:

The text is well-written and easy to follow. I particularly appreciate tables 1 and 2, which clearly define the goals of each module, the tunable parameters, and the input and outputs. I can see how the provided metrics would be useful to other groups studying organelle dynamics. Additionally, because the code is open-source, it should be possible for experienced coders to use this as a backbone and then customize it for their own purposes.

Weaknesses:

Unfortunately, I was not able to install the package to test it myself using any standard install method. This is likely fixable by the authors, but until a functional distribution exists, the utility of this tool is highly limited. I would be happy to re-review this work after this is fixed.

The authors claim that there is "AI-Assisted Execution and Natural-Language Interface". However, this is never defended in any of the figures, and from quickly reviewing the .py files, there does not seem to be any built-in support or interface for this. Without significantly more instructions on how to connect this package to a (free) LLM, along with data to prove that this works reproducibly to produce equivalent results, this section should be removed.

Additionally, I have a few suggestions/questions:

(1) Red-green images are difficult for colorblind readers. I recommend that the authors change all raw microscopy images to a different color combination.

(2) For all of the velocity vs displacement graphs (Figure 3C and subpart G of every supplemental figure), there is a diagonal line clearly defining a minimum limit of detected movement. Is this a feature of the dataset (drift /shakiness /etc) or some sort of minimum movement threshold in the tracking algorithm? This should be discussed in the text.

(3) Integrated Correlation Summary (Figure 5) - Pearson is likely the wrong metric for most of these metric pairs because even interesting relationships may be non-linear. Please replace with Spearman correlation, which is less dependent on linearity.

Reviewer #3 (Public review):

Summary:

Authors mapped monosynaptic inputs to dopamine, GABA, and glutamate neurons in the ventral tegmental area (VTA) under different anesthesia methods, and under drug (cocaine, morphine, methamphetamine, amphetamine, nicotine, fluoxetine). First, they propose an analysis method to separate the actual manipulation effects from the variability caused by experimental procedures. Using this method, they found differences in the anatomical location of monosynaptic inputs to dopamine neurons under different conditions, and identified some key brain areas for such separation. They also searched the database for gene expression patterns that are common across input brain areas, with some changes by anesthesia or drug administration.

Strengths:

The whole-brain approach to address drug effects is appealing, and their conclusion is clear. The methodology and motivation are clearly explained.

Weaknesses:

While gene expression analyses may not be related to their findings on the anatomical effects of drugs, this is a nice starting point for follow-up studies.

Reviewer #3 (Public review):

Summary:

The authors demonstrate that CRF neurons in the extended amygdala form GABAergic synapses on to cholinergic interneurons and that CRF can excite these neurons. The evidence is strong, however the authors lack to make a compelling connection showing CRF released from these extended amygdala neurons is mediating any of these effects. Further, they show that acute alcohol appears to modulate this action, although the effect size is not particularly robust.

Strengths:

This is an exciting connection from the extended amygdala to the striatum that provides a new direction for how these regions can modulate behavior. The work is rigorous and well done.

Weaknesses:

The effects of acute ethanol are modest but consistent, the potential role of this has yet to be determined. Further, the opto stim experiments are conducted in an ai32 mouse, so it is impossible to determine if that is from CEA and BNST, vs. another population of CRF containing neurons. This is an important caveat that was acknowledged.

Reviewer #3 (Public review):

Summary:

This study examines how dominance hierarchy influences innate defensive behaviors in pair-housed male mice exposed to two types of naturalistic threats: a transient looming stimulus and a sustained live rat. The authors show that social presence reduces fear-related behaviors and promotes active defense, with dominant mice benefiting more prominently. They also demonstrate that threat exposure reinforces social roles and increases group cohesion. The work highlights the bidirectional interaction between social structure and defensive behavior.

Strengths:

This study makes a valuable contribution to behavioral neuroscience through its well-designed examination of socially modulated fear. A key strength is the use of two ethologically relevant threat paradigms - a transient looming stimulus and a sustained live predator, enabling a nuanced comparison of defensive behaviors. The experimental design is robust, systematically comparing animals tested alone versus with their cage mate to cleanly isolate social effects. The behavioral analysis is sophisticated, employing detailed transition maps that reveal how social context reshapes behavioral sequences, going beyond simple duration measurements. The finding that social modulation is rank-dependent adds significant depth, linking social hierarchy to adaptive defense strategies. Furthermore, the demonstration that threat exposure reciprocally enhances social cohesion provides a compelling systems-level perspective. Together, these elements establish a strong behavioral framework for future investigations into the neural circuits underlying socially modulated innate fear.

Weaknesses:

The study exhibits several limitations. The neural mechanism proposed is speculative, as the study provides no causal evidence.

Reviewer #3 (Public review):

Summary:

The aim of this study was to investigate the temporal progression of the neural response to event boundaries in relation to uncertainty and error. Specifically, the authors asked 1. How neural activity changes before and after event boundaries 2. If uncertainty and error both contribute to explaining the occurrence of event boundaries and 3. If uncertainty and error have unique contributions to explaining the temporal progression of neural activity.

Strengths:

One strength of this paper is that it builds on an already validated computational model. It relies on straightforward and interpretable analysis techniques to answer the main question, with a smart combination of pattern similarity metrics and FIR. This combination of methods may also be an inspiration to other researchers in the field working on similar questions. The paper is well written and easy to follow. The paper convincingly shows that 1. There is a temporal progression of neural activity change before and after an event boundary 2. Event boundaries are predicted best by the combination of uncertainty and error signals.

Weaknesses:

Regarding question 3, the results are less convincing. Although the analyses in Figure S1 show that there are some unique contributions of uncertainty and error, it is unclear to what extent the results in Figure 7 are driven by shared variance. Therefore, it is not clear to what extent the main claim in the abstract is due to shared or unique variance. More specific comments are provided below.

The other issue is the distance between events is short compared to the pre-onset effects that are observed. Halfway the distance between two events there are already neural signatures of change relating to the upcoming event boundary. I wonder if methodological issues could explain this effect and if not, what could allow participants to notice the impending event boundary.

Impact:

If these comments can be addressed sufficiently, I expect that this work will impact the field in its thinking on what drives event boundaries and spur interest in understanding the mechanisms behind the temporal progression of neural activity around these boundaries.

Comments

(1) The correlation between uncertainly and prediction error is very high, which makes it challenging to disentangle the effects of both on the neural response. The analysis in Figure S1 shows that the two predictors indeed have dissociable contributions. However, the results mainly reported in the discussion section and abstract still rely on models where only one of these factors is included at a time. This makes it debatable whether these specific networks mentioned really reflect unique contributions of each of these components. I specifically refer to this statement in the abstract: "Error-driven boundaries were associated with early pattern shifts in ventrolateral prefrontal areas, followed by pattern stabilization in prefrontal and temporal areas. Uncertainty-driven boundaries were linked to shifts in parietal regions within the dorsal attention network, with minimal subsequent stabilization. ". I would encourage repeating all analyses (also the ones in figure 7) with a models that includes both predictors and showing both results in the manuscript, so it is clear which regions really show unique variance related to one of the predictors. I also wonder why it is necessary to look at model comparisons between the combined and unique models, rather than simply reporting the significance of each predictor in the combined model.

(2) The distance between event boundaries ranges between 20 and 30 seconds. The early pre-boundary effect that are observed in the manuscript occur at -12 seconds. This means that these effects occur roughly halfway between the previous and current event. This seems much earlier than expected. That is why I worry that the FIR analyses might not be able to distinguish effects of the previous event from effects of the upcoming event. What evidence is there that the FIR analyses can actually properly show the return to baseline? One way to address this might be to randomize the locations of the event boundaries while preserving the distance between them and rerun the models. This will give a null-model with the same event distances and should be able to distinguish this temporal overlap from the true effects of event boundaries.

(3) If the analyses in point 2 confirm that there is indeed an event-boundary related change that occurs 12 seconds before event onset, it is important to consider what might cause these changes. Are there cues in the movie that indicate that an event boundary is coming? It would be interesting to investigate whether uncertainty and error are higher than expected at 12 seconds pre-onset.

Reviewer #3 (Public review):

Summary:

In this study, the authors investigate single neuron activity in the subthalamic nucleus (STN) of two monkeys performing a perceptual decision-making task in which both perceptual evidence and reward were manipulated. They find rich representations of decision variables (such as choice, perceptual evidence and reward) in neural activity, and following prior work, cluster a subset of these neurons into subpopulations with varying activity profiles. Further, they relate the activity of neurons within these clusters to parameters of drift diffusion models (DDMs) fit to animal behaviour on trial subsets by neural firing rates, finding heterogeneous and temporally varying relationships between different clusters and DDM parameters, suggesting that STN neurons may play multiple roles in decision formation and evaluation.

Strengths:

The behavioural task used by the authors is rich and affords disambiguation between decision variables such as perceptual evidence, value and choice, by independently manipulating stimulus strength and reward size. Both their monkeys show good performance on the task, and their population of ~150 neurons across monkeys reveals a rich repertoire of decision-related activity in single neurons, with individual neurons showing strong tuning to choice, stimulus strength and reward bias. There is little doubt that neurons in the STN are tuned to several decision variables and show heterogeneous tuning profiles.

Weaknesses:

The primary weakness of the paper lies in the claim that STN contains multiple sub-populations with distinct involvements in decision making, which is inadequately supported by the paper's methods and analyses.

First, while it is clear that the ~150 recorded neurons across 2 monkeys (91, 59 respectively) display substantial heterogeneity in their activity profiles across time and across stimulus/reward conditions, the claim of sub-populations largely rests on clustering a *subset of less than half the population - 66 neurons (48, 15 respectively) - chosen manually by visual inspection*. The full population seems to contain far more decision-modulated neurons, whose response profiles seem to interpolate between clusters. Moreover, it is unclear if the 4 clusters hold for each of the 2 monkeys, and the choice of 4-5 clusters does not seem well supported by metrics such as silhouette score, etc, that peak at 3 (1 or 2 were not attempted). From the data, it is easier to draw the conclusion that the STN population contains neurons with heterogeneous response profiles that smoothly vary in their tuning to different decision variables, rather than distinct sub-populations.

Second, assuming the existence of sub-populations, it is unclear how their time- and condition-varying relationship with DDM parameters is to be interpreted. These relationships are inferred by splitting trials based on individual neurons' firing rates in different task epochs and reward contexts, and regressing onto the parameters of separate DDMs fit to those subsets of trials. The result is that different sub-populations show heterogeneous relationships to different DDM parameters over time - a result that, while interesting, leaves the computational involvement of these sub-populations/implementation of the decision process unclear.

Outlook:

This is a paper with a rich dataset of neural activity in the STN in a rich perceptual decision-making task, and convincing evidence of heterogeneity in choice, value and evidence tuning across the STN, suggesting the STN may be involved in several aspects of decision-making. However, the authors' specific claims about sub-populations in the STN, each having distinct relationships to decision processes, are not adequately supported by their analyses.

Reviewer #3 (Public review):

I read this manuscript with great interest. The purpose of this paper is to use human intracranial recordings in patients undergoing routine epilepsy surgery evaluation to investigate speech production and perception during five specific and controlled tasks (auditory perception, picture perception, reading perception, speech production, and baseline). Linear classifiers were used to decode specific states with a mean accuracy of 64.4%. The interpretation of these findings is that the classifiers reveal distinct network signatures "underlying auditory and visual perception as well as speech production." Perhaps the most interesting finding is that the network signatures, including both regions with robust local neuronal activity and those without. Further, this study addresses an important gap by examining functional connectivity during overt speech production.

The abbreviation ECoG is used throughout the manuscript, and the methods state that grids and strips were placed, though many epilepsy centers now employ intracerebral recordings. Does this manuscript only include patients with surface electrodes? Or are depth electrodes also included? The rendering maps show only the cortical surface, but depth recordings could be very interesting, given that this is a connectivity analysis.

Also interesting, given both the picture and reading task, is whether there is coverage of the occipitotemporal sulcus?

A major strength of the chosen paradigm is the combination of both perception (auditory or visual) and production (speech). Have the authors considered oculomotor EMG artifacts that can be associated with the change in visual stimuli during the task (see Abel et al. for an example PMID: 27075536, but see also PMID: 19234780 and PMID: 20696256).

I'm very interested in the findings in Figure 4D, with regard to the temporal pole. I would recommend that the authors unpack what it means that the ratio of electrodes with the strongest connections is highest, but active and discriminative is perhaps the lowest. We (I think many groups!) are interested in this region as a multimodal hub that provides feedback in various contexts (like auditory or visual perception).

Given the varieties of tasks and the fact that electrodes are always placed based on clinical necessity, are there concerns about electrode sampling bias?

This manuscript makes an important contribution by demonstrating that functional connectivity analysis reveals task-specific network signatures beyond what is captured by local neuronal activity measures (LFP). The finding that low-activity regions are engaged in task-specific classifications has important implications for future human LFP connectivity work.

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6 bids including h*3 at 41.00 and r1 at 72.02 without an actual purchase

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Reviewer #3 (Public review):

Summary:

Brands and colleagues investigate how temporal adaptation can aid object recognition, and what neural computations may underlie these effects. They employed a previously published experimental paradigm to study how adaptation to temporally constant distractor input facilitates the recognition of a newly appearing target object. Specifically, they studied how this effect is modulated by the contrast of the target object.

They found that adaptation enhances the recognition of high-contrast objects more than that of low-contrast objects. This behavioral effect was mirrored by a larger effect of adaptation on the response to the high-contrast objects in relatively higher visual areas.

To investigate what neural computations can support this interaction, they implement several candidate neural mechanisms in a deep convolutional neural network: additive suppression, divisive suppression, and lateral recurrence. The authors conclude that divisive and additive suppression, which are intrinsic to the neuron, best explain the interaction between contrast and adaptation in the human data. They further show that these mechanisms, and divisive suppression in particular, show increased robustness to spatial shifts of the adaptor stimulus, hinting and potential perceptual benefits.

Strengths:

(1) Overall, this is a well-written paper, supported by thorough analyses and illustrated with clear, well-designed figures that effectively show overall trends as well as data variance. The authors tell a compelling story while responsibly steering away from overreaching conclusions.

(2) What makes this paper stand out is its comprehensive approach to understanding the behavioral benefit of neural adaptation and its mechanistic underpinnings. The authors effectively achieve this through integrating new behavioral and neural data with simulations using neural network models.

(3) The findings convincingly demonstrate that neuronally intrinsic adaptation mechanisms are sufficient to explain the observed interaction between temporal adaptation, contrast, and object recognition. Furthermore, the paper highlights that these intrinsic mechanisms offer superior robustness compared to learned lateral recurrence mechanisms, which, while being more expressive, can also be more brittle.

Weaknesses:

While the results and conclusion are well supported, there were a few major points that need clarification for me.

(1) Divisive normalization

I was confused by the author's classification of divisive normalization as a neuronally intrinsic mechanism, that is, one that operates within a single neuron, independent of interactions with other neurons.

My understanding is that divisive normalization, as originally proposed by Heeger in the early nineties, describes a mechanism where neurons integrate pooled activity from neighboring cells to mutually inhibit one another. In this form, divisive normalization is fundamentally an interneuronal mechanism involving recurrence. Adding to the confusion, the authors highlight in the introduction their interest in divisive normalization for its relation to stimulus contrast, a relation likely linked to neuronal pooling.

However, my reading of the methods section (Equations 6 and 7) suggests the authors implemented only a temporal feedback component, leaving out the pooling across neurons (Equation 5). This distinction should be disambiguated early in the paper. I recommend choosing a less ambiguous term than "divisive normalization". Even "temporal divisive normalization" is still ambiguous, as lateral neuronal interactions are also inherently temporal.

(2) Parietal electrodes

The paper's adapter-specific effects are centered around the P9/P10 electrodes, which the authors identify as "parietal." However, it is unclear to me which part of the cortex drives these electrodes, particularly whether it is actually the parietal cortex. I am no expert in EEG, but based on the topomaps in Figures 4 and 5, it appears that these electrodes cover more posterior occipito-temporal regions rather than truly parietal regions. Given the central role of P9/P10 to the main findings, the paper would be significantly improved for non-EEG readers by clarifying which cortical regions are covered by these electrodes.

(3) Interpretation of non-significant statistical results

In some places, the authors attach relatively strong claims to non-significant statistical results. For example, in Figure 5D, they claim that there is no effect of contrast on occipital electrodes, based on a non-significant p-value. P-values do not quantify evidence for the null hypothesis, so the authors should be careful with such claims. In fact, Figure 5D shows such a clear negative slope, with variance comparable to Figure 5A, that I am surprised that the p-value for the slope of Figure 5D was in fact so large. A similar issue arises in the discussion for Figure 6, where the authors claim that the effect of contrast is adapter-specific. However, this claim is based on the observation that is significant for same-noise trials, but not for different-noise or blank trials. To statistically substantiate the claims that there is an adapter-specific effect, the authors should directly compare the slope for same-noise trials with the slope for different-noise/blank trials.

(4) The match between behavior and models

The authors' claim that models with intrinsic adaptation better match the interaction between contrast and temporal adaptation observed in human behavior is not fully substantiated. This conclusion appears to be based on a qualitative assessment of Figure 8, which, in my view, does not unambiguously rule out an interaction for lateral recurrence. Furthermore, a potential confounding factor is the ceiling effect that limits higher accuracy values. Indeed, conditions where the interaction was not/less (i.e., shorter time sequences and lateral inhibition) are also the conditions where accuracy values are closer to this ceiling, which may mask a potential interaction.

Reviewer #3 (Public review):

Summary:

The authors provide a highly valuable and thoroughly documented pipeline to accelerate the processing and spike sorting of high-density electrophysiology data, particularly from Neuropixels probes. The scale of data collection is increasing across the field, and processing times and data storage are growing concerns. This pipeline provides parallelization and benchmarking of performance after data compression that helps address these concerns. The authors also use their pipeline to benchmark different spike sorting algorithms, providing useful evidence that Kilosort4 performs the best out of the tested options. This work, and the ability to implement this pipeline with minimal effort to standardize and speed up data processing across the field, will be of great interest to many researchers in systems neuroscience.

Strengths:

The paper is very well written and clear in most places. The accompanying GitHub and ReadTheDocs are well organized and thorough. The authors provide many benchmarking metrics to support their claims, and it is clear that the pipeline has been very thoroughly tested and optimized by users at the Allen Institute for Neural Dynamics. The pipeline incorporates existing software and platforms that have also been thoroughly tested (such as SpikeInterface), so the authors are not reinventing the wheel, but rather putting together the best of many worlds. This is a great contribution to the field, and it is clear that the authors have put a lot of thought into making the pipeline as accessible as possible.

Weaknesses:

There are no major weaknesses. I have only a handful of very minor questions and suggestions that could clarify/generalize aspects of the pipeline or make the text more understandable to non-specialists.

(1) Could the authors please expand on the statement on line 274, that processing their test dataset serially "on a single GPU-capable cloud workstation... would take approximately 75 hours and cost over 90 USD." How were these values calculated? I was a bit surprised that this is a >4-fold slow-down from their pipeline, but only increases the cost by ~1.35x, if I understood correctly. More context on why this is, and maybe some context on what a g4dn.4xlarge is compared to the other instances, might help readers who are less familiar with AWS and cloud computing.

(2) One of the most commonly used preprocessing pipelines for Neuropixels data is the CatGT/ecephys pipeline from the developers of SpikeGLX at Janelia. It may be worth commenting very briefly, either in the preprocessing section or in the discussion, on how the preprocessing steps available in this pipeline compare to the steps available in CatGT. For example, is "destriping" similar to the "-gfix" option in catGT to remove high-amplitude artifacts?

(3) Why are there duplicate units (line 194), and how often is this an issue? I understand that this is likely more of a spike sorter issue than an issue with this pipeline, but 1-2 sentences elaborating why might be helpful for readers.

(4) It seems from the parameter files on GitHub that the cluster curation parameters are customizable - correct? If so, it may be worth explicitly saying so in the curation section of the text, as the presented recipe will not always be appropriate. A presence ratio of >0.8 could be particularly problematic for some recordings, for example, if a cell is only active during a specific part of the behavior, that may be a feature of the experiment, or the animal could be transitioning between sleep and wake states, in which different units may become active at different times.

(5) The axis labels in Figures 3d-e are too small to see, and Figure 3d would benefit from a brief description of what is shown.

(6) What is the difference between "neural" and "passing QC" in Figure 4?

(7) I understand the current paper is focused on spike data, so there may not be an answer to this, but I am curious about the NP2.0 probes that save data in wideband. Does the lossy compression negatively affect the LFP data? Is software filtering applied for the spike band before or after compression?

Reviewer #3 (Public review):

Summary:

This study uses high-resolution videography and a custom computer-vision pipeline to dissect the motor control of cephalopod chromatophores in Euprymna berryi and Sepia officinalis. By quantifying anisotropic chromatophore deformations and applying dimensionality reduction methods, the authors infer that individual chromatophores can be a part of multiple motor units. Clustering analyses reveal putative motor units that often span multiple chromatophores, with diverse and overlapping geometries. Chromatophore expansion dynamics are faster and more stereotyped than relaxation, consistent with active neural contraction followed by passive recoil. Together, the results show that chromatophores function not as uniform pixels but as fractionated, coordinately controlled elements that enable flexible pattern generation

Strengths:

The authors present compelling, direct evidence that a). chromatophore deformations are anisotropic, and indirect evidence that b) individual chromatophores can be split across multiple putative motor units. This evidence is provided through data collected over large spatial scales, but also at a sub-chromatophore resolution. This combination of scale and resolution is not possible using traditional neuroanatomical and physiological approaches alone.

The authors also develop a new non-invasive, image analysis approach to extract information about chromatophore deformation across large spatial scales on the organism's body. In principle, this approach is applicable across species and may allow for further comparative characterization of chromatophore motor control. It is therefore a promising new tool and useful resource for the community.

Weaknesses:

An important weakness of the work is that the methods the authors develop can only be applied during resting, spontaneous 'flickering' activity of chromatophores. The inability to reliably apply their technique during any kind of realistic camouflage is a large limitation, as it means this method cannot be used to study the dynamics of motor control during realistic camouflage behaviors.

Another weakness of this paper is the rather limited electrophysiological validation of the computational findings. The authors present only one electrophysiology experiment in E. berryi, the species that they used only for 'methodological development' and not for detailed characterization. A complementary electrophysiological experiment in S. officinalis, or some visualization of neuron morphology confirming that motor neurons do indeed project to multiple chromatophores, would strengthen the generalizability of their computational analysis. This would be particularly pertinent to validate the author's claim that some motor units contain chromatophores that are quite distant from one another on the animal.

Overall, the authors' technical contributions and method development are an important advance. This work serves as an excellent proof of concept that their method can extract useful information about chromatophore motor control. Further validation of their method is needed to fully trust the fine-scale conclusions drawn about the distribution and composition of multi-innervated chromatophores. Furthermore, the authors raise many interesting ideas about developmental constraints on circuit wiring and potential adaptive significance of multi-innervated chromatophores for certain features of camouflage patterning. Their method may be able to help resolve some of these questions in the future if it is refined and applied across developmental stages, regions of the animal, and across species

• Effect Size Comparison: The author presents research suggesting that Vitamin D and Omega-3 (specifically EPA) have larger effect sizes on depression than many standard antidepressants and psychotherapies.
• Recommended Dosages:
  • Vitamin D: ~4000 IU daily (the official safe limit), with a note that 10,000 IU has been used in trials without lasting adverse effects.
  • Omega-3: ~1500 mg daily of high-EPA Omega-3 (where EPA is at least 60% of the total).
• Stacking Interventions: The author emphasizes that supplements are not necessarily a "replacement" for medication but can be used alongside them ("stacked") to improve outcomes.
• Other High-Impact Factors: Beyond supplements, the post highlights other lifestyle interventions with significant effect sizes, including good sleep (1.10), aerobic exercise (0.79), and bright light therapy (0.48).
• The "Vicious Cycle": Depression is described as a negative feedback loop where low energy leads to low-effort choices, which further drains energy; intervention is needed to break this cycle.
• Personal Philosophy: The author views depression as a combination of biology and "mind-debugging," advocating for evidence-based tools like Cognitive Behavioral Therapy (CBT) and tracking progress with metrics like the Burns Depression Checklist.

Hacker News Discussion

• Skepticism of Effect Sizes: Many commenters warn that the "massive" effect sizes for supplements often come from small, low-quality studies and typically shrink or disappear in larger, more rigorous clinical trials.
• Causality Concerns: Users pointed out that low Vitamin D levels might be a symptom of depression (staying indoors, poor diet) rather than the primary cause, making supplementation less of a "magic bullet" than the data might suggest.
• Pharma vs. Supplements: Some criticized the "supplement-over-pharma" narrative, noting that while antidepressants have a smaller effect size on paper, they are life-saving for many, whereas supplements often fail to show significance in meta-analyses.
• Bioavailability and Specificity: Discussion touched on the importance of specific forms of supplements, such as D3 vs. D2 and the critical EPA/DHA ratio in fish oil, echoing the author's point about EPA-dominant Omega-3.
• The "Small Study Effect": Several comments highlighted the "file drawer problem," where only small studies with positive, outlier results get published, leading to an exaggerated perception of efficacy for natural remedies.

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h****3 $265 win (by $1) on 2026-01-26 on a brown Smith-Corona Galaxie typewriter with a script typeface

Cross reference: https://www.reddit.com/r/typewriters/comments/1qo4us4/why_is_this_going_for_so_much/

Reviewer #3 (Public review):

Summary:

This study by Griciunaite et al. investigates the function of the adhesion molecule Jam2 in initiating the formation of organ (intestinal)-specific vasculature in zebrafish. Their previous studies identified a group of late-forming vascular progenitors from the lateral plate mesoderm along the yolk extension termed the secondary vascular field (SVF), which can contribute to intestinal vasculature. Transcriptomic analysis of the zebrafish trunk region identified SVF-enriched marker genes, which include jam2b. They then performed expression analysis of jam2b using whole-mount in situ hybridization and Gal4 knock-in transgenic line analysis. These analyses show that jam2b is expressed in the SVF cells that correspond to etv2 and kdrl expression past 24 hours. Lineage tracing combining jam2b:Gal4 and UAS:Cre or UAS:CreERT2 show the contribution of jam2b in SVF and intestinal vasculature formation. jam2b mutations did not cause observable defects in the vasculature, but combined jam2a; jam2b mutations led to impaired ISV, PCV, SIA, SIV and thoracic duct lymphatic vasculature formation. Finally, the authors show that mutations in the transcription factor hand2 led to reduced jam2b expression and impaired SVF formation.

Strengths:

The authors accomplished many feats in generating new reporter lines and mutations that are valuable to the community. The study provided an interesting perspective on organ-specific vascular development and origin heterogeneity. The genetic aspects of the study are clean, and the mutational phenotypes are convincing.

Several suggestions and major comments that can improve the manuscript include:

(1) Overall molecular mechanisms of Jam2 function are not fully uncovered in the study. How do the adhesion molecules Jam2a and Jam2b regulate SVF cell formation? Are they responsible for migration, adhesion or fate determination of these structures? The authors should provide a more in-depth study of the jam2a, jam2b mutations and assess the processes affected in these mutants. Combining these mutants with etv2:Kaede can also provide a stronger causative link between their functions and defects in SVF formation.

(2) Have the authors tested the specificity of the jam2b knock-in reporter line? This is an important experiment, as many of the conclusions derive from lineage tracing and fluorescence reporting from this knock-in line. One suggestion is to cross the jam2b:GFP or jam2b:Gal4, UAS:GFP line to the generated jam2b mutants, and examine the expression pattern of these lines. Considering that the ISH experiment showed lack of jam2b expression, the reporter line should not be expressed in the jam2b mutants.

(3) The rationale behind the regeneration study is not clear, and the mechanisms underlying the phenotype are not well described. How do the authors explain the phenotype with the impaired regeneration, and what is the significance of this finding as it relates to SVF formation and function?

(4) The authors need to include representative images of jam2b>CreERT2 with 4-OH activation at different timepoints in Figure 3.

(5) The etv2:Kaede photoconversion experiment to show that the majority of intestinal vasculature derives after 24 hours needs to be supplemented with additional data on photoconverted post-24-hour-old endothelial cells, with the expectation that the majority of intestinal endothelial cells at 4 days will then be labeled with red Kaede. In addition, there have been data that show the red Kaede protein is not stable past several days in vivo, and 3 days might be sufficient for the removal or degradation of this photoconverted protein. Thus, the statement that intestinal vasculature forms largely by new vasculogenesis might be too strong based on existing data.

(6) To strengthen the claim that hand2 acts upstream of jam2b, the authors can perform combinatorial genetic epistatic analysis and examine whether jam2b mutations worsen hand2 homozygous or heterozygous effects on the SVF. Similarly, overexpressing jam2b might rescue the loss of SVF/etv2 expression in hand2 mutants.

Reviewer #3 (Public review):

Summary:

The manuscript by Poh et al. investigated whether dopamine release in the ventral medial striatum integrates information about action selection, controllability of reward pursuit, effort, and reward approach. Rats were implanted with FSCV probes and trained in four Go/No Go task variants:

(1) trials were self-initiated and had two trial types (Go vs. No Go) that were auditorily cued,

(2) trials were cue-initiated and had two trial types (Go vs. No Go) that were auditorily cued,

(3) trials were self-initiated and had three trial types (Go vs. No Go vs. free reward) that were auditorily cued, and effort was increased,

(4) trials were cue-initiated and had three trial types (Go vs. No Go vs. free reward) that were auditorily cued.

The authors report that dopamine levels rose during Go trials and slowly rose in No Go trials, but this pattern did not differ across task variants that modified effort and whether trials were cued or initiated. They also report that dopamine levels rose as rats approached the reward location and were greater in rats that bit the noseport while holding during the No Go response.

Strengths:

(1) Interesting task and variants within the task paradigm that would allow the authors to isolate specific behavioral metrics.

(2) The goal of determining precisely what VMS dopamine signals do is highly significant and would be of interest to many researchers.

Weaknesses:

(1) This Go/No-Go procedure is different from the traditional tasks, and this leads to several problems with interpreting the results:

(a) Go/No Go tasks typically require subjects to refrain from doing any action. In this task, a response is still required for the No Go trials (e.g., continue holding the nosepoke). The problem with this modified design is that failure to withhold a response on No Go trials could be because i) rats could not continue holding the response, as holding responses are difficult for rodents, or ii) rats could not suppress the prepotent go response. This makes interpreting the behavior and the dopamine signal in No Go trials very difficult.

(b) Most Go/No Go tasks bias or overrepresent Go trials so that the Go response is prepotent, and consequently, successful suppression of the Go response is challenging. I didn't see any information in the manuscript about how often each trial type was presented or how the authors ensured that No Go responses (or lack thereof) were reflecting a suppression of the Go response.

(2) The authors observe relatively consistent differences in the DA signal between Go and No Go trials after the action-cue onset. However, the response type was not randomized between trial type, so there is a confound between trial type (Go/No Go) and response (lever/nosepoke). The difference in DA signal may have nothing to do with the cue type, but reflects differences in DA signal elicited by levers vs. nosepokes.

(3) Both Go and No Go trials start with the rat having their nose in the noseport. One cue (Go cue) signals the rat to remove their nose from the noseport and make two lever responses in 5 seconds, whereas the other cue (No Go cue) signals the rat to keep their nose in the noseport for an additional 1.7-1.9 s. The authors state that the time between cue onset and reward delivery was kept the same for all trial types, and Figure 1 suggests this is 2 s, so was reward delivered before rats completed the two lever presses? I would imagine reward was only delivered if rats completed the FR requirement, but again, the descriptions in the text and figures are incongruent.

(4) The manuscript is difficult to understand because key details are not in the main text or are not mentioned at all. I've outlined several points below:

(a) The author's description in the manuscript makes it appear as a discrimination task versus a Go/No Go task. I suggest including more details in the main text that clarify what is required at each step in the task. Additionally, providing clarity regarding what task events the voltammetry traces are aligned to would be very useful.

(b) How many subjects were included in each task variant? The text makes it seem like all rats complete each task variant, but the behavioral data suggest otherwise. Moreover, it appears that some rats did more than one version. Was the order counterbalanced? If not, might this influence the DA signal?

(5) There is a major challenge in their design and interpretation of the dopamine signal. Both trial types (Go and No Go) start with the rat having their nose in the noseport. An auditory cue is presented for 2-3 s signaling to the rat to either leave the noseport and make a lever response (Go trial) or to stay in the noseport (No Go trial). The timing of these actions and/or decisions is entirely independent, so it is not clear to me how the authors would ever align these traces to the exact decision point for each trial type. They attempt to do this with the nose-port exit analysis, but exiting the noseport for a Go trial (a rat needs to make 2 lever presses and then get a reward) versus a No Go trial (a rat needs to go retrieve the reward) is very different and not comparable.

(6) The voltammetry analysis did not appear to test the hypotheses the authors outlined in the intro. All comparisons were done within task variants (DA dynamics in Go vs. No Go trials, aligned to different task events), but there were no comparisons across task variants to determine if the DA signal differed in cued vs self-initiated trials.

(7) Classification of No Go behaviors was interesting, but was not well integrated with the rest of the paper and was underdeveloped. It also raised more questions for me than answers. For example:

(a) Was the behavior classification consistent across rats for all No Go trials? If not, did the DA signal change within subjects between biting vs digging vs calm?

(b) If "biting rats" were not always biting rats on every No Go trial, then is it fair to collapse animals into a single measure (Figure 3C).

(c) Some of the classification groups only had 2 or fewer rats in them, making any statistical comparison and inference difficult.

Reviewer #3 (Public review):

Summary:

After salamander limb amputation, the cross-section of the stump has two major axes: anterior-posterior and dorsal-ventral. Cells from all axial positions (anterior, posterior, dorsal, ventral) are necessary for regeneration, yet the molecular basis for this requirement has remained unknown. To address this gap, Yamamoto et al. took advantage of the ALM assay, in which defined positional identities can be combined on demand and their effects assessed through the outgrowth of an ectopic limb. They propose a compelling model in which dorsal and ventral cells communicate by secreting Wnt10b and Fgf2 ligands respectively, with this interaction inducing Shh expression in posterior cells. Shh was previously shown to induce limb outgrowth in collaboration with anterior Fgf8 (PMID: 27120163). Thus, this study completes a concept in which four secreted signals from four axial positions interact for limb patterning. Notably, this work firmly places dorsal-ventral interactions upstream of anterior-posterior, which is striking for a field that has been focussed on anterior-posterior communication. The ligands identified (Wnt10b, Fgf2) are different to those implicated in dorsal-ventral patterning in the non-regenerative mouse and chick models. The strength of this study is in the context of ALM/ectopic limb engineering. Although the authors attempt to assay the expression of Wnt10b and Fgf2 during limb regeneration after amputation, they were unable to pinpoint the precise expression domains of these genes beyond 'dorsal' and 'ventral' blastema. Given that experimental perturbations were not performed in regenerating limbs - almost exclusively under ALM conditions - this author finds the title "Dorsoventral-mediated Shh induction is required for axolotl limb regeneration" a little misleading.

Strengths:

(1) The ALM and use of GFP grafts for lineage tracing (Figures 1-3) take full advantage of the salamander model's unique ability to outgrow patterned limbs under defined conditions. As far as I am aware, the ALM has not been combined with precise grafts that assay 2 axial positions at once, as performed in Figure 3. The number of ALMs performed in this study deserves special mention, considering the challenging surgery involved.

(2) The authors identify that posterior Shh is not expressed unless both dorsal and ventral cells are present. This echoes previous work in mouse limb development models (AER/ectoderm-mesoderm interaction) but this link between axes was not known in salamanders. The authors elegantly reconstitute dorsal-ventral communication by grafting, finding that this is sufficient to trigger Shh expression (Figure 3 - although see also section on Weaknesses).

(3) Impressively, the authors discovered two molecules sufficient to substitute dorsal or ventral cells through electroporation into dorsal- or ventral- depleted ALMs (Figure 5). These molecules did not change the positional identity of target cells. The same group previously identified the ventral factor (Fgf2) to be a nerve-derived factor essential for regeneration. In Figure 6, the authors demonstrate that nerve-derived factors, including Fgf2, are alone sufficient to grow out ectopic limbs from a dorsal wound. Limb induction with a 3-factor cocktail without supplementing with other cells is conceptually important for regenerative engineering.

(4) The writing style and presentation of results is very clear.

Overall appraisal:

This is a logical and well-executed study that creatively uses the axolotl model to advance an important framework for understanding limb patterning. The relevance of the mechanisms to normal limb regeneration are not yet substantiated, in the opinion of this reviewer. Additionally, Wnt10b and Fgf2 should be considered molecules sufficient to substitute dorsal and ventral identity (solely in terms of inducing Shh expression). It is not yet clear whether these molecules are truly necessary (loss of function would address this).

Comments on revisions:

Congratulations - I still find this an elegant and easy-to-read study with significant implications for the field! Linking your mechanisms to normal limb regeneration (i.e. regenerating blastema, not ALM), as well as characterising the cell populations involved, will be interesting directions for the future.

Reviewer #3 (Public review):

Summary:

The manuscript explores the dependence of ABC transporter activity on membrane curvature. The underlying concept being analysed here is whether membrane mechanics can regulate the conformation of the protein and thereby its activity.

Strengths:

The protein of choice here is BmrA, a bacterial transmembrane ABC transporter. This protein was previously found to exhibit two states: open conformation with Nucleotide Binding domains (NBDs) separated from each other and an ATP-bound closed conformation with dimerised NBDs. The protein was purified and reconstituted into liposomes of varying diameters, largely categorised as Small vesicles (SV) and Large vesicles (LV). The authors find that the activity of the protein is reduced with the changing curvature of the membrane vesicles used to make the proteoliposomes. This could be modulated by making vesicles at different temperatures, LV at high and SV at lower temperature (4 {degree sign}C), following which they perform biochemical measurement of activity or smFRET experiments at HT or RT. They use well-characterized single-molecule FRET-based measurements to assess the change in conformation of the protein during the ATPase cycle. They find that a significant fraction of the protein is in an open (inactive) conformation in vesicles of higher curvature (SVs) at a given temperature. The authors develop a simple yet elegant theoretical model based on the energy of protein configuration states and their coupling to membrane energetics (bending rigidity) and curvature to explain these findings. The model provides a parameter-free fit that predicts the open/closed state distributions as well as the ATPase activity differences between SV and LV. Using experimentally determined values of the protein conicity, the authors to extract reasonable values of membrane rigidity, consistent with available literature.

The data and theoretical model together convincingly support the claim that membrane mechanics via local curvature modulation may bias membrane protein conformation states and thereby modify the activity of membrane proteins. This is an important and general conclusion that the authors also elaborate on in their discussion.

Weaknesses:

The authors say that the protein activity is irreversibly inhibited by orthovanadate, but 50% of the proteins are still in open conformation, while being accessible to the analogue (Table 2). It is unclear what this means in the context of activity vs. conformation.

The difference in the fraction of proteins in closed conformation is quite similar between LV and SV treated with AMP-PNP at 20 {degree sign}C (Figure 2B), and it is not clear if the difference is significant. The presence of a much higher FRET tail in the plots of smFRET experiment in SVs at 20 {degree sign}C or 33 {degree sign}C in the apo conformation of the protein (Figure 3A-B) is cause of some concern since one would not expect BmrA to access the closed states more frequently in the Apo conformation especially when incorporated in the SV. This is because the subtraction of the higher fraction of closed states in the Apo conformation contributes directly to enhancing the bias between the closed states in SV versus LV membrane bilayers.

Reviewer #3 (Public review):

Summary:

This is a really nice manuscript with different lines of evidence to show that the IL encodes inhibitory memories that can then be manipulated by optogenetic stimulation of these neurons during extinction. The behavioral designs are excellent, with converging evidence using extinction/re-extinction, backwards/forwards aversive conditioning, and backwards appetitive/forwards aversive conditioning. Additional factors, such as nonassociative effects of the CS or US, also are considered, and the authors evaluate the inhibitory properties of the CS with tests of conditioned inhibition. The authors have addressed the prior reviews. I still think it is unfortunate that the groups were not properly balanced in some of the figures (as noted by the authors, they were matched appropriately in real time, but some animals had to be dropped after histology, which caused some balancing issues). I think the overall pattern of results is compelling enough that more subjects do not need to be added, but it would still be nice to see more acknowledgement and statistical analyses of how these pre-existing differences may have impacted test performance.

Strengths:

The experimental designs are very rigorous with an unusual level of behavioral sophistication.

Weaknesses:

The various group differences in Figure 2 prior to any manipulation are still problematic. There was a reliable effect of subsequent group assignment in Figure 2 (p<0.05, described as "marginal" in multiple places). Then there are differences in extinction (nonsignificant at p=.07). The test difference between ReExt OFF/ON is identical to the difference at the end of extinction and the beginning of Forward 2, in terms of absolute size. I really don't think much can be made of the test result. The authors state in their response that this difference was not evident during the forward phase, but there clearly is a large ordinal difference on the first trial. I think it is appropriate to only focus on test differences when groups are appropriately matched, but when there are pre-existing differences (even when not statistically significant) then they really need to be incorporated into the statistical test somehow.

The same problem is evident in Figure 4B, but here the large differences in the Same groups are opposite to the test differences. It's hard to say how those large differences ultimately impacted the test results. I suppose it is good that the differences during Forward conditioning did not ultimately predict test differences, but this really should have been addressed with more subjects in these experiments. The authors explore the interactions appropriately but with n=6 in the various subgroups, it's not surprising that some of these effects were not detected statistically.

It is useful to see the trial-by-trial test data now presented in the supplement. I think the discussion does a good job of addressing the issues of retrieval, but the ideas of Estes about session cues that the authors bring up in their response haven't really held up over the years (e.g., Robbins, 1990, who explicitly tested this; other demonstrations of within-session spontaneous recovery), for what it's worth.

Reviewer #3 (Public review):

Summary:

This study describes a computational model of the rat spinal locomotor circuit and how it could be reconfigured after lateral hemisection or contusion injuries to replicate gaits observed experimentally.

The model suggests the emergence of detour circuits after lateral hemisection whereas after a midline contusion, the model suggests plasticity of left-right and sensory inputs below the injury.

Strengths:

The model accurately models many known connections within and between forelimb and hindlimb spinal locomotor circuits.

The simulation results mirror closely gait parameters observed experimentally. Many gait parameters were studied as well as variability in these parameters in intact versus injured conditions.

A sensitivity analysis provides some sense of the relative importance of the various modified connectivity after injury in setting the changes in gait seen after the two types of injuries

Overall, the authors achieved their aims and the model provides solid support for the changes in connectivity after the two types of injuries modelled. This work emphasizes specific changes in connectivity after lateral hemisection or after contusion that could be investigated experimentally. The model is available to be used by the public and could be a tool used to investigate the relative importance of various highlighted or undiscovered changes in connectivity that could underlie the recovery of locomotor function in spinalized rats.

Comments on revisions:

The authors addressed the comments made by the reviewers. The sensitivity analysis adds insights to the manuscript

Reviewer #3 (Public review):

Summary:

In Monziani et al. paper entitled: "EPB41L4A-AS1 long noncoding RNA acts in both cis- and trans-acting transcriptional regulation and controls nucleolar biology", the authors made some interesting observations that EPB41L4A-AS1 lncRNA can regulate the transcription of both the nearby coding gene and genes on other chromosomes. They started by computationally examining lncRNA-gene pairs by analyzing co-expression, chromatin features of enhancers, TF binding, HiC connectome and eQTLs. They then zoomed in on four pairs of lncRNA-gene pairs and used LNA antisense oligonucleotides to knock down these lncRNAs. This revealed EPB41L4A-AS1 as the only one that can regulate the expression of its cis-gene target EPB41L4A. By RNA-FISH, the authors found this lncRNA to be located in all three parts of a cell: chromatin, nucleoplasm and cytoplasm. RNA-seq after LNA knockdown of EPB41L4A-AS1 showed that this increased >1100 genes and decreased >1250 genes, including both nearby genes and genes on other chromosomes. They later found that EPB41L4A-AS1 may interact with SUB1 protein (an RNA binding protein) to impact the target genes of SUB1. EPB41L4A-AS1 knockdown reduced the mRNA level of SUB1 and altered the nuclear location of SUB1. Later, the authors observed that EPB41L4A-AS1 knockdown caused increase of snRNAs and snoRNAs, likely via disrupted SUB1 function. In the last part of the paper, the authors conducted rescue experiments that suggested that the full-length, intron- and SNORA13-containing EPB41L4A-AS1 is required to partially rescue snoRNA expression. They also conducted SLAM-Seq and showed that the increased abundance of snoRNAs is primarily due to their hosts' increased transcription and stability. They end with data showing that EPB41L4A-AS1 knockdown reduced MCF7 cell proliferation but increased its migration, suggesting a link to breast cancer progression and/or metastasis.

Strengths:

The strength of the paper includes: it is overall well-written; the results are overall presented with good technical rigor and appropriate interpretation. The observation that a complex lncRNA EPB41L4A-AS1 regulates both cis and trans target genes, if fully proven, is interesting and important.

Weaknesses:

The weakness includes: the paper is a bit disjointed as it started from cis and trans gene regulation, but later it switched to a partially relevant topic of snoRNA metabolism via SUB1; the paper was limited in the mechanisms as to how these trans genes (including SUB1 or NPM1 genes themselves) are affected by EPB41L4A-AS1 knockdown; there are discrepancy of results upon EPB41L4A-AS1 knockdown by LNA versus by CRISPR activation, or by plasmid overexpression of this lncRNA.

Overall, the data is supportive of a role of this lncRNA in regulating cis and trans target genes, and thereby impacting cellular phenotypes.

Reviewer #3 (Public review):

Summary:

The authors describe a new structural biology framework termed "in extracto cryo-EM," which aims to bridge the gap between single-particle cryo-EM of purified complexes and in situ cryo-electron tomography (cryo-ET). By utilizing high-resolution 2D template matching (2DTM) on mammalian cell lysates, the authors sought to visualize the translational apparatus in a near-native environment while maintaining near-atomic resolution. The study identifies elongation factor 2 (eEF2) as a major hibernation factor bound to both 60S and 80S particles and describes a variety of hibernation scenarios involving factors such as SERBP1, LARP1, and CCDC124.

Strengths:

(1) The use of 2DTM effectively overcomes the signal-to-noise challenges posed by the dense and viscous nature of cellular extracts, yielding maps as high as 2.2 Å.

(2) The discovery of eEF2-GDP as a ubiquitous shield for ribosomal functional centers, particularly its unexpected stabilization on the 60S subunit, provides a compelling model for ribosome preservation during stress.

Weaknesses:

(1) Representative nature of cell samples and lower detection limit

The cells used in this study (MCF-7, BSC-1, and RRL) are either fast-growing cancer cell lines or specialized protein-synthetic systems. For cells with naturally low ribosomal abundance (such as quiescent primary cells), achieving the target concentration (e.g., A260 > 1000 ng/uL) would require an exponentially larger starting cell population.

Is there a defined lower limit of ribosomal concentration in the raw lysate below which the 2DTM algorithm fails to yield high-resolution classes? In ribosome-sparse lysates, A260 becomes an unreliable proxy for ribosome density due to the high background of other RNA species and proteins. How do the authors estimate specific ribosome abundance in such heterogeneous fields?

(2) Quantitation in heterogeneous lysates and crowding effects

The authors utilize A260 as a key quality control measure before grid preparation. However, if extreme physical concentration is required to see enough particles, the background concentration of other cytoplasmic components also increases. This may lead to molecular crowding or sample viscosity that interferes with the formation of optimal thin ice. How do the authors calculate or estimate the specific abundance of ribosomes in the cryo-EM field of view when they represent a much smaller percentage of the total cellular content?

(3) Optimization of sample preparation

The authors describe lysates as dense and viscous, requiring multiple blotting steps (2-3 times) for 3-8 seconds. Have the authors tested whether a larger molecular weight cutoff (e.g., 100 kDa) during concentration could improve the ribosome-to-background ratio without losing small factors like eIF5A (approx. 17 kDa)? Could repeated blotting of a concentrated, viscous lysate introduce shearing forces or increased exposure to the air-water interface that perturbs the native conformation of the complexes?

(4) The regulatory switch and mechanism of eEF2

The finding that eEF2-GDP occupies dormant ribosomes is striking. What drives eEF2 from its canonical role in translocation to this hibernation state? Is this transition purely driven by stoichiometry (lack of mRNA/tRNA) and the GDP/GTP ratio, or is there a role for post-translational modifications? How do these eEF2-bound dormant ribosomes rapidly re-enter the translation pool upon stress relief?

(5) Hibernation diversity and LARP1 contextualization

The study reveals that hibernation strategies vary across cell types. Does the high hibernation rate in RRL reflect a physiological state, or does it hint at "preparation-induced stress" due to resource exhaustion or mRNA degradation in the cell-free system? How do the authors reconcile their discovery of LARP1 on 80S particles with recent 2024 reports that primarily describe LARP1 as an SSU-bound repressor?

Reviewer #3 (Public review):

Summary:

It has been a long time since I enjoyed reviewing a paper as much as this one. In it, the authors generate an unprecedented view of the aboral organ of a 5-day old ctenophore. They proceed to derive numerous insights by reconstructing the populations and connections of cell types, with up to 150 connections from the main Q1-4 neuron.

Strengths:

The strengths of the analysis are the sophisticated imaging methods used, the labor-intensive reconstruction of individual neurons and organelles, and especially the mapping of synapses. The synaptic connections to and from the main coordinating neurons allow the authors to created a polarized network diagram for these components of the aboral organ. These connections give insight about the potential functions of the major neurons, which also giving some unexpected results, particularly the lack of connections from the balancer system to the coordinating system.

Weaknesses:

There were no significant weaknesses in the paper - only a slate of interesting unanswered questions to motivate future studies.

Comments on revisions:

This manuscript was already strong from the start, and I am fully satisfied with the revisions, which corrected a few glitches and points of clarification.

Reviewer #3 (Public review):

Summary:

This study investigated the role of AHL15 in the regulation of gene expression using AHL15 overexpression lines. Their results do show that more genes are downregulated when AHL15 is upregulated, and its binding does not affect the chromatin accessibility. Further, they investigated AHL15 binds in regions depleted in histone modifications and other epigenetic signatures. Subsequently, they investigated the presence of AHL15 in the gene chromatin loops. They found overlaps with both upregulated and downregulated genes. The methods are appropriately described, but could be improved to include the analysis of self-looping gene boundaries.

Strengths:

Their study clearly showed a lack of any specific sequence enrichment in the AHL15 binding sites, other than these being AT-rich, suggesting that AHL proteins do not recognize a specific DNA sequence but are recruited to their AT-rich target sites in another way. The study does suggest significant enrichment of AHL15 binding sites at TSS and TES, and AHL15 sites are depleted of any histone marks. They also identified that AHL15 binding sites overlap with self-looping gene boundaries.

Weaknesses:

The claim that AHL15 acts as a repressor and genes regulated by it are downregulated needs to be investigated based on AHL15 binding sites, to show enrichment/ depletion of AHL15 binding sites in overexpressing genes and repressed genes. The authors should provide data to support plant longevity with AHL15 overexpression using the DEX-induced system to support the claims in the title. Calculation of the enrichment score of AHL15 peaks in the self-looping genes that are upregulated or downregulated, and discussion about the different effects of AHL15 binding on self-looping regions to regulate gene expression may be helpful to understand the significance of the study. Motif enrichment in upregulated and downregulated genes separately to identify binding sequence preferences may be useful. It is not clear how the overlap of AHL15 peaks with self-looping genes has been carried out.

Reviewer #3 (Public review):

Summary:

The authors used genetic models and immunohistochemistry to identify how training in a spatial discrimination working memory task influences activity in the dentate gyrus subregion of the hippocampus. Finding that more cognitively challenging variants of the task evoked more and distinct patterns of activity, they then investigated whether newborn neurons in particular were important for learning this task and regulating the spatial activity patterns.

Strengths:

The focus on precise anatomical locations of activity is relatively novel and potentially important, given that little is known about how DG subregions contribute to behavior. The authors also use a task that is known to depend on this memory-related part of the brain.

Weaknesses:

Statistical rigor is insufficient. Many statistical results are not stated, inappropriate tests are used, and sample sizes differ across experiments (which appear to potentially underlie null results). The chemogenetic approach to inhibit adult-born neurons also does not appear to be targeting these neurons, as judged by their location in the DG.

Reviewer #3 (Public review):

The manuscript by Palo and colleagues demonstrates identification of FRG1 as a novel regulator of nonsense-mediated mRNA decay (NMD), showing that FRG1 inversely modulates NMD efficiency by controlling UPF1 abundance. Using cell-based models and a frg1 knockout zebrafish, the authors show that FRG1 promotes UPF1 ubiquitination and proteasomal degradation, independently of DUX4. The work further positions FRG1 as a structural component of the spliceosome and exon junction complex without compromising its integrity. Overall, the manuscript provides mechanistic insight into FRG1-mediated post-transcriptional regulation and expands understanding of NMD homeostasis. The authors should address the following issues to improve the quality of their manuscript.

(1) Figure 7A-D, appropriate positive controls for the nuclear fraction (e.g., Histone H3) and the cytoplasmic fraction (e.g., GAPDH or α-tubulin) should be included to validate the efficiency and purity of the subcellular fractionation.

(2) To strengthen the conclusion that FRG1 broadly impacts the NMD pathway, qRT-PCR analysis of additional core NMD factors (beyond UPF1) in the frg1⁻/⁻ zebrafish at 48 hpf would be informative.

(3) Figure labels should be standardized throughout the manuscript (e.g., consistent use of "Ex" instead of mixed terms such as "Oex") to improve clarity and readability.

(4) The methods describing the generation of the frg1 knockout zebrafish could be expanded to include additional detail, and a schematic illustrating the CRISPR design, genotyping workflow, and validation strategy would enhance transparency and reproducibility.

(5) As FRG1 is a well-established tumor suppressor, additional cell-based functional assays under combined FRG1 and UPF1 perturbation (e.g., proliferation, migration, or survival assays) could help determine whether FRG1 influences cancer-associated phenotypes through modulation of the NMD pathway.

(6) Given the claim that FRG1 inversely regulates NMD efficacy via UPF1, an epistasis experiment such as UPF1 overexpression in an FRG1-overexpressing background followed by an NMD reporter assay would provide stronger functional validation of pathway hierarchy.

Reviewer #3 (Public review):

Summary:

Eukaryotic gene transcription requires a large assemblage of protein complexes that govern the molecular events required for RNA Polymerase II to produce mRNAs. One of these complexes, TFIIH, comprises two modules, one of which promotes DNA unwinding at promoters, while the other contains a kinase (Kin28 in yeast) that phosphorylates the repeated motif at the C-terminal domain (CTD) of the largest subunit of Pol II. Kin28 phosphorylation of Ser5 in the YSPTSPS motif of the CTD is normally highly localized at promoter regions, and marks the beginning of a cycle of phosphorylation events and accompanying protein association with the CTD during the transition from initiation to elongation.

The two modules of TFIIH are linked by Tfb3. Tfb3 consists of two globular regions, an N-terminal domain that contacts the Core module of TFIIH and a C-terminal domain that contacts the kinase module, connected by a linker. In this paper, Giordano et al. test the role of Tfb3 as a connector between the two modules of TFIIH in yeast. They show that while no or very slow growth occurs if only the C-terminal or N-terminal region of Tfb3 is present, near normal growth is observed when the two unlinked regions are expressed. Consistent with this result, the separate domains are shown to interact with the two distinct TFIIH modules. ChIP experiments show that the Core module of TFIIH maintains its localization at gene promoters when the Tfb3 domains are separated, while localization of the kinase module and of Ser5 phosphorylation on the CTD of Pol II is disrupted. Finally, the authors examine the effect of separating the Tfb3 domains on another function of TFIIH, namely nucleotide excision repair, and find little or no effect when only the N-terminal region of Tfb3 or the two unlinked domains are present.

Strengths:

Experiments involving expression of Tfb3 domains in yeast are well-controlled, and the data regarding viability, interaction of the separate Tfb3 domains with TFIIH modules, genome-wide localization of the TFIIH modules and of phosphorylated Ser5 CTDs, and of effects on NER, are convincing. The experiments are consistent with current models of TFIIH structure and function and support a model in which Tfb3 tethers the kinase module of TFIIH close to initiation sites to prevent its promiscuous action on elongating Pol II.

Weaknesses:

(1) The work is limited in scope and does not provide any major insights into the mechanism of transcription. One indication of this limitation is that in the Discussion, published structural and functional results on transcription are used to support the interpretations of the results here more than current results inform previous models or findings.

(2) The first described experiment, which purports to show that three kinases cannot function in place of Kin28 when tethered (by fusion) to Tfb3, is missing the crucial control of showing that Kin28 can support viability in the same context. This result also does not connect with the rest of the manuscript.

(3) Finally, the authors present the interesting and reasonable speculation that the TFIIH complex and connecting Tfb3 found in mammals and yeast may have evolved from an earlier state in which the two TFIIH subdomains were present as unconnected, distinct enzymes. This idea is supported by a single example from the literature (T. brucei). A more thorough evolutionary analysis could have tested this idea more rigorously.

Reviewer #3 (Public review):

Summary:

This study investigates the role of BICC1 in the regulation of PKD1 and PKD2 and its impact on cytogenesis in ADPKD. By utilizing co-IP and functional assays, the authors demonstrate physical, functional, and regulatory interactions between these three proteins.

Strengths:

(1) The scientific principles and methodology adopted in this study are excellent, logical, and reveal important insights into the molecular basis of cystogenesis.

(2) The functional studies in animal models provide tantalizing data that may lead to a further understanding and may consequently lead to the ultimate goal of finding a molecular therapy for this incurable condition.

(3) In describing the patients from the Arab cohort, the authors have provided excellent human data for further investigation in large ADPKD cohorts. Even though there was no patient material available, such as HUREC, the authors have studied the effects of BICC1 mutations and demonstrated its functional importance in a Xenopus model.

Weaknesses:

This is a well-conducted study and could have been even more impactful if primary patient material was available to the authors. A further study in HUREC cells investigating the critical regulatory role of BICC1 and potential interaction with mir-17 may yet lead to a modifiable therapeutic target.

Conclusion:<br /> The authors achieve their aims. The results reliably demonstrate the physical and functional interaction between BICC1 and PKD1/PKD2 genes and their products.

The impact is hopefully going to be manifold:

(1) Progressing the understanding of the regulation of the expression of PKD1/PKD2 genes.

Comments on revision:

My comments have been addressed and sorted.

Reviewer #3 (Public review):

Summary:

In this study, Liu et al. analyze fMRI data collected during movie watching, applied an energy landscape method with pairwise maximum entropy models. They identify a set of brain states defined at the level of canonical functional networks and quantify how the brain transitions between these states. Transitions are classified as "easy" or "hard" based on changes in the inferred energy landscape, and the authors relate transition probabilities to inter-subject correlation. A major emphasis of the work is the role of the thalamus, which shows transition-linked activity changes and dynamic connectivity patterns, including differential involvement of parvalbumin- and calbindin-associated thalamic subdivisions.

Strengths:

The study is methodologically complex and technically sophisticated. It integrates advanced analytical methods into high-dimensional fMRI data. The application of energy landscape analysis to movie-watching data appears to be novel as well. The finding on the thalamus involved energy state transition and provides a strong linkage to several theories on thalamic control functions, which is a notable strength.

Weaknesses:

The main weakness is the conceptual clarity and advances that this otherwise sophisticated set of analyses affords. A central conceptual ambiguity concerns the energy landscape framework itself. The authors note that the "energy" in this model is not biological energy but a statistical quantity derived from the Boltzmann distribution. After multiple reads, I still have major trouble mapping this measure onto any biological and cognitive operations. BOLD signal is a measure of oxygenation as a proxy of neural activity, and correlated BOLD (functional connectivity) is thought to measure the architecture of information communication of brain systems. The energy framework described in the current format is very difficult for most readers to map onto any neural or cognitive knowledge base on the structure and function of brain systems. Readers unfamiliar with maximum entropy models may easily misinterpret energy changes as reflecting metabolic cost, neural effort, or physiological variables, and it is just very unclear what that measure is supposed to reflect. The manuscript does not clearly articulate what conceptual and mechanistic advances the energy formalism provides beyond a mathematical and statistical report. In other words, beyond mathematical description, it is very hard for most readers to understand the process and function of what this framework is supposed to tell us in regards to functional connectivity, brain systems, and cognition. The brain is not a mathematical object; it is a biological organ with cognitive functions. The impact of this paper is severely limited until connections can be made.

Relatedly, the use of metaphors such as "valleys," "hills," and "routes" in multidimensional measures lacks grounding. Valleys and hills of what is not intuitive to understand. Based on my reading, these features correspond to local minima and barriers in a probability distribution over binarized network activation patterns, but similar to the first point, the manuscript does not clearly explain what it means conceptually, neurobiologically, or computationally for the brain to "move" through such a landscape. The brain is not computing these probabilities; they are measurement tools of "something". What is it? To advance beyond mathematical description, these measurements must be mapped onto neurobiological and cognitive information.

This conceptual ambiguity goes back to the Introduction. At the level of motivation, the purpose and deliverables of the study are not defined in the Introduction. The stated goal is "Transitions between distinct cortical brain states modulate the degree of shared neural processing under naturalistic conditions". I do not know if readers will have a clear answer to this question at the end. Is the claim that state transitions cause changes in inter-subject correlation, that they index moments of narrative alignment, or that they reflect changes in attentional or cognitive mode? This level of explanation is largely dissociated from the methods in their current form.

Several methodological choices can use clarification. The use of a 21-TR window centered on transition offsets is unusually long relative to the temporal scale of fMRI dynamics and to the hypothesized rapidity of state transitions. On a related note, what is the temporal scale of state transition? Is it faster than 21 TRs?

The choice of movie-watching data is a strength. But, many of the analyses performed here, energy landscape estimation, clustering of states, could in principle be applied to resting-state data. The manuscript does not clearly articulate what is gained, mechanistically or cognitively, by using movie stimuli beyond the availability of inter-subject correlation.

Because of the above issues, a broader concern throughout the results is the largely descriptive nature of the findings. For example, the LASSO analysis shows that certain state transitions predict ISC in a subset of regions, with respectable R² values. While statistically robust, the manuscript provides little beyond why these particular transitions should matter, what computations they might reflect, or how they relate to known cognitive operations during movie watching. Similar issues arise in the clustering analyses. Clustering high-dimensional fMRI-derived features will almost inevitably produce structure, whether during rest, task, or naturalistic viewing. What is missing is an explanation of why these specific clusters are meaningful in functional or mechanistic terms.

Finally, the treatment of the thalamus, while very exciting, could use a bit more anatomical and circuit-level specificity. The manuscript largely treats the thalamus as a unitary structure, despite decades of work demonstrating big functional and connectivity differences across thalamic nuclei. A whole-thalamus analysis without more detailed resolution is increasingly difficult to justify. The subsequent subdivision into PVALB- and CALB-associated regions partially addresses this, but these markers span multiple nuclei with overlapping projection patterns.

Reviewer #3 (Public review):

Summary:

Luque et al. proposed stratifying chemotherapy response in triple-negative breast cancer based on spatial protein patterns from IMC data. This proposed method combines GNN with GNNexplainer to identify several important protein markers and cell types related to chemotherapy. As one of the most significant challenges in cancer research, this work holds great potential for translational medicine.

Strengths:

(1) Targeting the invention decision-making of TNBC, one of the prominent challenges in the field.

(2) Cutting-edge spatial proteomics data with enough cohort and clinical outcome.

(3) Appropriate usage of cutting-edge machine learning models and comprehensive analysis.

Weaknesses:

(1) More scientific rigor is needed for machine learning benchmarking.

(2) More depth is needed, comparing related works with using similar approaches.

Reviewer #3 (Public review):

Summary:

In the manuscript " Dynamics of mesoscale brain network during decision-making learning revealed by chronic, large-scale single-unit recording", Wang et al investigated mesoscale network reorganization during visual stimulus discrimination learning in mice using chronic, large-scale single-unit recordings across 10 cortical/subcortical regions. During learning, mice improved task performance mainly by suppressing licking on no-go trials. The authors found that learning induced restructuring of functional connectivity, with visual (V1, V2M) and frontal (OFC, M2) regions forming a task-relevant subnetwork during the acquisition of correct No-Go (CR) trials. Learning also compressed sequential neural activation and broadened stimulus encoding across regions. In addition, a region's network connectivity rank correlated with its timing of peak visual stimulus encoding. Optogenetic inhibition of orbitofrontal cortex (OFC) and high order visual cortex (V2M) impaired learning, validating its role in learning. The work highlights how mesoscale networks underwent dynamic structuring during learning.

Strengths:

The use of ultra-flexible microelectrode arrays (uFINE-M) for chronic, large-scale recordings across 10 cortical/subcortical regions in behaving mice represents a significant methodological advancement. The ability to track individual units over weeks across multiple brain areas will provide a rare opportunity to study mesoscale network plasticity.<br /> While limited in scope, optogenetic inhibition of OFC and V2M directly ties connectivity rank changes to behavioral performance, adding causal depth to correlational observations.

Weaknesses:

The weakness is also related to the strength provided by the method. While the method in principle enables chronic tracking of individual units, the authors have not showed chronically tracked neurons across learning. Without demonstrating that and taking advantage of analyzing chronically tracked neurons, this approach is not different from acute recording in individual days across learning, weaking the attractiveness of the methodology and this study.

Another weakness is that major results are based on analyses of functional connectivity. Functional connection strengthen across areas is ranked 1-10 based on relative strength. And the regional input/out is compared across learning. This approach reveals differential changes in some cortical and subcortical areas. In my view, learning-related changes should be validated using complementary methods.

Reviewer #3 (Public review):

Summary:

Use of reporter assays to understand the regulatory mechanisms controlling gene expression moves beyond simple correlations of cis-regulatory sequence accessibility, evolutionary sequence conservation, and epigenetic status with gene expression, instead quantifying regulatory sequence activity for individual elements. Tulloch et al., provide systematic characterization of two new reporter assay techniques (LS-MPRA and d-MPRA) to comprehensively identify cis-regulatory sequences contained within genomic loci of interest during retinal development. The authors then apply LS-MPRA and d-MPRA to identify putative cis-regulatory sequences controlling Olig2 and Ngn2 expression, including potential regulatory motifs that known retinal transcription factors may bind. Transcription factor binding to regulatory sequences is then assessed via CUT&RUN. The broader utility of the techniques are then highlighted by performing the assays across development, across species, and across tissues.

Strengths:

The authors validate the reporter assays on retinal loci for which the regulatory sequences are known (Rho, Vsx2, Grm6, Cabp5) mostly confirming known regulatory sequence activity but highlighting either limitations of the current technology or discrepancies of previous reporter assays and known biology. The techniques are then applied to loci of interest (Olig2 and Ngn2) to better understand the regulatory sequences driving expression of these transcription factors across retinal development within subsets of retinal progenitor cells, identifying novel regulatory sequences through comprehensive profiling of the region.

LS-MPRA provides broad coverage of loci of interest

d-MPRA identifies sequence features that are important for cis-regulatory sequence activity.

The authors take into account transcript and protein stability when determining the correlation of putative enhancer sequence activity with target gene expression.

Overall, the manuscript highlights the utility of the techniques to identify novel cis-regulatory sequence contributions to gene expression, including systematic characterizations of sequence motifs conferring activating or repressive functions.

Limitations:

Barcoding strategies have the potential to induce high collision rates (see Table S3) that may lead to misinterpretation of the data and/or high false positive/negative rates.

There are limited robust methods to distinguish differentially active versus inactive CRMs in the LS-MPRA.

Reviewer #3 (Public review):

Summary:

Histone variant H2A.Z is evolutionarily conserved among various species. The selective incorporation and removal of histone variants on the genome play crucial roles in regulating nuclear events, including transcription. Shih et al. aimed to address antagonistic mechanisms between histone variant H2A.Z deposition and DNA methylation. To this end, the authors reconstituted H2A.Z nucleosomes in vitro using methylated or unmethylated human satellite II DNA sequence and examined how DNA methylation affects H2A.Z nucleosome structure and dynamics. The cryo-EM analysis revealed that DNA methylation induces a more open conformation in H2A.Z nucleosomes. Consistent with this, their biochemical assays showed that DNA methylation subtly increases restriction enzyme accessibility in H2A.Z nucleosomes compared with canonical H2A nucleosomes. The authors identified genome-wide profiles of H2A.Z and DNA methylation using genomic assays and found their unique distribution between Xenopus sperm pronuclei and fibroblast cells. Using Xenopus egg extract systems, the authors showed SRCAP complex, the chromatin remodelers for H2A.Z deposition, preferentially deposit H2A.Z on unmethylated DNA.

Strengths:

The study is solid, and most conclusions are well-supported. The experiments are rigorously performed, and interpretations are clear. The study presents a high-resolution cryo-EM structure of human H2A.Z nucleosome with methylated DNA. The discovery that the SRCAP complex senses DNA methylation is novel and provides important mechanistic insight into the antagonism between H2A.Z and DNA methylation.

Weaknesses:

The study is already strong, and most conclusions are well supported. However, it can be further strengthened in several ways.

(1) It is difficult to interpret how DNA methylation alters the orientation of the H4 tail and leads to the additional density on the acidic patch. The data do not convincingly support whether DNA methylation enhances interactions with H2A.Z mono-nucleosomes, nor whether this effect is specific to methylated H2A.Z nucleosomes.

(2) It remains unclear whether DNA methylation alters global H2A.Z nucleosome stability or primarily affects local DNA end flexibility. Moreover, while the authors showed locus-specific accessibility by HinfI digestion, an unbiased assay such as MNase digestion would strengthen the conclusions.

Reviewer #3 (Public review):

Summary:

Bogdan et al. present an intriguing investigation into the spontaneous dynamics of prediction error (PE)-related brain states. Using two independent fMRI tasks designed to elicit prediction and prediction error in separate participant samples, alongside both fMRI and EEG data, the authors identify convergent brain network patterns associated with high versus low PE. Notably, they further show that similar patterns can be detected during resting-state fMRI, suggesting that PE-related neural states may recur outside of explicit task demands.

Strengths:

The authors use a well-integrated analytic framework that combines multiple prediction tasks and brain imaging modalities. The inclusion of several datasets probing PE under different contexts strengthens the claim of generalizability across tasks and samples. The open sharing of code and data is commendable and will be valuable for future work seeking to build on this framework.

Weaknesses:

A central challenge of the manuscript lies in interpreting the functional significance of PE-related brain network states during rest. Demonstrating that a task-defined cognitive state recurs spontaneously is intriguing, but without clear links to behavior, individual traits, or experiential content during rest, it remains difficult to interpret what such spontaneous brain states tell us about the mind and brain. For example, it is unclear whether these states support future inference or learning, reflect offline predictive processing, or instead suggest state reinstatement due to a more general form of neural plasticity and circuit dynamics in the brain. Demonstrating any one of these downstream relationships would be valuable since it has the potential to inform our understanding of cognitive function or more general principles of neural organization.

I appreciate the authors' position that establishing the existence of such states is a necessary first step, and that future work may clarify their behavioral relevance. However, the current form makes it challenging to assess the conceptual advance of the present work in isolation.

Relatedly, in my previous review I raised questions about both across- and within-individual variability-for example, whether individuals who exhibit stronger or more distinct PE-related fluctuations at rest also show superior performance on prediction-related tasks (across-individual), or whether momentary increases in PE-network expression during tasks relate to faster or more accurate prediction (within-individual). The authors thoughtfully addressed this suggestion by conducting an individual-differences analysis correlating each participant's fluctuation amplitude with approximately 200 behavioral and trait measures from the HCP dataset.

The reported findings-a negative association with age and card-sorting performance, alongside a positive association with age-adjusted picture sequence memory-are interesting but difficult to interpret within a coherent functional framework. As presented, these results do not clearly support the idea that spontaneous PE-state fluctuations are related to enhancement in prediction, inference, or broader cognitive function. Instead, they raise the possibility that fluctuation amplitude may reflect more general factors (e.g., age) rather than a functionally meaningful PE-related process.

Overall, while the methodological contribution is strong, the manuscript would benefit from a clearer articulation of what functional conclusions can or cannot be drawn from the presence of spontaneous PE-related states, as well as a more cautious framing of their potential cognitive significance.

Further comments:

I appreciate that the authors took my earlier suggestions seriously and incorporated additional analyses examining behavioral relevance and permutation tests in the revision.

Reviewer #3 (Public review):

Summary:

S. Keeley & collaborators propose a computational approach to infer time-varying latent variables directly from calcium traces (for instance, obtained with 2p imaging) without the need for deconvolving the traces into spike trains in a preliminary, independent step. Their approach rests on 1 of 3 families of latent models: GPFA, HMM and dynamical systems - which they augment with an observation model that maps latent variables to fluorescence traces. They validate their approach on simulated and real data, showing that the approach improves latent variable inference and model fitting, compared to more traditional approaches (although not directly compared with the 2-step one; see below). They provide a GitHub repository with code to fit their models (which I have not tested).

Strengths:

The approach is sound and well-motivated. The authors are specialists in latent variable models. The manuscript is succinct, well-written, and the figures are clear. I particularly liked the diversity of latent models considered, in particular latent models with continuous (GPFA) vs. discrete (HMM) dynamics, which are useful for characterizing different types of neural computations. The validation on both simulated and real data is convincing.

Weaknesses:

The main weakness that I see is that the approach is tested only on a single real dataset (odor response dataset). The other model fits are obtained from simulated data. While the results are convincing, it would be useful to see the approach tested on other datasets, for instance, datasets with different brain areas, different behavioral conditions, or different calcium indicators. This would help assess the generality of the approach and its robustness to different experimental conditions.

The other points below mostly pertain to clarifications and possible extensions of the approach, and to simple model recovery experiments that would help quantify the advantage of the proposed approach over more traditional ones.

I have a question related to interpretability and diagnosis of model fits. One advantage of the two-step approach: (1) deconvolution => (2) latent variance inference, is that one can inspect the quality of the deconvolution step independently from the latent variable inference step. In the proposed approach, it seems more difficult to diagnose potential problems with model fitting. For instance, if the inferred latent variables are not interpretable, how can one determine whether this is due to a poor choice of latent model (e.g., HMM with too few states), or a poor fit of the observation model (e.g., wrong parameters for the calcium dynamics)? Are there any diagnostic tools that could help identify potential problems with model fitting?

Could the authors comment on whether their approach allows for instance to compare different forms of latent models (e.g., HMM vs. GPFA) in terms of model evidence, cross-validated log-likelihood or other model comparison metrics? This would be useful to quantitatively determine which type of latent dynamics is more appropriate for a given dataset.

The HMM part reveals a pretty large number of states, with one state being interpretable (evoked response). Shouldn't we expect a simpler scenario, with 2 states? I know this is a difficult question that is more general and common with HMM approaches, but it would be useful to discuss this point. For instance, would a hierarchical HMM (with a smaller number of "super-states") be more appropriate here?

While it certainly makes sense that models accounting for the full transformation of latent => spikes => fluorescence data should outperform the two-step (1) deconvolution => (2) latent variance inference approach, the amount of improvement is not clear. A direct comparison (e.g., w/ parameter & model recovery metrics) between the two approaches on simulated data would be useful to quantify the advantage of the proposed approach over more traditional ones.

It would be useful to discuss the possible extension of the approach to other types of data that are related to neural activity but have different observation models, e.g., voltage imaging, or neuromodulator sensors (e.g., GRAB-NE, dLight, etc). Do the authors see any specific challenges that would arise in these cases and that would need to be addressed in the future (other than changing the Poisson spiking part)?

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Reviewer #3 (Public review):

Summary:

In this work, the authors present a chromatin polymer model with some specific pattern of transcription units (TUs) and diffusing TFs; they simulate the model and study TFclustering, mixing, gene expression activity, and their correlations. First, the authors designed a toy polymer with colored beads of a random type, placed periodically (every 30 beads, or 90kb). These colored beads are considered a transcription unit (TU). Same-colored TUs attract with each other mediated by similarly colored diffusing beads considered as TFs. This led to clustering (condensation of beads) and correlated (or anti-correlation) "gene expression" patterns. Beyond the toy model, when authors introduce TUs in a specific pattern, it leads to emergence of specialized and mixed cluster of different TFs. Human chromatin models with realistic distribution of TUs also lead to the mixing of TFs when cluster size is large.

Strengths:

This is a valuable polymer model for chromatin with a specific pattern of TUs and diffusing TF-like beads. Simulation of the model tests many interesting ideas. The simulation study is convincing and the results provide solid evidence showing the emergence of mixed and demixed TF clusters within the assumptions of the model.

Reviewer #3 (Public review):

Summary:

Aghabi et al set out to characterize a T. gondii transmembrane protein with a ZIP domain, termed ZFT. The authors investigate the consequences of ZFT downregulation and overexpression for parasite fitness. Downregulation of ZFT causes defects in the parasite's endosymbiotic organelles, the apicoplast and the mitochondrion. Specifically, lack of ZFT causes a decrease in mitochondrial respiration, consistent with its role as an iron transporter. This impact on the mitochondria appears to trigger partial differentiation to bradyzoites. The authors furthermore demonstrate that expression of TgZFT can rescue a yeast mutant lacking its zinc transporter and perform an array of direct metal ion measurements including X-ray fluorescence microscopy and inductively coupled mass spectrometry (ICP-MS). These reveal reduced metal ions in parasites depleted in ZFT. In the manuscript's revision, the authors performed additional transport assays in Xenopus oocysts, providing further evidence for the transporter trafficking iron. Overall, the data by Aghabi et al. convincingly support that ZFT is a major metal ion transporter in T. gondii, importing iron and zinc for diverse essential processes.

Strengths:

This study's strength lies in the thorough characterization of the transporter. The authors combine a number of techniques to measure the impact of ZFT depletion, ranging form the direct measurement of metal ions to determining the consequences for the parasite's metabolism (mitochondrial respiration) as well as performing a yeast mutant complementation and transport assays in Xenopus oocysts expressing the T. gondii protein. This work is very thorough and clearly presented, leaving little doubt about this protein's function.

Weaknesses:

None. The authors have addressed all my previous queries/ concerns.

Reviewer #3 (Public review):

Summary:

The paper from Hall et al. reports the effects of an altered function spx allele on the physiology of S. aureus. Since Spx is essential in this organism, the authors compare WT with a spx C10A allele that retains Spx functions that are independent of the formation of a C10-C13 disulfide. However, the major role of Spx in maintaining disulfide homeostasis in this organism appears to be reduced by this mutation, including a reduction (relative to WT) in the DIA-induction of thioredoxin, thioredoxin reductase, and BSH biosynthesis and reduction enzymes.

Strengths:

Based on a wide range of studies, the authors develop a model in which Spx is required for adaptation to disulfide stress, and this adaptation involves (in part) induction of both cystine/Cys uptake and the Fur regulon. Overall, the results are compelling, but further efforts to clarify the presentation will aid readers in being able to follow this very complicated story.

Weaknesses:

(1) More details are needed on how relative growth is defined and calculated (e.g., line 145 and Figure 1C). The raw data (growth curves) should be included when reporting relative growth so that readers can see what changed (lag, growth rate, final OD?). Later in the paper, the authors refer to "the diamide-induced growth delay of the spxC10A mutant" (line 379), but this is not apparent from the presented data.

(2) Are the spx C10A, spx C13A, and spx C10A,C13A all really equivalent? In all cases, the Spx protein is presumably made (as confirmed for C10A in panel 1D). However, the only evidence to suggest that they are equivalent is the similar growth effects in panel 1C, and (as noted above), this data presentation can mask differences in how the mutations affect protein activity.

(3) Figure 1D and Figure 1 Supplement 2 report results related to the effect of diamide treatment on protein half-life (t1/2). Only single results are shown for both panels, and the conclusions do not seem to be statistically robust. For example, in Figure 1, Supplement 2 concludes that Spx C10A has a t1/2 is 3.38 min (this should be labeled correctly in the Figure legend as the red line). and WT Spx is 8.69 min. However, Figure 1D suggests that the protein levels at time 0 may not be equivalent, and this is lost in the data processing. Indeed, there are significant differences in Spx levels between time 0 - and + DIA, which is curious. Further, the authors' conclusion relies very heavily on line-fitting that includes a final point that has very low signal intensity (as judged from Figure 1D) and therefore is likely the least reliable of all the data. It might be worth showing curve fitting for multiple gels. Regardless of the overfitting of the data, the general conclusion that Spx is partially stabilized against proteolysis by ClpXP, and that the C10A mutant is reduced in stabilization, is probably correct.

(4) Figure 2 concludes that despite differences in the mRNA profiles between WT and spx C10A after 15 min. of DIA treatment, the overall level of responsiveness of the bacillithiol pool is unchanged. The authors find it "surprising" that the BSH pool responds normally despite some differences in gene expression. This is not surprising. The major events visualized in panel 2D are the chemical oxidation of BSH to BSSB and, presumably, the re-reduction by Bdr(YpdA). While it is seen that BSH synthesis (bshC) and ypdA expression may be less induced by DIA in the C10A mutant (2C), there is no evidence that the basal levels are different prior to stress. Therefore, the chemical oxidation and enzymatic re-reduction might be expected to occur at similar rates, as observed.

(5) Line 215. For the reason stated above, there is no reason to invoke Cys uptake as needed for the reduction of BSSB. Further, since CySS (presumably an abbreviation for cystine) is imported, this itself can contribute to disulfide stress.

(6) Line 235. Following on the above point, "diamide-induced disulfide stress increased L-CySS uptake in the spxC10A mutant to re-establish the BSH redox equilibrium." This is counterintuitive since LCySS is itself a disulfide and is thought to be reduced to 2 L-Cys in cells by BSH (leading to an increase in BSSB, not a reduction). Is there a known cystine reductase? Could cystine or L-cys be affecting gene regulation? (e.g., through CymR or Spx or ?). Cystine can also lead to mixed disulfide formation (e.g., could it modify Spx on C13?).

(7) l. 247 "a functional Spx redox switch allows S. aureus to avoid this trade-off and maintain thiol homeostasis without excessive L-CySS uptake." Can the authors expand on how this is thought to work? Does Spx normally affect cystine uptake? I thought this was CymR? I am not following the logic here.

(8) Line 258. "The fur mutant, which is known to accumulate iron...". My understanding is that fur mutant strains typically have higher bioavailable (free) Fe pools. This is seen in E. coli, for example, using EPR methods. However, they also often have lower total Fe due to the iron-sparing response, which represses the expression of abundant, Fe-rich proteins. Please provide a reference that supports this statement that in S. aureus fur mutants have higher total iron per cell.

(9) Figure 4. For the reasons stated above (point 1), it is hard to interpret data presented only as "Rel. Growth". Perhaps growth curve data could be included in a supplement.

(10) The interpretation of Figure 4 is complicated. It is not clear that there is necessarily a change in bioavailable Fe pools, although it does seem clear that Fe homeostasis is perturbed. It has been shown that one strong effect of DIA on B. subtilis physiology is to oxidize the BSH pool to BSSB (as shown also here), and this leads to a mobilization of Zn (buffered by BSH). Elevated Zn pools can inactivate some Fe(II)-dependent enzymes, which could account for the rescue by Fe(II) supplementation. Zn(II) can also dysregulate PerR and likely Fur regulons.

Reviewer #3 (Public review):

Summary:

In this manuscript, Blanco-Ameijeiras and collaborators describe the 3D differentiation of human pluripotent stem cells into the posterior spinal cord. The authors first test the exposure of different combinations of extrinsic signals to generate human neural organoids with distinct antero-posterior identities, as shown by bulk transcriptome analysis. They show that neural organoids, whether anterior or posterior, display tissue architecture, organisation and dynamics resembling the in vivo situation. Increasing the size of initial cell aggregates leads to the formation of a single lumen through a multi-lumen stage and a process of cell intercalation, mimicking the situation that they recently described for chick secondary neurulation (Gonzalez-Gobartt et al. Dev Cell. 2021 PMID: 33878300). The authors go on to show that, as in chick, YAP is involved in the resolution of multiple lumens into a single lumen. They conclude that their human organoid approach faithfully models human secondary neurulation, which may be instrumental in unravelling the mechanisms of human neural tube defects.

Strengths:

Overall, this is an important study demonstrating that lumen formation in human spinal organoids recapitulates key aspects of secondary neurulation observed in animal models. This organoid approach may be instrumental in unravelling the mechanisms of human neural tube defects.

Weaknesses:

The significance of the findings is tempered by several limitations. While the authors show convincing evidence that organoids undergo lumen formation with similar morphological, cellular and molecular features as seen in chick in their previous work (Gonzalez-Gobartt et al. Dev Cell. 2021 PMID: 33878300), whether this is linked to their caudal spinal cord identity is unclear.

Reviewer #3 (Public review):

Summary:

In this study, Liu and colleagues utilize TRAP-seq to profile the repertoire of actively translated mRNAs in different intestinal cell types (anterior INT1 vs. posterior INT2-9 cells) in C. elegans. A key goal of this study was to identify transcripts differentially expressed/translated between these intestinal cell subtypes in the context of animals being well fed or subjected to acute (30 minutes) or chronic (3 hours) starvation, followed by refeeding.

The authors identify a number of differentially expressed genes across all of the conditions tested. They then provide an initial survey of the landscape of translatome changes through Weighted Gene Network Correlation Analysis (WGNA), and some high-level functional surveys via Gene Ontology (GO) term analysis and protein domain analysis. The authors validate the enriched expression patterns of some of their identified candidate genes using fluorescent promoter fusion reporters, confirming INT1-specific expression. The authors further implicate the role of several other candidate genes in pathogen avoidance and in response to nutritional cues by knocking them down specifically in INT1 cells by RNAi. Finally, the authors identify pyruvate as a major nutrient signal coming from the bacterial diet that suppresses the release of a key insulin peptide (INS-7), and identify some of the genes expressed in INT1 that are required for this response.

Strengths:

(1) Good use of and justification for TRAP-seq, because scRNA-seq would be difficult under the varied conditions used (starvation, refeeding).

(2) The manuscript is generally clear to read, and the data are generally well-presented with good supporting data that includes replicates, sample sizes, error measurements, and associated statistics.

(3) The dataset will be an interesting resource to mine for future studies focusing on mechanisms of how particular intestinal cell types respond to different environmental signals.

Weaknesses:

(1) A limitation of TRAP-seq, although powerful, is that only relative comparisons can be made between genotypes/conditions to identify differentially-expressed genes, rather than assessing whether a given gene is expressed at a certain level in a cell type under a certain condition. This limitation is due to the non-specific association of sticky RNA species with the beads during the immunoprecipitation step. This is a minor point, however, and the authors do a nice job of focusing their analysis on differentially expressed transcripts in the current study.

(2) Another limitation of the current study is that the experiments testing the role of candidate genes identified by their profiling experiments do not delve a bit deeper into providing a mechanistic understanding of the phenotypes being studied. At present, the results are thus viewed more as a genomics-based screen with some limited follow-up on interesting hits. However, this reviewer appreciates that when placed in the context of the work presented, a presentation of the profiling data along with some validation is an excellent starting point for future mechanistic studies elaborating on these interesting candidates.

Appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

The main goal of the study was to survey the dynamic responses at the level of actively translated mRNAs of the INT1 vs INT2-9 cells in response to metabolic challenge.

Overall, the authors use established methods to perform their genome-wide analysis, and the set of differentially regulated genes is enriched for expected molecular functions and forms coherent networks in anticipated pathways.

The validation experiments (promoter::GFP fusion reporters, INT1-specific knockdowns of highly regulated genes) further corroborate the quality of the TRAP-seq datasets generated.

I have a few points for the authors that would further strengthen this work:

(1) The authors rightfully focus on the top differentially-regulated candidates, but it's unclear at present how far down their fold change list would lead to expression pattern validations. It would be useful to test a few more promoter::GFP fusion reporters at different enrichment/fold-change/statistical cutoffs.

(2) Although the INT1-specific RNAi provides a convenient strategy for rapidly perturbing and testing genes of interest for phenotypes, independently validating the knockdowns with genetic mutants, or alternatively (if genes are essential), degron alleles.

Impact:

The TRAP-seq data and list of differentially-expressed candidate genes will form an interesting set of high-priority candidates to study for their role in the reception and transduction of nutritional cues in response to food status and pathogens. This data will thus benefit the C. elegans community of researchers studying the mechanisms governing these phenomena.

Reviewer #3 (Public review):

Summary:

The presented study describes the long journey towards the expression of members' SVMP toxins from snake venom, which are toxins of major importance in a snakebite scenario. As in the past, their functional analysis relied on challenging isolation; the toxins' heterologous expression offers a potential solution to some major obstacles hindering a better understanding of toxin pathophysiology. Through a series of laborious and elegantly crafted experiments, including the reporting of various failed attempts, the authors establish the expression of all three SVMP subtypes and prove their activity in bioassays. The expression is carried out as naturally occurring zymogens that autocleave upon exposure to zinc, which is a novel modus operandi for yielding fusion proteins and sheds also some new light on the potential mechanism that snakes use to activate enzymatic toxins from zymogenic preforms.

Strengths:

The manuscript draws from an extensive portfolio of well-reasoned and hypothesis-driven experiments that lead to a stepwise solution. The wetlands data generated is outstanding, although not all experiments along this rocky road to victory were successful. A major strength of the paper is that, translationally speaking, it opens up novel routes for biodiscovery since a first reliable platform for expression of an understudied, yet potent toxin class is established. The discovered strategy to pursue expression as zymogens could see broad application in venom biotechnology, where several toxin types are pending successful expression. The work further provides better insights into how snake toxins are processed.

Weaknesses:

The manuscript contains several chapters reporting failed experiments, which makes it difficult to follow in places. The reporting of experimental details, especially sample sizes and replicates, could be optimised. At the time of writing, it remains unclear whether the glycosilations detected at a pIII SVMP could have an impact on the bioactivities measured, which is a major aspect, and future follow-ups should clarify this. Finally, the work, albeit of critical importance, would benefit from a more down-to-earth evaluation of its findings, as still various persistent obstacles that need to be overcome.

Major comments to the manuscript:

(1) Lines 148-149: "indicating that expressing inactivated SVMPs could be a viable, although inefficient, approach". I think this text serves a good purpose to express some thoughts on the nature of how the current draft is set up. It is quite established that various proteases cause extreme viability losses to their expression host (whether due to toxicity, but surely also because of metabolic burden), which is why their expression as inactive fusion proteins is the default strategy in all cases I have thus far seen. I believe that, especially in venom studies, this is of importance given the increased toxicity often targeting cellular integrity, and especially here, because Echis are known to feed on arthropods at younger life history stages, making it very likely that some venom components are especially active against insects and other invertebrates. With that in mind, I would argue that exploring their production in inactive form is the obvious strategy one would come up with and not really the conclusion of a series of (well-conducted and scientifically sound!) experiments. For me, the insight of inactive expression is largely confirmatory of what is established, unless I miss something in the authors' rationale. If yes, it would be important to clarify that in the online version.

(2) Line 173: Here, Alphafold 3 was used, whereas in previous sections (e.g., line 153, line 210), it was Alphafold 2. I suggest using one release across the manuscript.

(3) Line 252-254: I fully agree, the PIII SVMP is glycosylated. Glycosylation is an important mediator of snake venom activity, and several works have described their importance in the field. This raises the question, which glycosylations have been introduced here in the SVMP, and to verify that these are glycosylations that belong to those found in snakes. This is important as insects facilitate thousands of N- and O- O-glycosylations to modulate the activity of their proteome, of which many are specific to insects. If some of these were integrated into the SVMP, this could have an impact on downstream produced bioassays and also antigenicity (the surface would be somewhat different from natural toxins, causing different selection).

(4) General comment for the bioassays: It would be good to specify the replicates again and report the data, including standard deviations.

Discussion:

I think the data generated in the study is very valuable and will be instrumental for pushing the frontiers in SVMP research, but still I would like to see a bit of modesty in their discussion. As I have pointed out above, it is unclear which effect the glycosilations may have (i.e., are the glycosilations found reminiscent of natural ones?), despite their being functionally important. Also, yes, isolation of SVMPs is challenging, but the reality is that their expression is equally challenging, as evidenced by the heaps of presented negative data (with which I have no problems, I think reporting such is actually important). So far, the "generic" protocol has been used to express one member per structural class of Echis SVMP, but no evidence is provided that it would work equally well on other members from taxonomically more distant snakes (e.g., the pIII known from Naja oxiana). It is very likely, but at the time of writing, purely speculative. Lastly, the reality is also that the expression in insect cells can only be carried out by highly specialized labs (even in the expression world, as most laboratories work with bacterial or fungal hosts), whereas the isolation can be attempted in most venom labs. That said, production in insect cells also has economic repercussions as it will be very challenging to generate yields that are economically viable versus other systems, which is pivotal because the authors talk about bioprospecting and the toxins used in snakebite agent research. Again, I believe the paper is highly important and excellently crafted, but I think especially the discussion should see some refinement to address the drawbacks and to evaluate the paper's findings with more modesty.

Reviewer #3 (Public review):

Summary:

This manuscript by Ampartzidis et al., significantly extends the human induced pluripotent stem cell system originally characterized by the same group as a tool for examining cellular remodeling during differentiation stages consistent with those of human neural tube closure (Ampartzidis et al., 2023). Given that there are no direct ways to analyze cellular activity in human neural tube closure in vivo, this model represents an important platform for investigating neural tube defects which are a common and deleterious human developmental disease. Here, the authors carefully test whether this system is robust and reproducible when using hiPSC cells from different donors and pluripotency induction methods and find that despite all these variables the cellular remodeling programs that occur during early neural differentiation are statistically equivalent, suggesting that this system is a useful experimental substrate. Additionally, the carefully selected donor populations suggest these aspects of human neural tube closure are likely to be robust to sexual dimorphism and to reasonable levels of human genetic background variation, though more fully testing that proposition would require significant effort and be beyond the scope of the current work. Subsequent to this careful characterization, the authors next tested whether this system could be used to derive specific insights into cell remodeling during early neural differentiation. First, they used a reverse genetics approach to knock in a human point mutation in the critical regulator of planar cell polarity and apical constriction, Vangl2. Despite being identified in a patient, this R353C variant has not been directly functionally tested in a human system. The authors find that this variant, despite showing normal expression and phospho-regulation, leads to defects consistent with a failure in apical constriction, a key cell behavior required to drive curvature change during cranial closure. Finally, the authors test the utility of their hiPSC platform to understand human patient-specific defects by differentiating cells derived from two clinical spina bifida patients. The authors identify that one of these patients is likely to have a significant defect in fully establishing early proneural identity as well as defects in apicobasal thickening. While early remodeling occurs normally in the other patient, the authors observe significant defects in later neuronal induction and maturation. In addition, using whole exome sequencing the authors identify candidate variant loci that could underly these defects.

Major comments:

(1) One of my few concerns with this work is that the relative constriction of the apical surface with respect to the basal surface is not directly quantified for any of the experiments. This worry is slightly compounded by the 3D reconstructions Figure 1h, and the observation that overall cell volume is reduced and cell height increased simultaneously to area loss. Additionally, the net impact of apical constriction in tissues in vivo is to create local or global curvature change, but all the images in the paper suggest that the differentiated neural tissues are an uncurved monolayer even missing local buckles. I understand that these cells are grown on flat adherent surfaces limiting global curvature change, but is there evidence of localized buckling in the monolayer? While I believe-along with the authors-that their phenotypes are likely failures in apical constriction, I think they should work to strengthen this conclusion. I think the easiest way (and hopefully using data they already have) would be to directly compare apical area to basal area on a cell wise basis for some number of cells. Given the heterogeneity of cells, perhaps 30-50 cells per condition/line/mutant would be good? I am open to other approaches; this just seems like it may not require additional experiments.

(2) Another slight experimental concern I have regards the difference in laser ablation experiments detailed in Figure 3h-i from those of Figure 2d-e. It seems like WT recoil values in 3h-I are more variable and of a lower average than the earlier experiments and given that it appears significance is reached mainly by impact of the lower values, can the authors explain if this variability is expected to be due to heterogeneity in the tissue, i.e. some areas have higher local tension? If so, would that correspond with more local apical constriction?

Significance:

Overall, I am enthusiastic about this work and believe it represents a significant step forward in the effort to establish precision medicine approaches for diagnoses of the patient-specific causative cellular defects underlying human neural tube closure defects. This work systematizes an important and novel tool to examine the cellular basis of neural tube defects. While other hiPSC models of neural tube closure capture some tissue level dynamics, which this model does not, they require complex microfluidic approaches and have limited accessibility to direct imaging of cell remodeling. Comparatively, the relative simplicity of the reported model and the work demonstrating its tractability as a patient-specific and reverse genetic platform make it unique and attractive. This work will be of interest to a broad cross section of basic scientists interested in the cellular basis of tissue remodeling and/or the early events of nervous system development as well as clinical scientists interested in modeling the consequences of patient specific human genetic deficits identified in neural tube defect pregnancies.

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Reviewer #3 (Public review):

Summary:

CTF18-RFC is an alternative eukaryotic PCNA sliding clamp loader which is thought to specialize in loading PCNA on the leading strand. Eukaryotic clamp loaders (RFC complexes) have an interchangeable large subunit which is responsible for their specialized functions. The authors show that the CTF18 large subunit has several features responsible for its weaker PCNA loading activity, and that the resulting weakened stability of the complex is compensated by a novel beta hairpin backside hook. The authors show this hook is required for the optimal stability and activity of the complex.

Relevance:

The structural findings are important for understanding RFC enzymology and novel ways that the widespread class of AAA ATPases can be adapted to specialized functions. A better understanding of CTF18-RFC function will also provide clarity into aspects of DNA replication, cohesion establishment and the DNA damage response.

Strengths:

The cryo-EM structures are of high quality enabling accurate modelling of the complex and providing a strong basis for analyzing differences and similarities with other RFC complexes. They use complementary pre-steady state FRET and polymerase primer extension assays to investigate the role of a unique structural element in CTF18.

Weaknesses:

The manuscript would have benefited from a more detailed biochemical analysis using mutagenesis and assays to tease apart the functional relevance of the many differences with the canonical RFC complex.

Overall appraisal:

Overall, the work presented here is solid and important. The data is sufficient to support the stated conclusions.

Reviewer #3 (Public review):

Summary:

The present paper by Shinoda et al. from the Miura group builds upon findings reported in an earlier study by the same team (Shinoda et al., PNAS, 2019), which identified a non-apoptotic role for the Drosophila executioner caspase Dcp-1 in promoting wing tissue growth. That earlier work attributed this function primarily to Dcp-1 and to Decay, a caspase structurally related to executioner caspases, but not to DrICE, the principal apoptotic executioner caspase. The authors further proposed that this non-apoptotic caspase activity operates independently of the initiator caspase Dronc.

In the current study, the authors both corroborate aspects of their previous findings and extend the investigation to mechanisms regulating Dcp-1 in this context. They identify roles for the giant IAP Bruce, two BCL-2 family members, and autophagy-related components in modulating non-apoptotic Dcp-1 activity. Moreover, they show that Bruce binds to a BIR-like peptide exposed upon Dcp-1 cleavage, but not to DrICE. The study further suggests that low levels of Dcp-1 activity promote wing tissue growth, whereas excessive activity induces cell death, as evidenced by impaired wing development following Dcp-1 overexpression. Overall, the manuscript provides several intriguing insights into the non-apoptotic regulation of the comparatively weak apoptotic executioner caspase Dcp-1 and complements the group's earlier work. However, several concerns remain regarding certain interpretations of the data and the experimental rigour of some of the results.

Strengths:

A major strength of the work is its systematic genetic and biochemical approaches, which combine tissue-specific manipulation with protein interaction mapping to explore how Dcp-1 is regulated. The identification of several regulatory factors, including an inhibitor of cell death protein and components linked to autophagy, provides a coherent framework for understanding how Dcp-1 activity might be tuned.

Weaknesses:

The evidence supporting some key claims remains incomplete. In particular, the type of cell death form induced when Dcp-1 is overexpressed is not clearly established, and additional tests would be needed to distinguish between the different cell death types.

Likely impact:

The study contributes to a growing body of work showing that proteins traditionally associated with cell death can have broader roles in tissue development. This conceptual advance is likely to be of interest to researchers studying growth control and tissue maintenance.

Specific points:

(1) Nature of the wing ablation phenotype

A central concern is whether the wing ablation phenotype observed upon Dcp-1 overexpression truly reflects apoptotic cell death. The authors show in Figure 1c that nuclei in cells overexpressing Dcp-1, but not DrICE, zymogens are highly condensed, which is suggestive of apoptosis. However, it is equally plausible that this phenotype reflects a form of non-apoptotic, Dcp-1-dependent cell death (e.g. autophagy-dependent cell death). This distinction could be readily addressed using TUNEL labelling and direct caspase activity assays. The latter would be particularly informative, as it remains unclear whether zymogen Dcp-1 is capable of cleaving standard effector caspase reporters in vivo. Does the anti-cleaved Dcp-1 antibody detect Dcp-1 activation following overexpression of the Dcp-1 zymogen?

(2) Role of Decay

In their earlier study, the authors identified Decay as another caspase influencing wing growth, albeit more modestly than Dcp-1. It is therefore unclear why this line of investigation was not pursued further in the current work. This omission is notable, as Decay is not implicated in apoptosis and, to date, no substantial physiological function has been assigned to this caspase in any system. At a minimum, this point should be discussed explicitly.

(3) Figure 2: Proximity labelling analysis

The authors use TurboID-mediated proximity labelling to reveal distinct Dcp-1- and DrICE-associated proteomes across tissues, with a particular focus on the wing disc. They further demonstrate that RNAi-mediated knockdown of the Dcp-1-associated proteins Sirt1 and Fkbp59 suppresses the wing ablation phenotype induced by Dcp-1 overexpression, suggesting that these factors are required for Dcp-1 activity. However, it should be clarified whether Bruce was identified as a Dcp-1 interactor in the proximity labelling dataset, given its proposed central regulatory role. In addition, further discussion of Fkbp59, its known functions and how it might mechanistically influence Dcp-1 activity would be valuable.

(4) Figure 3: Autophagy-related factors

Given that Sirt1 is known to promote autophagy, the authors next examine autophagy-related proteins and identify roles for Atg2, Atg8a, Debcl, and Buffy in Dcp-1 activation. Notably, these proteins do not promote cell death in the Hid-induced canonical apoptotic pathway. However, it is important to determine whether knockdown of Debcl, Buffy, Atg2, or Atg8a alone affects wing development in the absence of Dcp-1 overexpression, to exclude the possibility that these perturbations independently impair wing formation.

(5) Evidence for canonical autophagy

The involvement of autophagy would be more convincingly demonstrated by testing additional core autophagy genes, such as Atg7, Atg5, and Atg12, as well as performing a combined knockdown of Atg8a and Atg8b. Moreover, direct assessment of autophagy at the cellular level using established genetic reporters would substantially strengthen the conclusions.

(6) Figures 4-5: Functional consequences

It would be informative to determine whether Synr, Debcl, or Buffy influence wing size on their own and whether their overexpression enhances wing growth.

(7) Terminology and interpretation of cell death

Taken together, the results suggest that Dcp-1 zymogen overexpression induces a form of non-apoptotic cell death, potentially autophagy-dependent or related. The reviewer does not understand the authors' insistence on referring to this process as apoptosis. The authors should be more cautious in their terminology: there is no canonical versus non-canonical apoptosis; there is simply apoptosis. Without stronger evidence, these effects should not be described as apoptotic cell death.

Reviewer #3 (Public review):

Summary:

Lazimi and coworkers present an updated experimental protocol by which viral vectors can be used with live pancreas slices in order to efficiently transduce fluorescent protein biosensors. This is of high importance, given that live human pancreas slices provide a means to study islet function while maintaining the architecture of the local environment. Thus, efficiently delivering a wide range of fluorescent protein biosensors provides expanded capabilities to study the human islet and its dysfunction in type 1 and type 2 diabetes. The authors demonstrate the improved transduction provided by their revised protocol, which includes orbital culture, while retaining or, in some cases, improving cell viability, hormone release, and Ca2+ responses. Further, the authors demonstrate how a 'Ca2+ integrator', CAMPARI2, can be used to profile the Ca2+ response of large numbers of cells and islets, to capture the variability in islet responses in healthy and diabetic cases.

Strengths:

The data presented are generally robust, and the methods are well described, such that this protocol could be repeated by other investigators. All findings are representative of multiple donors. Importantly, the data is highly novel.

Weaknesses:

Weaknesses in the manuscript mainly include a lack of technical details by which data is presented or analyzed, as well as caveats by which certain data related to islet size are interpreted.