Reviewer #2 (Public review):

Summary:

A particular challenge in treating infections caused by the parasite Toxoplasma gondii is to target (and ultimately clear) the tissue cysts that persist for the lifetime of an infected individual. The study by Maus and colleagues leverages the development of a powerful in vitro culture system for the cyst-forming bradyzoite stage of Toxoplasma parasites to screen a compound library for candidate inhibitors of parasite proliferation and survival. They identify numerous inhibitors capable of inhibiting both the disease-causing tachyzoite and the cyst-forming bradyzoite stages of the parasite. To characterize the potential targets of some of these inhibitors, they undertake metabolomic analyses. The metabolic signatures from these analyses lead them to identify one compound (MMV1028806) that interferes with aspects of parasite mitochondrial metabolism. The authors claim that MV1028806 targets the bc1 complex of the mitochondrial electron transport chain of the parasite, although the evidence for this is indirect and speculative. Nevertheless, the study presents an exciting approach for identifying and characterizing much-needed inhibitors for targeting tissue cysts in these parasites.

Strengths:

The study presents convincing proof-of-principle evidence that the myotube-based in vitro culture system for T. gondii bradyzoites can be used to screen compound libraries, enabling the identification of compounds that target the proliferation and/or survival of this stage of the parasite. The study also utilizes metabolomic approaches to characterize metabolic 'signatures' that provide clues to the potential targets of candidate inhibitors, although falls short of identifying the actual targets.

Weaknesses:

(1) The authors claim to have identified a compound in their screen (MMV1028806) that targets the bc1 complex of the mitochondrial electron transport chain (ETC). The evidence they present for this claim is indirect (metabolomic signatures and changes in mitochondrial membrane potential) and could be explained by the compound targeting other components of the ETC or affecting mitochondrial biology or metabolism in other ways. In order to make the conclusion that MMV1028806 targets the bc1 complex, the authors should test specifically whether MMV1028806 inhibits bc1-complex activity (i.e. in a direct enzymatic assay for bc1 complex activity). Testing the activity of MMV1028806 against other mitochondrial dehydrogenases (e.g. dihydroorotate dehydrogenase) that feed electrons into the ETC might also provide valuable insights. The experiments the authors perform also do not directly measure whether MMV1028806 impairs ETC activity, and the authors could also test whether this compound inhibits mitochondrial O2 consumption (as would be expected for a bc1 inhibitor).

(2) The authors claim that compounds targeting bradyzoites have greater lipophilicity than other compounds in the library (and imply that these compounds also have greater gastrointestinal absorbability and permeability across the blood-brain barrier). While it is an attractive idea that lipophilicity influences drug targeting against bradyzoites, the effect seems pretty small and is complicated by the fact that the comparison is being made to compounds that are not active against parasites. If the authors are correct in their assertion that lipophilicity is a major determinant of bradyzoicidal compounds compared to compounds that target tachyzoites alone, you would expect that compounds that target tachyzoites alone would have lower lipophilicity than those that target bradyzoites. It would therefore make more sense to (statistically) compare the bradyzoicidal and dual-acting compounds to those that are only active in tachyzoites (visually the differences seem small in Figure S2B). This hypothesis would be better tested through a structure-activity relationship study of select compounds (which is beyond the scope of the study). Overall, the evidence the authors present that high lipophilicity is a determinant of bradyzoite targeting is not very convincing, and the authors should present their conclusions in a more cautious manner.

(3) Page 11 and Figure 7. The authors claim that their data indicate that ATP is produced by the mitochondria of bradyzoites "independently of exogenous glucose and HDQ-target enzymes." The authors cite their previous study (Christiansen et al, 2022) as evidence that HDQ can enter bradyzoites, since HDQ causes a decrease in mitochondrial membrane potential. Membrane potential is linked to the synthesis of ATP via oxidative phosphorylation. If HDQ is really causing a depletion of membrane potential, is it surprising that the authors observe no decrease in ATP levels in these parasites? Testing the importance of HDQ-target enzymes using genetic approaches (e.g. gene knockout approaches) would provide better insights than the ATP measurements presented in the manuscript, although would require considerable extra work that may be beyond the scope of the study. Given that the authors' assay can't distinguish between ATP synthesized in the mitochondrion vs glycolysis, they may wish to interpret their data with greater caution.

Reviewer #2 (Public review):

The present study by Le Gac et al. investigates the vasoconstriction of cerebral arteries during neurovascular coupling. It proposes that pyramidal neurons firing at high frequency lead to prostaglandin E2 (PGE2) release and activation of arteriolar EP1 and EP3 receptors, causing smooth muscle cell contraction. The authors further claim that interneurons and astrocytes also contribute to vasoconstriction via neuropeptide Y (NPY) and 20-hydroxyeicosatetraenoic acid (20-HETE) release, respectively. The study mainly uses brain slices and pharmacological tools in combination with Emx1-Cre; Ai32 transgenic mice expressing the H134R variant of channelrhodopsin-2 (ChR2) in the cortical glutamatergic neurons for precise photoactivation. Stimulation with 470 nm light using 10-second trains of 5-ms pulses at frequencies from 1-20 Hz revealed small constrictions at 10 Hz and robust constrictions at 20 Hz, which were abolished by TTX and partially inhibited by a cocktail of glutamate receptor antagonists. Inhibition of cyclooxygenase-1 (COX-1) or -2 (COX-2) by indomethacin blocked the constriction both ex vivo (slices) and in vivo (pial artery), and inhibition of EP1 and EP3 showed the same effect ex vivo. Single-cell RT-PCR from patched neurons confirmed the presence of the PGE2 synthesis pathway.

While the data are convincing, the overall experimental setting presents some limitations. How is the activation protocol comparable to physiological firing frequency? The delay (minutes) between the stimulation and the constriction appears contradictory to the proposed pathway, which would be expected to occur rapidly. The experiments are conducted in the absence of vascular "tone," which further questions the significance of the findings. Some of the targets investigated are expressed by multiple cell types, which makes the interpretation difficult; for example, cyclooxygenases are also expressed by endothelial cells. Finally, how is the complete inhibition of the constriction by the NPY Y1 receptor antagonist BIBP3226 consistent with a direct effect of PGE2 and 20-HETE in arterioles? Overall, the manuscript is well-written with clear data, but the interpretation and physiological relevance have some limitations. However, vasoconstriction is a rather understudied phenomenon in neurovascular coupling, and the present findings may be of significance in the context of pathological brain hypoperfusion.

Reviewer #2 (Public review):

The authors aim to investigate the role of spontaneous activity in shaping the development of multisensory integration in the brain, specifically focusing on the connections between primary visual and somatosensory sensory areas (V1 and S1) and a higher-order cortical area rostro-lateral to V1 (RL). They seek to understand how spontaneous activity guides the formation of aligned topographic maps and the emergence of bimodal neurons in RL.

First, the authors found that spontaneous activity in all three areas sparsifies over time, but S1 exhibits more mature patterns earlier than V1 and RL. They claimed that correlated activity among neighboring regions of these areas during development carries topographic information. These data were used to implement a computational model that employed Hebbian rules of synaptic plasticity. The model indicated that correlated spontaneous activity can generate topographic connectivity between S1/V1 and RL and bimodal neurons in RL. The model suggested that the more mature spontaneous activity in S1 can guide map alignment between V1 and RL. In addition, the model also suggested that a mixture of bimodal and unimodal neurons in RL is optimal for decoding information from V1 and S1.

While the data presented in the manuscript is promising and provides preliminary insights into the role of spontaneous activity in multisensory integration, it would be beneficial to strengthen the experimental foundation regarding the correlation between V1, S1, and RL. Incorporating more rigorous spatio-temporal analyses of spontaneous activity could enhance the robustness of these findings.

Here are some important concerns:

(1) The analysis of how spatial topography influences activity correlations in Figure 2 has several issues.<br /> 1a. While squares in V1 and S1 covered a small area of these sensory areas, the correlated territories in RL covered the entire area of RL. The topographic map in V1 continues caudally, so where is the rest of the map in RL? Something similar applies to the relationship between S1 and RL.<br /> 1b. It is essential to know how areas were drawn. High precision is required.<br /> 1c. It is not clear if correlated activity means different events in sync or large events that cover 2 or all 3 cortical areas of interest. The figure points to the second option, which contradicts the size of events at these stages, mainly in the oldest mice analyzed here.<br /> 1d. It is fundamental to know in detail and provide examples of how the detection of events was performed. For instance, could the dispersion of light from an event in V1 close to RL cause the detection of activity in RL?

(2) For the correlations among V1, S1, and RL, it is crucial to have a consistent method to delineate the borders of cortical areas. The authors mention in one sentence that areas were drawn according to a reference map. More details are needed to convince the reader that the borders are accurate, especially because their shape and position change with age.

(3) The results from the model seem to be based on the initial bias in connectivity between neighboring cells from the different areas. Then, it seems straightforward that implementing correlated activity with Hebbian and synaptic depression rules will force the strengthening of connections between spatially close cells. Despite this apparent predisposition of the model towards a defined outcome, the flaws in the experimental data used prevent a rigorous interpretation of the computational model.

(4) In the Introduction, the authors nicely and briefly explain the role of primary and higher-order sensory cortices in information processing. They also explain how spontaneous activity during development helps to build these circuits by refining connections or establishing hierarchies. They continue explaining the relevance of aligning different topographic maps to allow multisensory integration. Then they provide some examples of sites of multisensory integration. This provides a general context for the data presented in the Results section; however, and importantly, there is no specific introduction of why they are interested in RL and its interaction with V1 and S1. The authors should introduce the RL area and explain why it is an interesting site for multisensory processing.

(5) The results shown in Figure 1 corroborate published data from Golshani et al, Rochefort et al, Murakami et al. While the reproduction of data is more than welcome, the authors should specify which part of the data is completely new and acknowledge clearly the rest as corroboration of previous data. The sentence "As described in previous experiments ..." partially acknowledges this fact but is not clear enough. In addition, the transition between this part of the manuscript and the next data is not smooth. Data seems to be used to feed the model so perhaps the organization of the manuscript leaves room for improvement.

Reviewer #2 (Public review):

Summary

Briola and co-authors have performed a structural analysis of the human CTF18 clamp loader bound to PCNA. The authors purified the complexes and formed a complex in solution. They used cryo-EM to determine the structure to high resolution. The complex assumed an auto-inhibited conformation, where DNA binding is blocked, which is of regulatory importance and suggests that additional factors could be required to support PCNA loading on DNA. The authors carefully analysed the structure and compared it to RFC and related structures.

Strength & Weakness

Their overall analysis is of high quality, and they identified, among other things, a human-specific beta-hairpin in Ctf18 that flexibly tethers Ctf18 to Rfc2-5. Indeed, deletion of the beta-hairpin resulted in reduced complex stability and a reduction in a primer extension assay with Pol ε. This is potentially very interesting, although some more work is needed on the quantification. Moreover, the authors argue that the Ctf18 ATP-binding domain assumes a more flexible organisation, but their visual representation could be improved.

The data are discussed accurately and relevantly, which provides an important framework for rationalising the results.

All in all, this is a high-quality manuscript that identifies a key intermediate in CTF18-dependent clamp loading.

Reviewer #2 (Public review):

Summary:

This paper describes the molecular characterisation of a new isolate of the giant virus Jyvaskylavirus, a member of the Marseilleviridae family infecting Acanthamoeba castellanii. The isolate comes from a boreal environment in Finland, showcasing that giant viruses can thrive in this ecological niche. The authors came up with a non-trivial isolation procedure that can be applied to characterise other members of the family and will be beneficial for the virology field. The genome shows typical Marseilleviridae features and phylogenetically belongs to their clade B. The structural characterisation was performed on the level of isolated virion morphology by negative stain EM, virions associated with cells either during the attachment or release by helium microscopy, the visualisation of the virus assembly inside cells using stained thin sections, and lastly on the protein secondary structure level by reconstructing ~6 A icosahedral map of the massive virion using cryoEM. The cryoEM density combined with gene product structure prediction enabled the identification and functional assessment of various virion proteins.

Strengths:

The detailed description of the virus isolation protocol is the largest strength of the paper and this reviewer believes it can be modified for isolating various viruses infecting small eukaryotes. The cryoEM map allows us to understand how exceptionally large virions of these viruses are stabilised by minor capsid proteins and nicely demonstrates the integration of medium-resolution cryoEM with protein structure prediction in deciphering virion protein function. The visualisation of ongoing virus assembly inside virus factories brings interesting hypotheses about the process that; however, needs to be verified in the next studies.

Weaknesses:

The conclusions from helium microscopy images are overinterpreted, as the native membrane structure cannot be preserved in a fixed and dehydrated sample. In the image, there are many other parts of the curved membrane and a lot of virions, to me it seems the specific position of the highlighted virion could arise by a random chance. The claim that the cells were imaged in the near-original state by this method should be therefore omitted. Also, no mass spectrometry data are presented that would supplement and confirm the identity of virion proteins which predicted models were fitted into the cryoEM density. For a general virology reader outside of the giant virus field, the results presented in the current state might not have enough influence and the section should be rewritten to better showcase the novelty of findings.

Reviewer #2 (Public review):

Summary:

The authors recently uncovered a novel nested gene, Aff3ir, and this work sets out to study its function in endothelial cells further. Based on differences in expression correlating with areas of altered shear stress, they investigate a role for the isoform Aff3ir-ORF2 in endothelial activation and development of atherosclerosis downstream of disturbed shear stress. Using a knockout mouse model and in vivo overexpression experiments, they demonstrate a strong potential for Aff3ir-ORF2 to alleviate atherosclerosis. They find that Aff3ir-ORF2 interacts with the pro-inflammatory transcription factor IRF5 and retains it in the cytoplasm, hence preventing upregulation of inflammation-associated genes. The data expands our knowledge of IRF5 regulation which could be relevant to researchers studying various inflammatory diseases as well as adding to our understanding of atherosclerosis development.

Strengths:

The in vivo data is solid using immunofluorescence staining to assess AFF3ir-ORF2 expression, a knockout mouse model, overexpression and knockdown studies, and rescue experiments in combination with two atherosclerotic models to demonstrate that Aff3ir-ORF2 can lessen atherosclerotic plaque formation in ApoE-/- mice.

Weaknesses:

While the in vivo data is generally convincing, a few data panels have issues and will need addressing. Also, the knockout mouse model will need to be described, since the paper referred to in the manuscript does not actually report any knockout mouse model. Hence it is unclear how Aff3ir-ORF2 is targeted, but Figure S2B shows that targeting is partial, since about 30% expression remains at the RNA level in MEFs isolated from the knockout mice.

While the effect on atherosclerosis is clear, the conclusion that this is the result of reduced endothelial cell activation is not supported by the data. The mouse model is described as a global knockout and the shRNA knockdowns (Figure 5) and overexpression data in Figure 2 are not cell type-specific. Only the overexpression construct in Figure 6 uses an ICAM-2 promoter construct, which drives expression in endothelial cells, though leaky expression of this promoter has been reported in the literature. Therefore, other cell types such as smooth muscle cells or macrophages could be responsible for the effects observed.

The weakest part of the manuscript is the in vitro experiments. While they are solidly executed, all experiments are performed in MEFs, and results are interpreted as being equivalent to endothelial cell responses. There is also an RNA-seq experiment performed on MEFs from the Aff3ir-ORF2 knockout and control mice, but the data is not disclosed other than showing some non-identifiable expression differences. The data is used to hypothesise on a role for IRF5 in the effects observed with Aff3ir-ORF2 knockout.

Overall, the paper succeeds in demonstrating a link between Aff3ir-ORF2 and atherosclerosis, but the cell types involved and mechanisms remain unclear. The study also shows a functional interaction between Aff3ir-ORF2 and IRF5 in embryonic fibroblasts, but any relevance of this mechanism for atherosclerosis or any cell types involved in the development of this disease remains largely speculative.

Reviewer #2 (Public review):

Summary:

In their study 'AI-Based Discovery and CryoEM Structural Elucidation of a KATP Channel Pharmacochaperone', ElSheikh and colleagues undertake a computational screening approach to identify candidate drugs that may bind to an identified binding pocket in the SUR1 subunit of KATP channels. Other KATP channel inhibitors such as glibenclamide have been previously shown to bind in this pocket, and in addition to inhibition of KATP channel function, these inhibitors can very effectively rescue cell surface expression of trafficking deficient KATP mutations that cause excessive insulin secretion (Congenital Hyperinsulinism). However, a challenge for their utility for the treatment of hyperinsulinism has been that they are powerful inhibitors of the channels that are rescued to the channel surface. In contrast, successful therapeutic pharmacochaperones (eg. CFTR chaperones) permit the function of the channels rescued to the cell membrane. Thus, a key criterion for the authors' approach, in this case, was to identify relatively low-affinity compounds that target the glibenclamide binding site (and be washed off) - these could potentially rescue KATP surface expression but also permit KATP function.

Strengths:

The main findings of the manuscript include:

(1) Computational screening of a large virtual compound library, followed by functional screening of cell surface expression, which identified several potential candidate pharmacochaperones that target the glibenclamide binding site.

(2) Prioritization and functional characterization of Aekatperone as a low-affinity KATP inhibitor which can be readily 'washed off' in patch clamp and cell-based efflux assays. Thus the drug clearly rescues cell surface expression but can be manipulated experimentally to permit the function of rescued channels.

(3) Determination of the binding site and dynamics of this candidate drug by cryo-EM, and functional validation of several residues involved in drug sensitivity using mutagenesis and patch clamp.

The experiments are well-conceived and executed, and the study is clearly described. The results of the experiments are very straightforward and clearly support the conclusions drawn by the authors. I found the study to provide important new information about the KATP chaperone effects of certain drugs, with interesting considerations in terms of ion channel biology and human disease.

Weaknesses:

I don't have any major criticisms of the study as described, but I had some remaining questions that could be addressed in a revision.

(1) The chaperones can effectively rescue KATP trafficking mutants, but clearly not as strongly as the higher affinity inhibitor glibenclamide. Is this relationship between inhibitory potency, and efficacy of trafficking an intrinsic challenge of the approach? I suspect that it may be an intractable problem in the sense that the inhibitor-bound conformation that underlies the chaperone effect cannot be uncoupled from the inhibited gating state. But this might not be true (many partial agonist drugs with low efficacy can be strongly potent, for example). In this case, the approach is really to find a 'happy medium' of a drug that is a weak enough inhibitor to be washed away, but still strong enough to exert some satisfactory chaperone effect. Could some additional clarity be added in the discussion on whether the chaperone and gating effects can be 'uncoupled'?

(2) Based on the western blots in Figure 2B, the rescue of cell surface expression appears to require a higher concentration of AKP compared to the concentration-response of channel inhibition (~9 microM in Figure 3, perhaps even more potent in patch clamp in Figure 2C). Could the authors clarify/quantify the concentration response for trafficking rescue?

(3) A future challenge in the application of pharmacochaperones of this type in hyperinsulinism may be the manipulation of chaperone concentration in order to permit function. In experiments, it is straightforward to wash off the chaperone, but this would not be the case in an organism. I wondered if the authors had attempted to rescue channel function with diazoxide in the presence of AKP, rather than after washing off (ie. is AKP inhibition insurmountable, or can it be overcome by sufficient diazoxide).

(4) Do the authors have any information about the turnover time of KATP after the wash-off of the chaperone (how stable are the rescued channels at the cell surface)? This is a difficult question to probe when glibenclamide is used as a chaperone, but may be much simpler to address with a lower affinity chaperone like AKP.

Reviewer #2 (Public review):

Summary:

The regulation of kinesin is fundamental to cellular morphogenesis. Previously, it has been shown that OSM-3, a kinesin required for intraflagellar transport (IFT), is regulated by autoinhibition. However, it remains totally elusive how the autoinhibition of OSM-3 is released. In this study, the authors have shown that NEKL-3 phosphorylates OSM-3 and releases its autoinhibition.

The authors found NEKL-3 directly phosphorylates OSM-3 (although the method is not described clearly) (Figure 1). The phophorylated residue is the "elbow" of OSM-3. The authors introduced phospho-dead (PD) and phospho-mimic (PM) mutations by genome editing and found that the OSM-3(PD) protein does not form cilia, and instead, accumulates to the axonal tips. The phenotype is similar to another constitutive active mutant of OSM-3, OSM-3(G444A) (Imanishi et al., 2006; Xie et al., 2024). osm-3(PM) has shorter cilia, which resembles with loss of function mutants of osm-3 (Figure 3). The authors did structural prediction and showed that G444E and PD mutations change the conformation of OSM-3 protein (Figure 3). In the single-molecule assays G444E and PD mutations exhibited increased landing rate (Figure 4). By unbiased genetic screening, the authors identified a suppressor mutant of osm-3(PD), in which A489T occurs. The result confirms the importance of this residue. Based on these results, the authors suggest that NEKL-3 induces phosphorylation of the elbow domain and inactivates OSM-3 motor when the motor is synthesized in the cell body. This regulation is essential for proper cilia formation.

Strengths:

The finding is interesting and gives new insight into how the IFT motor is regulated.

Weaknesses:

The methods section has not presented sufficient information to reproduce this study.

Reviewer #2 (Public review):

Summary:

In this manuscript, Farber and colleagues have performed single-cell RNAseq analysis on bone marrow-derived stem cells from DO Mice. By performing network analysis, they look for driver genes that are associated with bone mineral density GWAS associations. They identify two genes as potential candidates to showcase the utility of this approach.

Strengths:

The study is very thorough and the approach is innovative and exciting. The manuscript contains some interesting data relating to how cell differentiation is occurring and the effects of genetics on this process. The section looking for genes with eQTLs that differ across the differentiation trajectory (Figure 4) was particularly exciting.

Weaknesses:

The manuscript is in parts hard to read due to the use of acronyms and there are some questions about data analysis that need to be addressed.

Reviewer #2 (Public review):

Summary:

The present study described GEARBOCS, an adeno-associated virus tool for in vivo gene editing in astrocytes. This tool is timely and important for glial biologists who often are troubled by efficient gene targeting in astrocytes. Overall the significance of the finding is valuable, and the strength of the evidence is solid. Presumably, there will be great potential associated with GEARBOCS applications in the future.

Strengths:

As efficient tools for targeting non-neuronal cells in the brains are rather limited for astrocytes and microglia, GEARBOCS adds to the small pool of currently available tools and will provide new options for glial biologists studying these tools. As the study revealed, GEARBOCS are capable of knockout and knockin manipulations for genes of interest, also ascribed with reporter tracking and gene-trap strategy. The promising multi-functional tool will advance our understanding of astrocytes and help to further elucidate the mechanism of neuron-glia interaction.

Weaknesses:

Even though the tool seems promising and powerful. the authors failed to provide more evidence on the robustness and specificity of GEARBOCS. Also, the advantages of GEARBOCS over some of the traditional methods were not clearly stated. Some of these concerns are described below.

Reviewer #2 (Public review):

Summary:

Wang et al. developed a set of optical sensors to monitor Rab protein activity. Their investigation into Rab activity in dendritic spines during structural long-term plasticity (sLTP) revealed sustained Rab10 inactivation (>30min) and transient Rab4 activation (~5 min). Through pharmacological and genetic manipulation to constitutively activate or inhibit Rab proteins, they found that Rab10 negatively regulates sLTP and AMPA receptor insertion, while Rab4 positively influences sLTP but only in the transient phase. The optical sensors provide new tools for studying Rab activity in cells and neurobiology. However, a full understanding of the timing of Rab activity will require a detailed characterization of sensor kinetics.

Strengths:

(1) Introduction of a series of novel sensors that can address numerous questions in Rab biology.

(2) Multiple methods to manipulate Rab proteins to reveal the roles of Rab10 and rab4 in LTP.

(3) Discovery of Rab4 activation and Rab10 inhibition with different kinetics during sLTP, correlating with their functional roles in the transient (Rab4) and both transient and sustained (Rab10) phases of sLTP.

Weaknesses:

(1) Lack of characterization of sensor kinetics, making it difficult to determine if the observed Rab kinetics during sLTP were due to sensor behavior or actual Rab activity.

(2) It is crucial to assess whether the overexpression of Rab proteins as reporters, affects Rab activity and cellular structure and physiology (e.g. spine number and size).

(3) The paper does not explain the apparently different results between NMDA receptor activation and glutamate uncaging. NMDA receptor activation increased Rab10 activity, while glutamate uncaging decreased it. NMDA receptor activation resulted in sustained Rab4 activation, whereas glutamate uncaging caused only brief activation of about 5 minutes. A potential explanation, ideally supported by data, is needed.

(4) There is a discrepancy between spine phenotype and sLTP potential with Rab10 perturbation. Rab10 perturbation affected spine density but not size, suggesting a role in spinogenesis rather than sLTP. However, glutamate uncaging affected sLTP, and spinogenesis was not examined. Explaining the discrepancy between spine size and sLTP potential is necessary. Exploring spinogenesis with glutamate uncaging would strengthen these results. Additionally, Figure 4j shows no change in synaptic transmission with Rab10 knockout, despite an increase in spine density. An explanation, ideally supported by data, is needed for the unchanged fEPSP slope despite an increase in spine density.

(5) Spine volume was imaged using acceptor fluorophores (mCherry, or mCherry/Venus) at 920nm, where the two-photon cross-section of mCherry is minimal. 920nm was also used to excite the donor fluorophore, hence the spine volume measurement based on total red channel fluorescence is the sum of minimal mCherry fluorescence from direct 920nm excitation, bleed-through from the green channel, and FRET. This confounded measurement requires correction and clarification.

Reviewer #2 (Public review):

This manuscript examines network mechanisms that allow networks of neurons to perform context-dependent decision-making.

In a recent study, Pagan and colleagues identified two distinct mechanisms by which recurrent neural networks can perform such computations. They termed these two mechanisms input-modulation and selection-vector modulation. Pagan and colleagues demonstrated that recurrent neural networks can be trained to implement combinations of these two mechanisms, and related this range of computational strategies with inter-individual variability in rats performing the same task. What type of structure in the recurrent connectivity favors one or the other mechanism however remained an open question.

The present manuscript addresses this specific question by using a class of mechanistically interpretable recurrent neural networks, low-rank RNNs.

The manuscript starts by demonstrating that unit-rank RNNs can only implement the input-modulation mechanism, but not the selection-vector modulation. The authors then build rank three networks that implement selection-vector modulation and show how the two mechanisms can be combined. Finally, they relate the amount of selection-vector modulation with the effective rank, ie the dimensionality of activity, of a trained full-rank RNN.

Strengths:

(1) The manuscript is written in a straightforward manner.<br /> (2) The analytic approach adopted in the manuscript is impressive.<br /> (3) Very clear identification of the mechanisms leading to the two types of context-dependent modulation.<br /> (4) Altogether this manuscript reports remarkable insights into a very timely question.

Weaknesses:

- The introduction could have been written in a more accessible manner for any non-expert readers.

Reviewer #2 (Public review):

Summary:

Scott and Frank propose a new method for controlling generalization and interference in neural networks that undergo continual learning. Their method called coordinated eligibility models (CEM), relies on the factorization of synaptic updates into input-driven and output-driving factors. They subsequently employ the fact that it is sufficient to orthogonalize any one of these two factors across different data points to nullify the interference during learning. They exemplify this on a number of toy tasks while comparing their result to vanilla gradient.

Strengths:

The specific mechanism proposed here is novel (while, as authors acknowledge, there is a large number of other mechanisms for the selective recruitment of synapses for the prevention of catastrophic forgetting). Furthermore, it is simple, elegant, and to a large extent biologically plausible, potentially pointing to specific and testable aspects of learning dynamics.

Weaknesses:

(1) Scope and toy nature of experiments: the model was only applied to very simple problems tailored specifically to demonstrate the strengths of the CEM method. Furthermore, single hyperparameter setting is presented for every scenario which leaves it questionable how general the numerical results are. The selection of input, output dimensionality and data set size also seems to be underexplored. Will a larger curriculum, smaller or larger dimension, compromise any of the CEM ingredients? Restriction to linear models seems arbitrary (it should be a no-time test to add non-linearity within a pytorch framework that authors used), and applicability for any non-synthetic problem is not obvious.

It is also unclear to what extent of domain knowledge is needed for surprise signals to be successfully generated. Can the authors make a stronger case about novel curriculum entries being easily recognizable by cosine distance, either in the brain or in machine learning? Can they alternatively demonstrate their method on a less toy benchmark (e.g. permuted MNIST from Kirkpatrick et al 2017 that they cite)?

Another limitation is that unlike smoother models of plasticity budgets (e.g. Kirkpatrick et al 17, Zenke et al 17), here eligibility seems to be lost forever, once surprise is applied. What happens to the model if more data from a previously visited task becomes available? Will the system be able to continue learning within the right context and how does CEM perform compared to other catastrophic-forgetting-prevention strategies?

(2) The clarity and organization must be improved. Specifically, the balance between verbal descriptions, equations, figures, and their captions needs to be improved. For example - two full-size equations are dedicated to the application of linear regression (around lines 183 and 236) while by far less obvious math such as settings for fig 7, including 'feature loadings', 'demands', etc., is presented in a hardly readable mixture figure and main text. Similarly, the surprise mechanism which is a key ingredient for the model is presented in a very non-straightforward fashion, scattered between the main text, figure, and methods. The figure legends are poorly informative in many cases as well (see minor comments for examples).

Reviewer #2 (Public review):

The author investigates the role of background noise on EEG-assessed speech tracking in a series of five experiments. In the first experiment, the influence of different degrees of background noise is investigated and enhanced speech tracking for minimal noise levels is found. The following four experiments explore different potential influences on this effect, such as attentional allocation, different noise types, and presentation mode.

The step-wise exploration of potential contributors to the effect of enhanced speech tracking for minimal background noise is compelling. The motivation and reasoning for the different studies are clear and logical and therefore easy to follow. The results are discussed in a concise and clear way. While I specifically like the conciseness, one inevitable consequence is that not all results are equally discussed in depth.

Based on the results of the five experiments, the author concludes that the enhancement of speech tracking for minimal background noise is likely due to stochastic resonance. Given broad conceptualizations of stochastic resonance as a noise benefit this is a reasonable conclusion.

This study will likely impact the field as it provides compelling support questioning the relationship between speech tracking and speech processing.

Reviewer #2 (Public review):

This paper examines how structural plasticity in neural circuits, particularly in dopaminergic systems, is regulated by Drosophila neurotrophin-2 (DNT-2) and its receptors, Toll-6 and Kek-6. The authors show that these molecules are critical for modulating circuit structure, dopaminergic neuron survival, synaptogenesis, and connectivity. They demonstrate that the loss of DNT-2 or Toll-6 function leads to the loss of dopaminergic neurons, reduced dendritic arborization, and synaptic impairment, whereas overexpression of DNT-2 increases dendritic complexity and synaptogenesis. Additionally, DNT-2 and Toll-6 influence dopamine-dependent behaviors, including locomotion and long-term memory, suggesting a link between DNT-2 signaling, structural plasticity, and behavior.

A major strength of this study is the impressive cellular resolution achieved. By focusing on specific dopaminergic neurons, such as the PAM and PPL1 clusters, and using a range of molecular markers, the authors were able to clearly visualize intricate details of synapse formation, dendritic complexity, and axonal targeting within defined circuits. Given the critical role of dopaminergic pathways in learning and memory, this approach provides a valuable foundation for exploring the role of DNT-2, Toll-6, and Kek-6 in experience-dependent structural plasticity. While the manuscript hints at a connection to experience-induced plasticity, the study does not establish a direct causal link between neurotrophin signaling and experience-driven changes. To support this idea, it would be necessary to observe experience-induced structural changes and demonstrate that downregulation of DNT-2 signaling prevents these changes. The closest attempt in this study was the artificial activation of DNT-2 neurons using TrpA1, which resulted in overgrowth of axonal arbors and an increase in synaptic sites in both DNT-2 and PAM neurons. However, whether the observed structural changes were dependent on DNT-2 signaling remains unclear.

In conclusion, this study demonstrates that DNT-2 and its receptors play a role in regulating the structure of dopaminergic circuits in the adult fly brain. Whether DNT-2 signaling contributes to experience-dependent structural plasticity within these circuits remains an exciting open question and warrants further investigation.

Comments on revisions:

I appreciate the authors' responses to my previous comments and have no further suggestions.

Reviewer #3 (Public review):

Summary:

In this manuscript, Qin and colleagues study the role of Malat1 in bone biology. This topic is interesting given the role of lncRNAs in multiple physiologic processes. A previous study (PMID 38493144) suggested a role for Malat1 in osteoclast maturation. However, the role of this lncRNA in osteoblast biology was previously not explored. Here, the authors note osteopenia with increased bone resorption in mice lacking Malat1 globally and in osteoblast lineage cells. At the mechanistic level, the authors suggest that Malat1 controls beta-catenin activity. These result advance the field regarding the role of this lncRNA in bone biology.

Strengths:

The manuscript is well-written and data are presented in a clear and easily understandable manner. The bone phenotype of osteoblast-specific Malat1 knockout mice is of high interest. The role of Malat1 in controlling beta-catenin activity and OPG expression is interesting and novel.

Weaknesses:

The lack of a bone phenotype when Malat1 is deleted with LysM-Cre is of interest given the previous report suggesting a role for this lncRNA in osteoclasts, especially in light of satisfactory deletion efficiency in this model. The data in the fracture model in Figure 8 is enhanced with quantitative data. The role of Malat1 and OPG in chondrocytes is unclear since the osteocalcin-Cre mice (which should retain normal Malat1 levels in chondrocytes) have similar bone loss as the global mutants.

Comments on revised version:

All previous comments have been addressed in a satisfactory manner.

Reviewer #2 (Public review):

Summary:

Mutations in SUFU are implicated in SHH medulloblastoma (MB). SUFU modulates Shh signaling in a context-dependent manner, making its role in MB pathology complex and not fully understood. This study reports that elevated FGF5 levels are associated with a specific subtype of SHH MB, particularly in pediatric cases. The authors demonstrate that Sufu deletion in a mouse model leads to abnormal proliferation of granule cell precursors (GCPs) at the secondary fissure (region B), correlating with increased Fgf5 expression. Notably, pharmacological inhibition of FGFR restores normal cerebellar development in Sufu mutant mice.

Strengths:

The identification of increased FGF5 in subsets of MB is novel and a key strength of the paper.

Weaknesses:

The study appears incomplete despite the potential significance of these findings. The current paper does not fully establish the causal relationship between Fgf5 and abnormal cerebellar development, nor does it clarify its connection to SUFU-related MB. Some conclusions seem overstated, and the central question of whether FGFR inhibition can prevent tumor formation remains untested.

Comments on revisions:

In this revised version, many of the concerns and comments raised by this and other reviewers remain unaddressed and require attention in future studies. The manuscript does not demonstrate significant improvement.

Specific Comments:

(1) In the figure provided by the authors, FGF5 appears to be highly expressed beneath the GCPs. Regarding our comment and Reviewer 1's Comment 7, it is essential to identify the cell types secreting FGF5 and clarify whether it functions in a paracrine or autocrine manner. This should be incorporated into the working model illustrated in Figure 5.<br /> (2) Contrary to the authors' claim that their results align completely with Jiwani et al. (2020), there is a discrepancy in the data. Jiwani et al. reported an increase in Gli2 levels in the Sufu mutant, whereas the current study shows the opposite result. This inconsistency needs to be addressed.

Reviewer #2 (Public review):

Summary:

If synaptic input is functionally clustered on dendrites, nonlinear integration could increase the computational power of neural networks. But this requires the right synapses to be located in the right places. This paper aims to address the question of whether such synaptic arrangements could arise by chance (i.e. without special rules for axon guidance or structural plasticity), and could therefore be exploited even in randomly connected networks. This is important, particularly for the dendrites and biological computation communities, where there is a pressing need to integrate decades of work at the single-neuron level with contemporary ideas about network function.

Using an abstract model where ensembles of neurons project randomly to a postsynaptic population, back-of-envelope calculations are presented that predict the probability of finding clustered synapses and spatiotemporal sequences. Using data-constrained parameters, the authors conclude that clustering and sequences are indeed likely to occur by chance (for large enough ensembles), but require strong dendritic nonlinearities and low background noise to be useful.

Strengths:

- The back-of-envelope reasoning presented can provide fast and valuable intuition. The authors have also made the effort to connect the model parameters with measured values. Even an approximate understanding of cluster probability can direct theory and experiments towards promising directions, or away from lost causes.

- I found the general approach to be refreshingly transparent and objective. Assumptions are stated clearly about the model and statistics of different circuits. Along with some positive results, many of the computed cluster probabilities are vanishingly small, and noise is found to be quite detrimental in several cases. This is important to know, and I was happy to see the authors take a balanced look at conditions that help/hinder clustering, rather than just focus on a particular regime that works.

- This paper is also a timely reminder that synaptic clusters and sequences can exist on multiple spatial and temporal scales. The authors present results pertaining to the standard `electrical' regime (~50-100 µm, <50 ms), as well as two modes of chemical signaling (~10 µm, 100-1000 ms). The senior author is indeed an authority on the latter, and the simulations in Figure 5, extending those from Bhalla (2017), are unique in this area. In my view, the role of chemical signaling in neural computation is understudied theoretically, but research will be increasingly important as experimental technologies continue to develop.

Weaknesses:

- The paper is mostly let down by the presentation. In the current form, some patience is needed to grasp the main questions and results, and it is hard to keep track of the many abbreviations and definitions. A paper like this can be impactful, but the writing needs to be crisp, and the logic of the derivation accessible to non-experts. See, for instance, Stepanyants, Hof & Chklovskii (2002) for a relevant example.

It would be good to see a restructure that communicates the main points clearly and concisely, perhaps leaving other observations to an optional appendix. For the interested but time-pressed reader, I recommend starting with the last paragraph of the introduction, working through the main derivation on page 7, and writing out the full expression with key parameters exposed. Next, look at Table 1 and Figure 2J to see where different circuits and mechanisms fit in this scheme. Beyond this, the sequence derivation on page 17 and biophysical simulations in Figures 5 and 6 are also highlights.

- The analysis supporting the claim that strong nonlinearities are needed for cluster/sequence detection is unconvincing. In the analysis, different synapse distributions on a single long dendrite are convolved with a sigmoid function and then the sum is taken to reflect the somatic response. In reality, dendritic nonlinearities influence the soma in a complex and dynamic manner. It may be that the abstract approach the authors use captures some of this, but it needs to be validated with simulations to be trusted (in line with previous work, e.g. Poirazi, Brannon & Mel, (2003)).

- It is unclear whether some of the conclusions would hold in the presence of learning. In the signal-to-noise analysis, all synaptic strengths are assumed equal. But if synapses involved in salient clusters or sequences were potentiated, presumably detection would become easier? Similarly, if presynaptic tuning and/or timing was reorganized through learning, the conditions for synaptic arrangements to be useful could be relaxed. Answering these questions is beyond the scope of the study, but there is a caveat there nonetheless.

Reviewer #2 (Public review):

Summary:

This manuscript builds from the interesting observation that local recruitment of the DHPH domain of the RhoGEF PRG can induce local retraction, protrusion, or neither. The authors convincingly show that these differential responses are tied to the level of expression of the PRG transgene. This response depends on the Rho-binding activity of the recruited PH domain and is associated with and requires (co?)-activation of Cdc42. This begs the question of why this switch in response occurs. The use a computational model to predict that the timing of protein recruitment can dictate the output of the response in cells expressing intermediate levels and found that, "While the majority of cells showed mixed phenotypes irrespectively of the activation pattern, in few cells (3 out of 90) we were able to alternate the phenotype between retraction and protrusion several times at different places of the cell by changing the frequency while keeping the same total integrated intensity (Figure 6F and Supp Movie)."

Comments on the revised manuscript:

The authors have addressed the previous points and they have convincingly demonstrated that an optogenetically recruited PRG-GEF acts, as expected, as a GEF for RhoA. However, if this fragment is strongly over-expressed, it activates Cdc42, instead of RhoA. This appears to be due to sequestration of active RhoA by the overexpressed PRG-GEF.

Reviewer #3 (Public review):

Summary:

The authors characterized the cardiovascular effects of acute and repetitive taVNS as an index of safety and concluded that taVNS treatment does not induce adverse cardiovascular effects such as bradycardia or QT prolongation.

Strengths:

This study contributes important information about the clinical utility of taVNS as a safe immunomodulatory treatment approach for SAH patients.

Comments on revised version:

A number of limitations were identified previously: https://elifesciences.org/reviewed-preprints/100088/reviews#peer-review-2. These major concerns were largely addressed by the authors.

Reviewer #2 (Public review):

In this study, Wada et al. investigate the low potential ferredoxin from Bacillus thermoproteolyticus (BtFd) using a combination of neutron crystallography, x-ray crystallography, DFT and spectroscopy to determine the influence of hydrogen bonding networks on the redox potential of ferredoxin's 4Fe-4S cluster. The use of neutron diffraction allowed the authors to probe the precise location of hydrogens around the 4Fe-4S cluster, which was not possible from prior studies, even with the previously reported high-resolution (0.92 Å) structure of BtFd. This allowed the authors to revise prior models of the proposed H bonding network theorized from earlier x-ray crystallography studies ( for example, showing that there is not in fact a H bond formed between the Thr63-O𝛾1 and the [4Fe-4S]-S4 atoms). With this newly described H-bonding network established, the electronic structure of the 4Fe-4S cluster was then investigated using DFT methodology, revealing a startling role of the deprotonated surface residue Asp64, which bears substantial electronic density in the LUMO which is otherwise localized to the 4Fe-4S cluster. While aspartate is usually deprotonated at physiological pH, the authors provide compelling evidence that this aspartate has a much higher pKa than is usual, and is able to act as a protonation-dependent switch which controls the stability of the reduced state of the 4Fe-4S cluster, and thus the redox potential.

The findings of this study and the conclusions drawn from them are well supported by the data and computational work. Their findings have implications for similar control mechanisms in other, non-ferredoxin 4Fe-4S bearing electron transport proteins which have yet to be explored, providing great value to the metalloprotein community. One change that the authors may consider to enhance the clarity of the manuscript regards the nomenclature used for the varying models discussed (CM, CMNA, CMH and so forth). It would be beneficial to the reader if the nomenclature included the redox state (ox. vs red.) of the model in the model's name.

Comments on revisions:

I'm satisfied with their revisions, it looks good to me.

Reviewer #2 (Public review):

Summary:

Scx is a well-established marker for tenocytes, but the expression in myogenic-lineage cells was unexplored. In this study, the authors performed lineage-trace and scRNA-seq analyses and demonstrated that Scx is expressed in activated SCs. Further, the authors showed that Scx is essential for muscle regeneration using conditional KO mice and identified the target genes of Scx in myogenic cells, which differ from those of tendons.

Strengths:

Sometimes, lineage-trace experiments cause mis-expression and do not reflect the endogenous expression of the target gene. In this study, the authors carefully analyzed the unexpected expression of Scx in myogenic cells using some mouse lines and scRNA-seq data.

Weaknesses:

Scx protein expression has not been verified.

Comments on revisions:

The authors sincerely addressed all concerns, excluding the protein expression of Scx. There is convincing evidence from other experiments that indirectly indicate the protein expression of Scx. In addition, the importance of this study is solid. So, this reviewer doesn't require the authors to make more revisions.

Reviewer #2 (Public review):

Summary:

The manuscript reports results of a combined experimental and simulation study of magnetotactic bacteria in microfluidic channels containing sediment-mimicking obstacles. The obstacles were produced based on micro-computer tomography reconstructions of bacteria-rich sediment samples. The swimming of bacteria through these channels is found experimentally to display the highest throughput for physiological magnetic fields. Computer simulations of active Brownian particles, parameterized based on experimental trajectories are used to quantify the swimming throughput in detail. Similar behavior as in experiments is obtained, but also considerable variability between different channel geometries. Swimming at strong field is impeded by the trapping of bacteria in corners, while at weak fields the direction of motion is almost random. The trapping effect is confirmed in the experiments, as well as the escape of bacteria with reducing field strength.

Strengths:

This is a very careful and detailed study, which draws its main strength from the fruitful combination of construction of novel microfluidic devives, their use in motility experiments, and simulations of active Brownian particles adapted to the experiment.<br /> Based on their results, the authors hypothesize that magnetotactic bacteria may have evolved to produce magnetic properties that are adapted to the geomagnetic field in order to balance movement and orientation in such crowded environments. They provide strong arguments in favor of such a hypothesis.

Weaknesses:

Some of the issues touched upon here have been studied also in other articles. It would be good to extend the list of references accordingly and discuss the relation briefly in the text.

Comments on revisions:

In their rebuttal letter, the authors have responded in detail to all points raised in my previous report. They have revised their manuscript accordingly.

Reviewer #2 (Public review):

Summary:

This work addresses the question of how human macrophages restrict intracellular replication of Salmonella.

Strengths:

Through a series of genetic knockouts and using specific inhibitors, Egan et al. demonstrated that the inflammasome components caspase-1, caspase-4, gasdermin D (GSDMD), and the final lytic death effector ninjurin-1 (NINJ1) are required for control of Salmonella replication in human macrophages. Interestingly, caspase-1 proved crucial in restricting Salmonella early during infection, whereas caspase-4 was essential in the later stages of infection. Furthermore, using a chloroquine resistance assay and state-of-the-art microscopy, the authors found that NAIP receptor and caspase-1 mostly regulate replication of cytosolic bacteria, with smaller, yet significant, impact on the vacuolar bacteria.

The finding that inflammasomes are critical in the restriction of replication of intracellular Salmonella in human macrophages contrasts with the published minimal role of inflammasomes in restriction of replication of intracellular Salmonella in murine macrophages. Some of these differences could be due to differences in the methodologies used in the two studies. However, the findings suggest yet another example of interspecies and intercellular differences in regulation of bacterial infections by the immune system.

Comments on revisions:

The authors may wish to comment that the measurements of released cytokines by ELISA do not discriminate between active and full-length forms of the cytokines.

Reviewer #2 (Public review):

Summary:

The manuscript "Quantifying microbial fitness in high-throughput experiments" provides a comprehensive analysis of the various approaches to quantifying fitness in microbial evolution, focusing on three primary factors: encoding of relative abundance, time scale of measurement, and the choice of reference subpopulation. The authors systematically explore how these choices impact fitness statistics and provide recommendations aimed at standardizing practices in the field. This manuscript aims to highlight the impact of differing fitness definitions and the methodologies utilized for analysis and how that can significantly alter interpretations of mutant fitness, affecting evolutionary predictions and the overall understanding of genetic interactions in the experiments. Although this manuscript focuses on a critical issue in the quantification of fitness in high throughput experiments, it heavily relies on only one experimental dataset (Warringer et al 2003) and one organism i.e, Yeast (Saccharomyces cerevisiae) grown in a defined medium, the environmental influence is not completely captured. While the theoretical framework is strong, more experimental examples with more organisms (i.e., more datasets) in their analysis and comparison would enhance the manuscript, especially its conclusion.

Strengths:

The choices for quantifying fitness in evolution experiments are critical and highly relevant given the increasing prevalence of high-throughput experiments in evolutionary biology. The authors methodically categorize fitness statistics and their implications, providing clarity on a complex subject. This structured approach aids in understanding the nuances of fitness measurement. The manuscript effectively highlights how different choices in fitness measurement can influence fitness rankings and the understanding of epistasis, which is important for modeling evolutionary dynamics.

Weaknesses:

The theoretical framework is robust, but the manuscript could benefit from more empirical examples to illustrate how different fitness quantification methods lead to varied conclusions in experiments. The discussion on the choice of reference subpopulation could be expanded with the influence of the environment or the condition. Different types of reference groups might yield different implications for fitness calculations, and further elaboration would enhance this section. The authors overgeneralize some findings; for instance, the implications of fitness measurement choices could vary significantly across different microbes or experimental conditions. A more detailed discussion would strengthen the conclusion.

Overall, this manuscript is a significant contribution to the field of evolutionary biology, addressing a critical issue in the quantification of fitness but lacks more experimental support to make it a wider claim. By systematically exploring the factors that influence fitness measurements, the authors provide valuable insights that can guide future research - the framework is computationally thorough but needs a more detailed explanation of concepts instead of generalizing. Further work is needed, particularly to incorporate empirical examples and expand certain discussions to include environmental variation and their impact, which would improve clarity and applicability.

Reviewer #2 (Public review):

In this manuscript, Cao et al. evaluated the efficacy and safety of 12 months pyrotinib after trastuzumab-based adjuvant therapy in patients with high-risk, HER2-positive early or locally advanced breast cancer. Notably, the 2-year iDFS rate reached 94.59% (95% CI: 88.97-97.38) in all patients, and 94.90% (95% CI: 86.97-98.06) in patients who completed 1-year treatment of pyrotinib. This is an interesting and uplifting results, given that in ExteNET study, the 2-year iDFS rate was 93.9% (95% CI 92·4-95·2) in the 1-year neratinib group, and the 5-year iDFS survival was 90.2%, and 1-year treatment of neratinib in ExteNET study did not translate into OS benefit after 8-year follow-up. In this case, readers will be eagerly anticipating the long-term follow-up results of the current PERSIST study, as well as the results of the phase III clinical trial (NCT03980054).

I have the following comments:

(1) The introduction of the differences between pyrotinib and neratinib in terms of mechanism, efficacy, resistance, etc. is supposed to be included in the text so that authors could better highlight the clinical significance of the current trial.

(2) Please make sure that a total of 141 patients were enrolled in the study, 38 patients had a treatment duration of less than or equal to 6 months, and a total of 92 and 31 patients completed 1-year and 6-month treatment of extended adjuvant pyrotinib, respectively, which means 7 patients had a treatment duration of fewer than 6 months.

(3) The previous surgery history should be provided, and how many patients received lumpectomy, and mastectomy.

Reviewer #2 (Public review):

This is a nice article that presents interesting findings. One main concern is that I don't think the predictions from the simulation are overlaid on the animal data at any point - I understand the match is qualitative, which is fine, but even that is hard to judge without at least one figure overlaying some of the data. Second is that it's not clear how the lateral coupling strengths of the model were trained/set, so it's hard to judge how important this hemi-split-belt paradigm is. The model's predictions match the data qualitatively, which is good; but does the comparison using the hemi-split-belt paradigm not offer any corrections to the model? The discussion points to modeling plasticity after SCI, which could be good, but does that mean the fit here is so good there's no point using the data to refine?

The manuscript is well-written and interesting. The putative neural circuit mechanisms that the model uncovers are great, if they can be tested in an animal somehow.

Page 2, lines 75-6: Perhaps it belongs in the other paper on the model, but it's surprising that in the section on how the model has been revised to have different regimes of operation as speed increases, there is no reference to a lot of past literature on this idea. Just one example would be Koditschek and Full, 1999 JEB Figure 3, where they talk about exactly this idea, or similarly Holmes et al., 2006 SIAM review Figure 7, but obviously many more have put this forward over the years (Daley and Beiwener, etc). It's neat in this model to have it tied down to a detailed neural model that can be compared with the vast cat literature, but the concept of this has been talked about for at least 25+ years. Maybe a review that discusses it should be cited?

Page 2, line 88: While it makes sense to think of the sides as supraspinal vs afferent driven, respectively, what is the added insight from having them coupled laterally in this hemisection model? What does that buy you beyond complete transection (both sides no supra) compared with intact? I can see how being able to vary cycle frequencies separately of the two limbs is a good "knob" to vary when perturbing the system in order to refine the model. But there isn't a ton of context explaining how the hemi-section with split belt paradigm is important for refining the model, and therefore the science. Is it somehow importantly related to the new "regimes" of operation versus speed idea for the model?

Page 5, line 212: For the predictions from the model, a lot depends on how strong the lateral coupling of the model is, which, in turn, depends on the data the model was trained on. Were the model parameters (especially for lateral coupling of the limbs) trained on data in a context where limbs were pushed out of phase and neuronal connectivity was likely required to bring the limbs back into the same phase relationship? Because if the model had no need for lateral coupling, then it's not so surprising that the hemisected limbs behave like separate limbs, one with surpaspinal intact and one without.

Page 8, line 360: The discussion of the mechanisms (increased influence of afferents, etc) that the model reveals could be causing the changes is exciting, though I'm not sure if there is an animal model where it can be tested in vivo in a moving animal.

Page 9, line 395: There are some interesting conclusions that rely on the hemi-split-belt paradigm here.

Reviewer #2 (Public review):

Summary:

I have reviewed a manuscript submitted by Wang et al., which is entitled "Shc1 cooperates with Frs2 and Shp2 to recruit Grb2 in FGF-induced lens development". In this paper, the authors first examined lens phenotypes in mice with Le-Cre-mediated knockdown (KD) of all four FGFR (FGFR1-4), and found that pERK signals, Jag1, and foxe3 expression are absent or drastically reduced, indicating that FGF signaling is essential for lens induction. Next, the authors examined lens phenotypes of FGFR1/2-KD mice and found that lens fiber differentiation is compromised and that proliferative activity and cell survival are also compromised in lens epithelium. Interestingly, Kras activation rescues defects in lens growth and lens fiber differentiation in FGFR1/2-KD mice, indicating that Ras activation is a key step for lens development. Next, the authors examined the role of Frs2, Shp2, and Grb2 in FGF signaling for lens development. They confirmed that lens fiber differentiation is compromised in FGFR1/3-KD mice combined with Frs2-dysfunctional FGFR2 mutants, which is similar to lens phenotypes of Grb2-KD mice. However, lens defects are milder in mice with Shp2YF/YF and Shp2CS mutant alleles, indicating that the involvement of Shp2 is limited for the Grb2 recruitment for lens fiber differentiation. Lastly, the authors showed new evidence on the possibility that another adapter protein, Shc1, promotes Grb2 recruitment independent of Frs2/Shp2-mediated Grb2 recruitment.

Strengths:

Overall, the manuscript provides valuable data on how FGFR activation leads to Ras activation through the adapter platform of Frs2/Shp2/Grb2, which advances our understanding of complex modification of the FGF signaling pathway. The authors applied a genetic approach using mice, whose methods and results are valid to support the conclusion. The discussion also well summarizes the significance of their findings.

Weaknesses:

The authors eventually found that the new adaptor protein Shc1 is involved in Grb2 recruitments in response to FGF receptor activation. however, the main data for Shc1 are histological sections and statistical evaluation of lens size. So, my major concern is that the authors need to provide more detailed data to support the involvement of Shc1 in Grb2 recruitment of FGF signaling for lens development.

Reviewer #2 (Public review):

Summary:

This manuscript by Kumar and Zhang presents compelling evidence that Edc3 and Scd6 decapping activators present a high degree of redundancy that can only be overcome by a double mutant. In addition, the authors provide strong evidence of these complexes in regulating starvation-induced pathways as evidenced by measurements of mitochondrial membrane potential, metabolomics, and analysis of the flux of Krebs cycle intermediates.

Strengths:

Kumar and Zhang et al provide multiple sources of evidence of the direct mechanism of Edc3 and Scd6 function, by using and comparing different approaches such as mRNA-seq, ribosome occupancies, and translational efficiencies. By extensive analysis, the authors show that this complex can also regulate genes outside the Environmental Stress Response (non-iESR), which are significantly up-regulated in all three mutants. Remarkably, the gene ontology analysis of these non-iESR genes identifies enrichment for mitochondrial proteins that are implicated in the Krebs cycle. Overall, this study adds novel mechanistic insight into how nutrients control gene expression by modulating decapping and translational repression.

Weaknesses:

The authors show very nicely in Figure S1A that growth phenotypes from scd6Δedc3∆ can be rescued by transformation of EDC3 (pLfz614-7) or SCD6 (pLfz615-5). The manuscript might benefit from using these rescue strategies in the analysis performed (e.g. RNA-seq, ribosome occupancies, and translational efficiencies). Also, these rescue assays could provide a good platform to further characterise the protein-protein interactions between Edc3, Scd6, and Dhh1.

Reviewer #2 (Public review):

Summary:

By combining bioinformatical and experimental approaches, the authors address the question of why several vertebrate lineages lack specific genes of the necroptosis pathway or those that regulate the interplay between apoptosis and necroptosis. The lack of such genes was already known from previous publications, but the current manuscript provides a more in-depth analysis and also uses experiments in human cells to address the question of the functionality of the remaining genes and pathways. A particular focus is placed on RIPK3/RIPK1 and their dual roles in inducing NFkB and/or necroptosis.

Strengths:

The well-documented bioinformatical analyses provide a comprehensive data basis of the presence/absence of RIP-kinases, other RHIM proteins, apoptosis signaling proteins (FADD, CASP8, CASP10), and some other genes involved in these pathways. Several of these genes are known to be missing in certain animal lineages, which raises the question of why their canonical binding partners are present in these species. By expressing several such proteins (both wildtype and mutants destroying particular interaction regions) in human cells, the authors succeed in establishing a general role of RIPK3 and RIPK1 in NFkB activation. This function appears to be better conserved and more universal than the necroptotic function of the RHIM proteins. The authors also scrutinize the importance of the kinase function and RHIM integrity for these separate functionalities.

Weaknesses:

A major weakness of the presented study is the experimental restriction to human HEK293 cells. There are several situations where the functionality of proteins from distant organisms (like lampreys or even mussels) in human cells is not necessarily indicative of their function in the native context. In some cases, these problems are addressed by co-expressing potential interaction partners, but not all of these experiments are really informative.

A second weakness is that the manuscript addresses some interesting effects only superficially. By using host cells that are deleted for certain signaling components, a more focussed hypothesis could have been tested.

Thus, while the aim of the study is mostly met, it could have been a bit more ambitious. The limited conclusions drawn by the authors are supported by convincing evidence. I have no doubts that this study will be very useful for future studies addressing the evolution of necroptosis and its regulation by NFkB and apoptosis.

Promover la participación pública y comunitaria efectiva

Invertir en el desarrollo de capacidades entre los grupos marginados

Legislar los derechos de participación pública ex ante

Proteger los datos colectivos y los derechos de la IA

Resultados esperados con metas claras

Puntos de acción

Incluir a grupos marginados, alineándolos con recomendaciones de políticas transformadoras de igualdad de género y diversidad.

Traducir estos objetivos en planes de acción detallados que incorporen responsabilidades, indicadores de monitoreo, presupuestos y directrices, asegurando la evaluación de políticas institucionales para identificar mejoras.

Definir hitos y métricas claras para medir el progreso, considerando la sostenibilidad y relevancia de las soluciones para prevenir y abordar daños relacionados con la IA.

Identificar las principales causas de exclusión de grupos marginados y cómo la IA puede amplificarlas, analizando el contexto social, económico, político, histórico y cultural, así como los marcos legales y normativos relevantes para proteger los derechos en riesgo.

Reviewer #2 (Public review):

Summary:

Casas-Tinto et al., provide new insight into glial plasticity using a crush injury paradigm in the ventral nerve cord (VNC) of adult Drosophila. The authors find that both astrocyte-like glia (ALG) and ensheating glia (EG) divide under homeostatic conditions in the adult VNC and identify ALG as the glial population that specifically ramps up proliferation in response to injury, whereas the number of EGs decreases following the insult. Using lineage-tracing tools, the authors interestingly observe interconversion of glial subtypes, especially of EGs into ALGs, which occurs independent of injury and is dependent on the availability of the transcription factor Prospero in EGs, adding to the plasticity observed in the system. Finally, when tracing the progeny of glia, Casas-Tinto and colleagues detect cells of neuronal identity and provide evidence that such glia-derived neurogenesis is specifically favored following ventral nerve cord injury, which puts forward a remarkable way in which glia can respond to neuronal damage.

Strengths:

This study highlights a new facet of adult nervous system plasticity at the level of the ventral nerve cord, supporting the view that proliferative capacity is maintained in the mature CNS and stimulated upon injury.

The injury paradigm is well chosen, as the organization of the neuromeres allows specific targeting of one segment, compared to the remaining intact and with the potential to later link observed plasticity to behavior such as locomotion.

Numerous experiments have been carried out in 7-day old flies, showing that the observed plasticity is not due to residual developmental remodeling or a still immature VNC.

By elegantly combining different methods, the authors show glial divisions including with mitotic-dependent tracing and find that the number of generated glia is refined by apoptosis later on.

The work identifies prospero in glia as an important coordinator of glial cell fate, from development to the adult context, which draws further attention to the upstream regulatory mechanisms.

Weaknesses:

The authors observe consistent inter-conversion of EG to ALG glial subtypes that is further stimulated upon injury. The authors conclude that these findings have important consequences for CNS regeneration and potentially for memory and learning. However, it remains somewhat unclear how glial transformation could contribute to regeneration and functional recovery.

The signal of the Fucci cell cycle reporter seems more complex to interpret based on the panels provided compared to the other methods employed by the authors to assess cell divisions.

Elav+ cells originating from glia do not express markers for mature neurons at the analysed time-point. If they will eventually differentiate<br /> or what type of structure is formed by them will have to be followed up in future studies.

Context/Discussion

There is some lack of connecting or later comparing the observed forms of glial plasticity in the VNC with respect to plasticity described in the fly brain.<br /> Highlighting some differences in the reactiveness of glia in the VNC compared to the brain could point to relevant differences in repair capacity in different areas of the CNS.

Based on the assays employed, the study points to a significant amount of glial "identity" changes or interconversions under homeostatic conditions. The potential significance of this rather unexpected "baseline" plasticity in adult tissues is not explicitly pointed out and could improve the understanding of the findings.<br /> Some speculations if "interconversion" of glia is driven by the needs in the tissue could enrich the discussion.

Reviewer #2 (Public review):

The present study focuses on calcium pools and fluxes in the unicellular parasite Toxoplasma gondii, and in particular on the role of the endoplasmic reticulum (ER) calcium pump TgSERCA in sequestering and redistributing calcium to other intracellular organelles following influx at the plasma membrane. Calcium sequestration by the ER and its interactions with other intracellular organelles, including the concept of tunneling through the ER, have been extensively characterized in mammalian cells and a number of other higher eukaryotes. However, these pathways are still not well understood in many organisms, including protist pathogens such as Toxoplasma. In addition, T. gondii has unique organelles not found in most other organisms, including the apicoplast and the plant-like vacuolar compartment (PLVAC). Moreover, the fact that T. gondii transitions through life cycle stages within and exterior to the host cells, with very different exposures to calcium, adds significance to the current investigation of the role of the ER in redistributing calcium following exposure to physiological levels of extracellular calcium.

The authors have provided significant new information on the T. gondii SERCA, including its ATP- and calcium-dependence, subcellular localization, and role in taking up calcium from the cytosol when cells are exposed to high extracellular calcium. They also use a conditional knockout of TgSERCA to investigate its role in ER calcium store-filling and the ability of other subcellular organelles to sequester and release calcium. These knockout experiments provide important evidence that ER calcium uptake plays a significant role in maintaining the filling state of other intracellular compartments.

While it is clearly demonstrated, and not surprising, that the addition of 1.8 mM extracellular CaCl2 to intact T. gondii parasites preincubated with EGTA leads to an increase in cytosolic calcium and subsequent enhanced loading of the ER and other intracellular compartments, there is a caveat to the quantitation of these increases in calcium loading. The authors rely on the amplitude of cytosolic free calcium increases in response to thapsigargin, GPN, nigericin, and CCCP, all measured with fura2. This likely overestimates the changes in calcium pool sizes because the buffering of free calcium in the cytosol is nonlinear, and fura2 (with a Kd of 100-200 nM) is a substantial, if not predominant, cytosolic calcium buffer. Indeed, the increases in signal noise at higher cytosolic calcium levels (e.g. peak calcium in Figure 1C) are indicative of fura2 ratio calculations approaching saturation of the indicator dye.

Another caveat, not addressed, is that loading of fura2/AM can result in compartmentalized fura2, which might modify free calcium levels and calcium storage capacity in intracellular organelles.

The finding that the SERCA inhibitor cyclopiazonic acid (CPA) only mobilizes a fraction of the thapsigargin-sensitive calcium stores in T. gondii coincides with previously published work in another apicomplexan parasite, P. falciparum, showing that thapsigargin mobilizes calcium from both CPA-sensitive and CPA-insensitive calcium pools (Borges-Pereira et al., 2020, DOI: 10.1074/jbc.RA120.014906). It would be valuable to determine whether this reflects the off-target effects of thapsigargin or the differential sensitivity of TgSERCA to the two inhibitors.

The authors interpret the residual calcium mobilization response to Zaprinast observed after ATc knockdown of TgSERCA (Figures 4E, 4F) as indicative of a target calcium pool in addition to the ER. While this may well be correct, it appears from the description of this experiment that it was carried out using the same conditions as Figure 4A where TgSERCA activity was only reduced by about 50%.

The data in Figures 4A vs 4G and Figures 4B vs 4H indicate that the size of the response to GPN is similar to that with thapsigargin in both the presence and absence of extracellular calcium. This raises the question of whether GPN is only releasing calcium from acidic compartments or whether it acts on the ER calcium stores, as previously suggested by Atakpa et al. 2019 DOI: 10.1242/jcs.223883. Nonetheless, Figure 1H shows that there is a robust calcium response to GPN after the addition of thapsigargin.

An important advance in the current work is the use of state-of-the-art approaches with targeted genetically encoded calcium indicators (GECIs) to monitor calcium in important subcellular compartments. The authors have previously done this with the apicoplast, but now add the mitochondria to their repertoire. Despite the absence of a canonical mitochondrial calcium uniporter (MCU) in the Toxoplasma genome, the authors demonstrate the ability of T. gondii mitochondrial to accumulate calcium, albeit at high calcium concentrations. Although the calcium concentrations here are higher than needed for mammalian mitochondrial calcium uptake, there too calcium uptake requires calcium levels higher than those typically attained in the bulk cytosolic compartment. And just like in mammalian mitochondria, the current work shows that ER calcium release can elicit mitochondrial calcium loading even when other sources of elevated cytosolic calcium are ineffective, suggesting a role for ER-mitochondrial membrane contact sites. With these new tools in hand, it will be of great value to elucidate the bioenergetics and transport pathways associated with mitochondrial calcium accumulation in T. gondi.

The current studies of calcium pools and their interactions with the ER and dependence on SERCA activity in T. gondi are complemented by super-resolution microscopy and electron microscopy that do indeed demonstrate the presence of close appositions between the ER and other organelles (see also videos). Thus, the work presented provides good evidence for the ER acting as the orchestrating organelle delivering calcium to other subcellular compartments through contact sites in T. gondi, as has become increasingly clear from work in other organisms.

Reviewer #2 (Public review):

The authors trained monkeys to discriminate peripheral visual cues and associate them with planning future saccades of an indicated direction. At the same time, the authors recorded single-unit neural activity in the cerebellar dentate nucleus. They demonstrated that substantial fractions of DN cells exhibited sustained modulation of spike rates spanning task epochs and carrying information about stimulus, response, and trial outcome. Finally, tracer injections demonstrated this region of the DN projects to a large number of targets including several known to interconnect the visual attention network. The data compellingly demonstrate the authors' central claims, and the analyses are well-suited to support the conclusions. Importantly, the study demonstrates that DN cells convey many motor and nonmotor variables related to task execution, event sequencing, visual attention, and arguably decision-making/working memory.

Reviewer #2 (Public review):

This work explores the advancement of awake mouse BOLD-fMRI at 14 Tesla. The study introduces custom-implanted RF coils aimed at improving signal-to-noise ratio (SNR) and assesses their performance in detecting responses to stimuli in awake mice. The coils show significant SNR improvements and are a noteworthy innovation. Detailed descriptions of the coil design, including parts lists and diagrams, enhance the reproducibility of the methods. A thorough 5-week acclimation protocol was used to minimize stress and motion during imaging. Stress was primarily evaluated using eye tracking which, in an fMRI setting, is novel and could help move the field forward with further validation (within the context of fMRI experiments). Overall, the authors successfully demonstrate high-resolution awake mouse fMRI with enhanced SNR; thus achieving their primary aim.

This work is likely to significantly impact the field by demonstrating the feasibility of high-quality awake mouse fMRI, potentially leading to more accurate and artifact-free studies of brain function. The detailed methods shared will facilitate replication and adoption by other researchers, promoting standardized practices. The methods and data provided serve as valuable resources for the neuroscience community.

Reviewer #3 (Public review):

The authors propose a mechanistic model of how the interplay between activity-independent growth and an activity-dependent synaptic strengthening/weakening model influences the dendrite shape, complexity, and distribution of synapses. The authors focus on a model for stellate cells with multiple dendrites emerging from a soma. The activity-independent component is provided by a random pool of presynaptic sites representing potential synapses and releasing a diffusible signal promoting dendritic growth. Then, a spontaneous activity pattern with some correlation structure is imposed at those presynaptic sites. The strength of these synapses follows a learning rule previously proposed by the lab: synapses strengthen when there is correlated firing across multiple sites, and synapses weaken if there is uncorrelated firing with the relative strength of these processes controlled by available levels of BDNF/proBDNF. Once a synapse is weakened below a threshold, the dendrite branch at that site retracts and loses its sensitivity to the growth signal.

This revised version of the manuscripts contains clarifications and additional experiments that better reflect the robustness of the model. I continue to maintain my favorable review. I am glad the research persevered the long delays with changing trainees.

Reviewer #2 (Public review):

This study provides an experimental and computational framework to behavioral biology that helps examine and understand how C. elegans make decisions while foraging in environments with patches of food. The authors show that worms actively reject or accept food patches depending on a number of internal and external factors.

The key novelty and strength of this paper is the explicit demonstration of behavior analysis and quantitative modeling to elucidate the decision-making process. In particular, the description of the exploring vs. exploiting phases, and sensing vs. non-sensing categories of C. elegans foraging behavior based on the clustering of behavioral states defined in a multi-dimensional behavior-metrics space, and the implementation of a generalized linear model (GLM) whose parameters can provide quantitative biological interpretations.

While the concept is interesting, there are many flaws in the experimental, analysis, and models that weaken what one can conclude from the work.

Reviewer #2 (Public review):

Summary:

The manuscript investigates the role of the Mid1 gene in hippocampal (HPC) development and its contribution to Opitz G/BBB syndrome (OS), which is characterized by neurological deficits and structural abnormalities. The authors use a knockout mouse model (Mid1-/y) to elucidate the underlying molecular mechanisms that contribute to learning and memory impairments. They demonstrate that Mid1 gene deletion leads to reduced synaptic plasticity, abnormal neural rhythms, and decreased cognitive functions, providing a mechanistic explanation for the neurological deficits seen in OS patients. This study addresses an important gap in understanding the neural mechanisms underlying Opitz G/BBB syndrome and provides substantial evidence that the Mid1 gene plays a critical role in hippocampal function and cognition.

Strengths:

Understanding the role of Mid1 in HPC development could have broader implications for neurodevelopmental disorders beyond OS, particularly in conditions associated with synaptic dysfunction or memory impairments. The study's focus on the impact of Mid1 on the cAMP signaling pathway, BDNF expression, and synaptic plasticity offers novel mechanisms relevant to both neurodevelopment and neurodegeneration. Moreover, the combination of RNA-seq, electrophysiological measurements, and histological staining provides a multidimensional approach to understanding how Mid1 influences neuronal function and structure.

Weaknesses:

(1) The introduction is insufficient, and the number of references is too low. With only nine references, there isn't enough context to adequately explain the background and previous evidence.

(2) The specificity of behavioral deficits is lacking. The authors indicate learning and memory dysfunction, yet the Y-maze and Morris water maze primarily assess spatial memory. Additional behavioral tests, such as the novel object recognition test for recognition memory or fear conditioning for associative learning, should be included to provide a more comprehensive assessment.

(3) The manuscript mentions decreased synaptic plasticity but lacks thorough investigation; a more detailed analysis of long-term potentiation (LTP) or depression (LTD) would strengthen the claims. Additionally, while spine morphology is analyzed, incorporating electrophysiological measurements of synaptic strength would better correlate structural changes with functional outcomes.

(4) The authors performed H&E staining to count the number of hippocampal pyramidal neurons; however, H&E lacks specificity for identifying pyramidal neurons. Neuronal-specific IHC staining would be more appropriate for this quantification. Additionally, the manuscript does not mention the counting method used, which should be clarified.

(5) Information on the knockout mice used in the study is missing from the Methods section. Additionally, the sex of the mice should be specified, as exploring potential sex-specific differences in the impact of Mid1 deletion could significantly enhance the study's findings.

Reviewer #2 (Public review):

Summary:

In this exciting study, Cesar and co-authors perform a meta-analysis on the influence of arthropod symbionts on the fitness of their hosts when they are exposed or not to natural enemies. These so-called defensive symbionts are increasingly recognized as key elements in arthropod survival against natural enemies, with effects that ripple through entire terrestrial ecosystems. The topic is timely, the approach is sound, and the manuscript is well-written. I believe this manuscript will attract the attention of entomologists and of microbiologists interested in symbiosis. This study builds on a previous meta-analysis that I was involved in, which was based on phloem-feeding insects. This novel data set is much larger and includes flies (including the model system Drosophila) and mosquitoes (a group of high medical interest). While the previous meta-analysis considered only parasitoids as natural enemies, this study also includes fungi, bacteria, and viruses.

Strengths:

The authors compile a very large dataset and provide a broad quantitative overview of the effects of defensive symbionts in insects. By measuring symbiont effects in the presence and absence of natural enemies, the authors are able to infer whether a trade-off between defense and the costs of mutualism in the absence of enemy pressure exists. Defensive symbioses are an important research topic that had its initial "momentum" a decade ago, so the timing for such a systematic review is very appropriate.

Weaknesses:

I think the manuscript could be improved by clarifying several sections, particularly the introduction and methods. The introduction section is too specific and heavily reliant on particular examples. In my view, the theoretical background of the study could be made clearer, and the knowledge gap identified more explicitly. A focus on how widespread defensive symbioses are, along with a brief, up-to-date review of the groups possessing such symbionts, would help. This lack of focus is also observed in the methods section, where more details are needed in many instances to better understand how data was collected and analyzed. Regarding the analyses, the multi-level analysis contains many moderators, but it's unclear why these moderators were included. While this may seem a minor issue, it highlights a disconnection between the analyses, the conceptual background, and the hypotheses tested. Another important weakness is that the analyses are too general, and much-hidden information is not immediately apparent. For instance, readers cannot easily identify which species of symbionts are studied (and the effects they have), or which natural enemies are involved. Although this information is found in the supplementary material, including it in the main body would significantly improve the manuscript.

Reviewer #2 (Public review):

Summary:

The Flower protein is expressed in various cell types, including neurons. Previous studies in flies have proposed that Flower plays a role in neuronal endocytosis by functioning as a Ca2+ channel. However, its precise physiological roles and molecular mechanisms in neurons remain largely unclear. This study employs C. elegans as a model to explore the function and mechanism of FLWR-1, the C. elegans homolog of Flower. This study offers intriguing observations that could potentially challenge or expand our current understanding of the Flower protein. Nevertheless, further clarification or additional experiments are required to substantiate the study's conclusions.

Strengths:

A range of approaches was employed, including the use of a flwr-1 knockout strain, assessment of cholinergic synaptic activity via analyzing aldicarb (a cholinesterase inhibitor) sensitivity, imaging Ca2+ dynamics with GCaMP3, analyzing pHluorin fluorescence, examination of presynaptic ultrastructure by EM, and recording postsynaptic currents at the neuromuscular junction. The findings include notable observations on the effects of flwr-1 knockout, such as increased Ca2+ levels in motor neurons, changes in endosome numbers in motor neurons, altered aldicarb sensitivity, and potential involvement of a Ca2+-ATPase and PIP2 binding in FLWR-1's function.

Weaknesses:

(1) The observation that flwr-1 knockout increases Ca2+ levels in motor neurons is notable, especially as it contrasts with prior findings in flies. The authors propose that elevated Ca2+ levels in flwr-1 knockout motor neurons may stem from "deregulation of MCA-3" (a Ca2+ ATPase in the plasma membrane) due to FLWR-1 loss. However, this conclusion relies on limited and somewhat inconclusive data (Figure 7). Additional experiments could clarify FLWR-1's role in MCA-3 regulation. For instance, it would be informative to investigate whether mutations in other genes that cause elevated cytosolic Ca2+ produce similar effects, whether MCA-3 physically interacts with FLWR-1, and whether MCA-3 expression is reduced in the flwr-1 knockout.

(2) In silico analysis identified residues R27 and K31 as potential PIP2 binding sites in FLWR-1. The authors observed that FLWR-1(R27A/K31A) was less effective than wild-type FLWR-1 in rescuing the aldicarb sensitivity phenotype of the flwr-1 knockout, suggesting that FLWR-1 function may depend on PIP2 binding at these two residues. Given that mutations in various residues can impair protein function non-specifically, additional studies may be needed to confirm the significance of these residues for PIP2 binding and FLWR-1 function. In addition, the authors might consider explicitly discussing how this finding aligns or contrasts with the results of a previous study in flies, where alanine substitutions at K29 and R33 impaired a Flower-related function (Li et al., eLife 2020).

(3) A primary conclusion from the EM data was that FLWR-1 participates in the breakdown, rather than the formation, of bulk endosomes (lines 20-22). However, the reasoning behind this conclusion is somewhat unclear. Adding more explicit explanations in the Results section would help clarify and strengthen this interpretation.

(4) The aldicarb assay results in Figure 3 are intriguing, indicating that reduced GABAergic neuron activity alone accounts for the flwr-1 mutant's hyposensitivity to aldicarb. Given that cholinergic motor neurons also showed increased activity in the flwr-1 mutant, one might expect the flwr-1 mutant to display hypersensitivity to aldicarb in the unc-47 knockout background. However, this was not observed. The authors might consider validating their conclusion with an alternative approach or, at the minimum, providing a plausible explanation for the unexpected result. Since aldicarb-induced paralysis can be influenced by factors beyond acetylcholine release from cholinergic motor neurons, interpreting aldicarb assay results with caution may be advisable. This is especially relevant here, as FLWR-1 function in muscle cells also impacts aldicarb sensitivity (Figure S3B). Previous electrophysiological studies have suggested that aldicarb sensitivity assays may sometimes yield misleading conclusions regarding protein roles in acetylcholine release.

(5) Previous studies have suggested that the Flower protein functions as a Ca²⁺ channel, with a conserved glutamate residue at the putative selectivity filter being essential for this role. However, mutating this conserved residue (E74Q) in C. elegans FLWR-1 altered aldicarb sensitivity in a direction opposite to what would be expected for a Ca²⁺ channel function. Moreover, the authors observed that E74 of FLWR-1 is not located near a potential conduction pathway in the FLWR-1 tetramer, as predicted by Alphafold3. These findings raise the possibility that Flower may not function as a Ca2+ channel. While this is a potentially significant discovery, further experiments are needed to confirm and expand upon these results.

(6) Phrases like "increased excitability" and "increased Ca2+ influx" are used throughout the manuscript. However, there is no direct evidence that motor neurons exhibit increased excitability or Ca2+ influx. The authors appear to interpret the elevated Ca2+ signal in motor neurons as indicative of both increased excitability and Ca2+ influx. However, this elevated Ca2+ signal in the flwr-1 mutant could occur independently of changes in excitability or Ca2+ influx, such as in cases of reduced MCA-3 activity. The authors may wish to consider alternative terminology that more accurately reflects their findings.

Reviewer #2 (Public review):

Summary:

This study introduces a set of genetically encoded tools for the selective and reversible ablation of excitatory and inhibitory synapses. Previously, the authors developed GFE3, a tool that efficiently ablates inhibitory synapses by targeting an E3 ligase to the inhibitory scaffolding protein Gephyrin via GPHN.FingR, a recombinant, antibody-like protein (Gross et al., 2016). Building on this work, they now present three new ablation tools: PFE3, which targets excitatory synapses, and two new versions of GFE3-paGFE3 and chGFE3-that are photoactivatable and chemically inducible, respectively. These tools enable selective and efficient synapse ablation in specific cell types, providing valuable methods for disrupting neural circuits. This approach holds broad potential for investigating the roles of specific synaptic input onto genetically determined cells.

Strengths:

The primary strength of this study lies in the rational design and robust validation of each tool's effectiveness, building on previous work by the authors' group (Gross et al., 2016). Each tool serves distinct research needs: PFE3 enables efficient degradation of PSD-95 at excitatory synapses, while paGFE3 and chGFE3 allow for targeted degradation of Gephyrin, offering spatiotemporal control over inhibitory synapses via light or chemical activation. These tools are efficiently validated through robust experiments demonstrating reductions in synaptic markers (PSD-95 and Gephyrin) and confirming reversibility, which is crucial for transient ablation. By providing tools with both optogenetic and chemical control options, this study broadens the applicability of synapse manipulation across varied experimental conditions, enhancing the utility of E3 ligase-based approaches for synapse ablation.

Weaknesses:

While this study provides valuable tools and addresses many critical points for validation, examining potential issues with specificity and background effects in further detail could strengthen the paper. For instance, PFE3 results in reductions in both PSD-95 and GluA1. In previous work, GFE3 selectively reduced Gephyrin without affecting major Gephyrin interactors or other PSD proteins. Clarifying whether PFE3 affects additional PSD proteins beyond GluA1 would be important for accurately interpreting results in experiments using PFE3. Additionally, further insight into PFE3's impact on inhibitory synapses would be valuable.

For paGFE3 and chGFE3, the E3 ligase (RING domain of Mdm2) is overexpressed throughout cells as a separate construct. Although the authors show that Gephyrin is not significantly reduced without light or chemical activation, it remains possible that other proteins could be ubiquitinated due to the overexpressed E3 domain. Addressing these points would clarify the strengths and limitations of tools, providing users with valuable information.

Reviewer #2 (Public review):

Summary:

This paper describes a number of aspects of transcranial ultrasound stimulation (TUS) including a generic review of what TUS might be used for; a meta-analysis of human studies to identify ultrasound parameters that affect directionality; a comparison between one postulated mechanistic model and results in humans; and a description of a database for collecting information on studies.

Strengths:

The main strength was a meta-analysis of human studies to identify which ultrasonic parameters might result in enhancement or suppression of modulation effects. The meta-analysis suggests that none of the US parameters correlate significantly with effects. This is a useful result for researchers in the field in trying to determine how the parameter space should be further investigated to identify whether it is possible to indeed enhance or suppress brain activity with ultrasound.

The database is a good idea in principle but would be best done in collaboration with ITRUSST, an international consortium, and perhaps should be its own paper.

Weaknesses:

The paper tries to cover too many topics and some of the technical descriptions are a bit loose. The review section does not add to the current literature. The comparison with a mechanistic model is limited to comparing data with a single model at a time when there is no general agreement in the field as to how ultrasound might produce a neuromodulation effect. The comparison is therefore of limited value.

Reviewer #2 (Public review):

Park et al. set out to test two competing hypotheses about the role of the medial prefrontal cortex (PFC) in cognitive control, the ability to use task-relevant cues and ignore task-irrelevant cues to guide behavior. The "central computation" hypothesis assumes that cognitive control relies on computations performed by the PFC, which then interacts with other brain regions to accomplish the task. Alternatively, the "local computation" hypothesis suggests that computations necessary for cognitive control are carried out by other brain regions that have been shown to be essential for cognitive control tasks, such as the dorsal hippocampus and the thalamus. If the central computation hypothesis is correct, PFC lesions should disrupt cognitive control. Alternatively, if the local computation hypothesis is correct, cognitive control would be spared after PFC lesions. The task used to assess cognitive control is the active place avoidance task in which rats must avoid a section of a rotating arena using the stationary room cues and ignoring the local olfactory cues on the rotating platform. Performance on this task has previously been shown to be disrupted by hippocampal lesions and hippocampal ensembles dynamically represent the room and arena depending on the animal's proximity to the shock zone. They found no group (lesion vs. sham) differences in the three behavioral parameters tested: distance traveled, latency to enter the shock zone, and number of shock zone entries for both the standard task and the "conflict" task in which the shock zone was rotated by 180 degrees. The only significant difference was the savings index; the lesion group entered the new shock zone more often than the sham group during the first 5 minutes of the second conflict session. This deficit was interpreted as a cognitive flexibility deficit rather than a cognitive control failure. Next, the authors compared cytochrome oxidase activity between sham and lesion groups in 14 brain regions and found that only the amygdala showed significant elevation in the lesion vs. sham group. Pairwise correlation analysis revealed a striking difference between groups, with many correlations between regions lost in the lesion group (between reuniens and hippocampus, reuniens and amygdala and a correlation between dorsal CA1 and central amygdala that appeared in the lesion group and were absent in the sham group. Finally, the authors assessed dorsal hippocampal representations of the spatial frame (arena vs. room) and found no differences between lesion and sham groups. The only difference in hippocampal activity was reduced overdispersion in the lesion group compared to the sham group on the pretraining session only and this difference disappeared after the task began. Collectively, the authors interpret their findings as supporting the local computation hypothesis; computations necessary for cognitive control occur in brain regions other than the PFC.

Strengths:

(1) The data were collected in a rigorous way with experimental blinding and appropriate statistical analyses.

(2) Multiple approaches were used to assess differences between lesion and sham groups, including behavior, metabolic activity in multiple brain regions, and hippocampal single-unit recording.

Weaknesses:

(1) Only male rats were used with no justification provided for excluding females from the sample.

(2) The conceptual framework used to interpret the findings was to present two competing hypotheses with mutually exclusive predictions about the impact of PFC lesions on cognitive control. The authors then use mainly null findings as evidence in support of the local computation hypothesis. They acknowledge that some people may question the notion that the active place avoidance task indeed requires cognitive control, but then call the argument "circular" because PFC has to be involved in cognitive control. This assertion does not address the possibility that the active place avoidance task simply does not require cognitive control.

(3) The authors did not link the CO activity with the behavioral parameters even though the CO imaging was done on a subset of the animals that ran the behavioral task nor did they make any attempt to interpret these findings in light of the two competing hypotheses posed in the introduction. Moreover, the discussion lacks any mechanistic interpretations of the findings. For example, there are no attempts to explain why amygdala activity and its correlation with dCA1 activity might be higher in the PFC lesioned group.

(4) Publishing null results is important to avoid wasting animals, time, and money. This study's results will have a significant impact on how the field views the role of the PFC in cognitive control. Whether or not some people reject the notion that the active place avoidance task measures cognitive control, the findings are solid and can serve as a starting point for generating hypotheses about how brain networks change when deprived of PFC input.

Reviewer #2 (Public review):

Summary:

In this article, Ruyle and colleagues assessed the contribution of central and peripheral mu opioid receptors in mediating fentanyl-induced respiratory depression using both naloxone and naloxone methiodide, which does not cross the blood-brain barrier. Both compounds prevented and reversed fentanyl-induced respiratory depression to a comparable degree. The advantage of peripheral treatments is that they circumvent the withdrawal-like effects of naloxone. Moreover, neurons located in the nucleus of the solitary tract are no longer activated by fentanyl when nalaxone methiodide is administered, suggesting that these responses are mediated by peripheral mu opioid receptors. The results delineate a role for peripheral mu opioid receptors in fentanyl-derived respiratory depression and identify a potentially advantageous approach to treating overdoses without inflicting withdrawal on the patients.

Strengths:

The strengths of the article include the intravenous delivery of all compounds, which increase the translational value of the article. The authors address both the prevention and reversal of fentanyl-derived respiratory depression. The experimental design and data interpretation are rigorous and appropriate controls were used in the study. Multiple doses were screened in the study and the approaches were multipronged. The authors demonstrated the activation of NTS cells using multiple techniques and the study links peripheral activation of mu opioid receptors to central activation of NTS cells. Both males and females were used in the experiments. The authors demonstrate the peripheral restriction of naloxone methiodide.

Weaknesses:

Nalaxone is already broadly used to prevent overdoses from opioids so in some respects, the effects reported here are somewhat incremental.

Reviewer #2 (Public review):

Summary:

Reeves et al explore a model of bone healing in the context of aging. They show that intermittent fasting can improve bone healing, even in aged animals. Their study combines a 'bone bandage' which delivers a canonical Wnt signal with intermittent fasting and shows impacts on the CD90 progenitor cell population and the healing of a critical-sized defect in the calvarium. They also explore potential regulators of this process and identify mitochondrial dysfunction in the age-related decline of stem cells. In this context, by modulating NAD+ pathways or the gut microbiota, they can also enhance healing, hinting at an effect mediated by complex impacts on multiple pathways associated with cellular metabolism.

Strengths:

The study shows a remarkable finding: that age-related decreases in bone healing can be restored by intermittent fasting. There is ample evidence that intermittent fasting can delay aging, but here the authors provide evidence that in an already-aged animal, intermittent fasting can restore healing to levels seen in younger animals. This is an important finding as it may hint at the potential benefits of intermittent fasting in tissue repair.

Weaknesses:

The authors explore potential mechanisms by which the intermittent fasting protocol might impact bone healing. However, they do not identify a magic bullet here that controls this effect. Indeed, the fact that their results with intermittent fasting can be replicated by changing the gut microbiota or modulating fundamental pathways associated with NAD, suggests that there is no single mechanism that drives this effect, but rather an overall complex impact on metabolic processes, which may be very difficult to untangle.

Reviewer #2 (Public review):

Summary:

This paper addresses mixed findings regarding levels of uncertainty-seeking/avoidance in past reinforcement learning studies. Using computational modelling and a novel variant of a bandit task performed across two sessions, the authors investigate the extent to which uncertainty-driven behaviour can be distinguished from heuristic-like behaviours (e.g., repetition, win-stay/lose-switch). They demonstrate that heuristics account for a significant and stable portion of the variance in choice behaviour, which might otherwise be misattributed to uncertainty-driven parameters. Additionally, they find that relative uncertainty explains additional variance and provides some evidence of stability across sessions.

Strengths:

The task is well-designed to tease apart multiple different factors contributing to choice during a bandit task, including separating those tied to uncertainty per se versus other policies. They validate a Bayesian model to account for learning and choice behaviour, as well as subjective estimates of learned value and confidence in these values. The work employs comprehensive model comparison to characterise behaviour in this task, and points to important risks within research on uncertainty preferences using bandit-like tasks when failing to fully account for heuristic-like drivers of such behaviour.

Weaknesses:

Part of this work seeks to relate individual differences in various choice parameters across sessions and to relate those to self-report scales. The estimates of cross-session reliability are valuable, particularly when comparing across the different parameters (e.g., heuristic ones being most robust), but the uncertainty-related parameters are interpreted too liberally (i.e., as being stable across sessions when both were weak and one was not significant). Moreover, the correlations with external scales are very hard to interpret given the number of comparisons that were run without correction. The findings overall will have value to people interested in modelling uncertainty preferences in learning tasks -- some of whom have considered heuristic factors less than others -- but perhaps be of more moderate impact beyond this group.

Reviewer #2 (Public review):

Using a variety of experimental manipulations, the authors show that the membrane estrogen receptor G protein-coupled estrogen receptor (GPER/GPR30) expressed in CCK+ excitatory spinal interneurons plays a major role in the pain symptoms observed in the chronic constriction injury (CCI) model of neuropathic pain. Intrathecal application of selective GPR30 agonist G 1induced mechanical allodynia and thermal hyperalgesia in male and female mice. Downregulation of GPR30 in CCK+ interneurons prevented the development of mechanical and thermal hypersensitivity during CCI. They also show the up modulation of AMPA receptor expression by GPR30.

Generally, the conclusions are supported by the experimental results. I also would like to see significant improvements in the writing and the description of results.

Methodological details for some of the techniques are rather sparse. For example, when examining the co-localization of various markers, the authors do not indicate the number of animals/sections examined. Similarly, when examining the effect of shGper1, it is unclear how many cells/sections/animals were counted and analyzed.

In other sections, there is no description of the concentration of drugs used (for example, Figure 4H). In Figures 4C-E, there is no indication of the duration of the recordings, the ionic conditions, the effect of glutamate receptor blockers, etc

Some results appear anecdotal in the way they are described. For example, in Figure 5, it is unclear how many times this experiment was repeated.

Reviewer #2 (Public review):

Summary:

This study employs a combination of state-of-the-art experimental approaches in mice to identify components of the brainstem circuits involved in the cough reflex in a freely behaving mouse model. The cough reflex is an important respiratory airway defense mechanism, and there has been longstanding interest in defining the neural circuits involved in the mammalian brainstem. Consistent with other recent studies, the present results provide multiple lines of evidence indicating that mice are a suitable model for studying neural mechanisms generating cough behavior. The main novel finding of this study is the authors' results indicating that the caudal spinal trigeminal nucleus (SP5C) nucleus plays a role in generating cough-like behaviors in response to inhaled tussigen. The supporting data presented for this role includes the authors' findings that: (1) neural activity in the SP5C is strongly correlated with tussigen-evoked cough-like behaviors, (2) impairing synaptic outputs or chemogenetic inhibition of SP5C neurons effectively abolished these cough-like reflexes, (3) optogenetically activating a specific subpopulation of excitatory neurons in the SP5C triggers cough-like behaviors, (4) SP5C neurons project monosynaptically to ventral medullary regions containing respiratory circuits that exhibit cough-related neural activity, and (5) specific activation of the SP5C-ventral respiratory circuitry induces robust cough-like behavior without tussive stimuli. This study will be valuable to respiratory neurobiologists studying mechanosensory control of breathing in mammals.

Strengths:

(1) The authors developed an experimental paradigm in mice that combines whole-body plethysmography (WBP), audio, and video tracking to assess breathing and putative cough-like behaviors in conscious animals.

(2) The mouse model enables optogenetic, chemogenetic, virus-based circuit tracing and manipulation, and in vivo fiber photometry to analyze neural activity and define circuity in the medulla-producing cough-like behavior.

(3) Multiple lines of evidence from these experimental approaches support the conclusion that the SP5C nucleus plays a role in the respiratory reflex behaviors studied in mice, but there is uncertainty that these behaviors are definitively cough.

Weaknesses:

(1) This paper lacks essential quantitative details about the number of animals studied explicitly for many of the experimental paradigms presented and for statistical analyses as well as to verify replication of the neuroanatomical data presented.

(2) The authors' evidence is incomplete that the reflex behavior produced in their mouse model is definitively cough, limiting functional interpretation of the putative circuit identified and requiring more thorough experimental interrogation of the behavior studied.

(3) The medullary circuit described conveys afferent sensorimotor signals to downstream respiratory circuits to coordinate cough-like motor behavior, but how the circuits that typically mediate the cough reflex, which involve airway-related vagal sensory neurons, operate in conjunction or parallel with the SP5C circuit described has not been determined, which is a significant gap in understanding how the present results fit into the neural control of the cough reflex.

Reviewer #2 (Public review):

Summary:

The basal ganglia is often understood within a reinforcement learning (RL) framework, where dopamine neurons convey a reward prediction error that modulates cortico-striatal connections onto spiny projection neurons (SPNS) in the striatum. However, current models of plasticity rules are inconsistent with learning in a reinforcement learning framework.

This paper proposes a new model that describes how distinct learning rules in direct and indirect pathway striatal neurons allow them to implement reinforcement learning models. It proposes that two distinct components of striatal activity affect action selection and learning. They show that the proposed implementation allows learning in simple tasks and is consistent with experimental data from calcium imaging data in direct and indirect SPNs in freely moving mice.

Strengths:

Despite the success of reward prediction errors at characterizing the responses of dopamine neurons as the temporal difference error within an RL framework, the implementation of RL algorithms in the rest of the basal ganglia has been unclear. A key missing aspect has been the lack of a RL implementation that is consistent with the distinction of direct- and indirect SPNs. This paper proposes a new model that is able to learn successfully in simple RL tasks and explains recent experimental results.

The author shows that their proposed model, unlike previous implementations, this model can perform well in RL tasks. The new model allows them to make experimental predictions. They test some of these predictions and show that the dynamics of dSPNs and iSPNs correspond to model predictions.

More generally, this new model can be used to understand striatal dynamics across direct and indirect SPNs in future experiments.

Weaknesses:

The authors could characterize better the reliability of their experimental predictions and the description of the parameters of some of the simulations

The authors propose some ideas about how the specificity of the striatal efferent inputs but should highlight better that this is a key feature of the model whose anatomical implementation has yet to be resolved.

Reviewer #2 (Public review):

Summary:

The authors analyze parameters related to anisotropy and gyrification in the developing human brain and describe an increase in tissue fraction (TF) across development. They correlate TF and sulcal depth in the CP and SP across local neighborhoods, describing a negative correlation. Also, they perform age-mismatched correlation of tissue fraction at early stages with sulcal depth at later ones and show correlation inside sulci, which they interpret as indicating the presence of minor structural changes in the brain that precede the development of sulci.

Strengths:

The study compiles a large cohort of cases through different developmental ages and performs sophisticated data analysis. Overall, the work is interesting.

Weaknesses:

I have some questions. What is the potential meaning of TF? It seems to be an estimator of anisotropy highly related to fractional anisotropy (FA), but it behaves in a complementary manner, increasing along gestation, in sharp contrast with the decrease observed in FA in this study (suppl. fig 3) and by others. Please clarify how it is calculated, what is the potential biological meaning of TF and how it differs from FA.

The correlations between TF and sulcal depth do not seem to provide much novelty, since as mentioned by the authors, previous evidence has pointed in that direction. The other concept in the paper relates to detecting structural changes in prospective sulcal areas in the cortex, which the authors analyze through the age-mismatched correlation of TF and subsequent sulcation. However, the results do not show a robust correlation as detailed below and do not seem particularly useful, as they require the inclusion of post-hoc information in the model, limiting the strength of the relationship and the predictive value. My main point of criticism is that if TF is a good marker of the structural modifications that will favor the development of sulci later in development, TF should show a map predictive of those sulci (e.g. at GW 25), that is however not the case. It is not necessary to correlate with future sulcal depth, as we know exactly where the primary sulci will develop. Conversely, it seems that TF decreases along the gyrification process, and it might just be a measure of the structural changes accompanying it.

In Figure 2 it illustrates the increase in TF across GA, but no R values or significance values are provided. Please add them to evaluate the robustness of the correlation.

In previous work of the authors, the subplate is not clearly distinguished from the subcortical white matter after 31 GW, as it starts to disintegrate (Kostovic et al., 2002; Calixto et al., 2024). However, in this manuscript, the SP is differentiated at those later ages. The methods section describes a 2 mm thick compartment below the cortical plate. However, if that is the case, it seems quite arbitrary (to coincide with the resolution of the diffusion imaging) and risks analyzing a compartment that is no longer present. Please explain the criteria followed for such distinction and more importantly, how such distinction is reliable considering the low detectability described in previous studies. In this regard, the discussion described that a rapid increase in TF was only seen in the SP after 30 GW, but maybe this increase would reflect the dissipation of the SP and the transformation of that space in subcortical white matter, with a much more expected anisotropy. The authors should review this.

The analysis describes a negative correlation between tissue fraction and sulcal depth when gyrification proceeds and the authors find that an age-mismatched correlation between tissue fraction in young embryos and sulcal depth in older embryos also shows a negative correlation in future sites of sulcation. However, for the correlation to exist, the tissue fraction in young individuals should already show low values in the prospective sulci, but no clear changes can be seen at GW 25 or 27 in lissencephalic areas that will bear sulci later on, as is the case of the central sulcus at GW 25 or the STS at GW 27, the latter showing very high tissue fraction (instead of the expected low).

Another question refers to Figures 3b and c. The graphs represent specific neighborhoods in the central sulcus at 27 and 35 GW. It can be argued that those neighborhoods might not be representative of the brain or of the whole sulcus. Please show the graph with all neighborhoods, which will provide more definitive evidence of the existence of the correlation. In this regard, the average graphs represented in Figure 3F seem to show a clear correlation at 27 GW in the subplate, but the correlation seems to fade at later stages (in both SP and CP), with both sulci and gyri exhibiting a negative correlation while other sulcal areas do not exhibit correlation. I think all points should be included in the correlation to better support the hypothesis.

Figure 4 shows the age-mismatched correlations, but they do not seem convincing particularly when they should be more useful, at 25 GW. Indeed, as seen in both Figures A and C, the central sulcus shows a negative correlation only in a few spots on one hemisphere, while (in C) most of the prospective sulcus shows a positive correlation, contrary to the hypothesis.

Lastly, the authors performed an age-mismatched correlation between TF at different ages and the sulcal depth at 35W, when it is maximal. This maximal depth might be "pushing" the correlation to significant territory. The authors should provide correlation also with the sulcal depth at other GAs, such as P29, P31, or P33, and analyze how the correlations hold.

Disease: Von-willebrand Disorder Type 2M

Patient1: 13 YO male

Patient2: 16 YO female

Note they are siblings

Variant: VWF NM_000552.5: c.5192C>T p.(Ser1731Leu), heterozygous variant, in A3 domain in exon 30

Phenotype patient 1: several bleedings after tosillectomy, recurrent epistaxis, decreased VWF:CB ratio, decreased VWF:CB/VWF:Ag ratio

Phenotype patient 2: menorrhagia, intermittent gum bleeding, easy bruising, decreased VWF:CB ratio, decreased VWF:CB/VWF:Ag ratio

For both patients: VWF:CB ratio corresponds to collagen type 1, multimer analysis was normal, VWF:Ag, VWF:Ac, and factor VIII activity was normal.

Family: Not listed

Present in dbSNP (rs764077750)

Present in gnomAD, rare MAF (ALL: 0.0012%)

Predictions:

SIFT- predicted deleterious (Score 0.01, median 3.34)

MutationTaster- Predicted disease causing (probability score 1)

PolyPhen2- Predicted probably damaging (Score 0.983)

CADD score- 26.5

Authors mention similar AA substitutions at the p.1731 position which leads to reduced binding of VWF to collagen in other patients.

Authors also cite a functional human cell experiment in COS7 cell line which resulted in functional VWF defect.

Authors conclude this novel variant as likely pathogenic.

Reviewer #3 (Public review):

Summary:

This manuscript introduces POST-IT (Pup-On-target for Small molecule Target Identification Technology), a novel non-diffusive proximity tagging system for identifying target proteins in live cells and organisms. This technology preserves cellular context essential for capturing specific drug-protein interactions, including transient complexes and membrane-associated proteins. Using an engineered fusion of proteasomal accessory factor A (PafA) and HaloTag, POST-IT specifically labels proximal proteins upon binding to a small molecule, with extensive optimization to enhance specificity and efficiency.

Strengths:

The study successfully identifies known targets and discovers new binders, such as SEPHS2 for dasatinib and VPS37C for hydroxychloroquine, advancing our understanding of their mechanisms. Additionally, its application in live zebrafish embryos demonstrates POST-IT's potential for widespread use in biological research and drug development.

Comments on revisions:

The authors have addressed most of the issues I raised in my review. I have no further comments.

Reviewer #2 (Public review):

Summary:

This study aimed to test experimentally a theoretical framework that aims to explain the perception of tinnitus, i.e., the perception of a phantom sound in the absence of external stimuli, through differences in auditory predictive coding patterns. To this aim, the researchers compared the neural activity preceding and following the perception of a sound using MEG in two different studies. The sounds could be highly predictable or random, depending on the experimental condition. They revealed that individuals with tinnitus and controls had different anticipatory predictions. This finding is a major step in characterizing the top-down mechanisms underlying sound perception in individuals with tinnitus.

Strengths:

This article uses an elegant, well-constructed paradigm to assess the neural dynamics underlying auditory prediction. The findings presented in the first experiment were partially replicated in the second experiment, which included 80 participants. This large number of participants for an MEG study ensures very good statistical power and a strong level of evidence. The authors used advanced analysis techniques - Multivariate Pattern Analysis (MVPA) and classifier weights projection - to determine the neural patterns underlying the anticipation and perception of a sound for individuals with or without tinnitus. The authors evidenced different auditory prediction patterns associated with tinnitus. Overall, the conclusions of this paper are well supported, and the limitations of the study are clearly addressed and discussed.

Reviewer #2 (Public review):

Fruchard et al. investigate the role of the queuosine (Q) modification of the tRNA (Q-tRNA) in the human pathogen Vibrio cholerae. First, the authors state that the absence of Q-modified tRNAs (tgt mutant) increases the translation of TAT codons and proteins with a high TAT codon bias. Second, the absence of Q increases rsxA translation, because rsxA gene has a high TAT codon bias. Third, increased RsxA in the absence of Q inhibits SoxR response, reducing resistance towards the antibiotic tobramycin (TOB). Authors also predict in silico which genes harbor a higher TAT bias and found that among them are some involved in DNA repair, experimentally observing that a tgt mutant is more resistant to UV than the wt strain. It is worth noting that authors employ a wide variety of techniques, both experimental and bioinformatics.

The authors have satisfactorily responded to most of the comments that needed clarification. Particularly interesting was the addition of the new results section "Q modification impacts decoding fidelity in V. cholerae", after the suggestion to explore the role of Q in prevention of stop codon readthrough. Although it is not a major problem, since the article is very complete and interesting, the interpretation of the results of RiboSeq data carried out in this work remains controversial. This technique, at least when it has been used in eukaryotes to investigate whether there is a bias in the translation of certain codons affected by Q (Tuorto et al., EMBO J. 2018; doi: 10.15252/embj.201899777), has been interpreted as meaning that ribosomes spend less time in the optimal codons and therefore there is an increase in occupancy at codons where translation slows down. On the other hand, it has been observed that "in ribosome profiling experiments conducted without cycloheximide pretreatment, there is a clear inverse relationship between tRNA abundance and ribosome occupancy, showing that ribosomes spend less time at optimal codons and specifically this has been observed in experiments in which a translation inhibitor such as cycloheximide is not used (see review: Hanson G & Coller J. Nat Rev Mol Cell Biol. doi: 10.1038/nrm.2017.91, and experiments in yeast: Hussmann JA et al. PLoS Genet. doi: 10.1371/journal.pgen.1005732). On the other hand, we believe that the comparison between RiboSeq and proteomic data would be interesting to check whether this interpretation of the RiboSeq data is correct. It should not be a problem that the proteomics data could be incomplete, it would just be a more limited study. If the correct interpretation of the RiboSeq results is as proposed by the authors, a correlation should be observed between the abundance of TAT-enriched RNA fragments and the most abundant proteins. Therefore, it would be interesting to perform this comparison and see if significant results are obtained that help to understand the correct interpretation of the RiboSeq experiments.

Reviewer #2 (Public review):

Summary:

The authors propose a methodology to perform causal (temporal) discovery. The approach appears to be robust and is tested in the different scenarios: one related to live-cell imaging data, and another one using synthetic (mathematically defined) time series data. They compare the performance of their findings against another well-known method by using metrics like F-score, precision and recall,

Strengths:

--Performance, robustness, the text is clear and concise, The authors provide the code to review.

Comments on revisions:

The authors have addressed my concerns properly providing the needed explanations.

Reviewer #2 (Public review):

Summary:

This manuscript seeks to reconcile observations in multisensory perception - from behavior and from neural responses. It is intuitively obvious that perceiving a stimulus via two senses results in better performance than one alone. However, the nature of this interaction is complicated and relating different measures (behavioural, neural) is challenging.

It is not uncommon to observe that for a perceptual task the percentage of correct responses seen with two senses is higher than the sum of the percentage correct obtained with each modality individually. i.e. the gains are "superadditive". The gains of adding a second sense are typically larger when the performance with the first sense is relatively poor - this effect is often called the principle inverse effectiveness. More generally, what this tells us is that performance in a multisensory perceptual task is a non-linear sum of performance for each sensory modality alone. In invasive recordings from single neurons, a wide range of non-linear interactions is observed - some superadditive, and some sub-additive.

Despite this abundance evidence of non-linearity in some measures of multisensory integration, evoked responses (EEG) to such sensory stimuli often show little evidence of it - and this is the problem this manuscript tackles. The key assertion made is that a univariate analysis of the EEG signal is likely to average out non-linear effects of integration. This is a reasonable assertion, and their analysis does indeed provide evidence that a multivariate approach can reveal non-linear interactions in the evoked responses.

Strengths:

It is of great value to understand how the process of multisensory integration occurs, and despite a wealth of observations of the benefits of perceiving the world with multiple senses, we still lack a reasonable understanding of how the brain integrates information. For example - what underlies the large individual differences in the benefits of two senses over one? One way to tackle this is via brain imaging, but this is problematic if important features of the processing - such as non-linear interactions are obscured by the lack of specificity of the measurements. The approach they take to analysis of the EEG data allows the authors to look in more detail at the variation in activity across EEG electrodes, which averaging across electrodes cannot.

This version of the manuscript is well written and for the most part clear and the report of non-linear summation of neural responses is convincing. A particular strength of the paper is their use of a statistical model of multisensory integration as their "null" model of neural responses, and the "inverted-encoder" which infers an internal representation of the stimulus which can explain the EEG responses. This encoder generates a prediction of decoding performance, which can be used to generate predictions of multisensory decoding from unisensory decoding, or from a sum of the unisensory internal representations.

In behavioural performance, it is frequently observed that the performance increase from two senses is close to what is expected from the optimal integration of information across the senses, in a statistical sense. It can be plausibly explained by assuming that people are able to weight sensory inputs according to their reliability - and somewhat optimally. Critically the apparent "superadditive" effect on performance described above does not require any non-linearity in the sum of information across the senses, but can arise from correctly weighting the information according to reliability.

The authors apply a similar model to predict the neural responses expected to audiovisual stimuli from the neural responses to audio and visual stimuli alone, assuming optimal statistical integration of information. The neural responses to audiovisual stimuli exceed the predictions of this model and this is the main evidence supporting their conclusion, and it is convincing.

Weaknesses:

The main weakness of the manuscript is that their behavioural data show no evidence of performance that exceeds the predictions of these statistical models. In fact, the models predict multisensory performance from unisensory performance pretty well. This makes it hard to interpret their results, as surely if these nonlinear neural interactions underlie the behaviour, then we should be able to see evidence of it in the behaviour. I cannot offer an easy explanation for this.

Overall, therefore, I applaud the motivation and the sophistication of the analysis method and think it shows great promise for tackling these problems.

Reviewer #2 (Public review):

Summary:

The paper introduces Ukiyo-e-Seq, a novel method integrating microscopy with single-cell transcriptomics to study individual, uncultured eukaryotic plankton cells. By combining microscopic imaging with transcriptomic analysis, the approach links plankton morphology to gene expression, enabling taxonomic identification and functional protein exploration. Ukiyo-e-Seq was tested on 66 microbial eukaryotic cells, revealing taxonomic diversity across four superkingdoms and allowing analysis of protein complexes and developmental genes in individual species. According to the authors, this method has the potential to advance single-cell marine biodiversity studies by addressing limitations in traditional taxonomy and metatranscriptomics, especially for rare or uncultured organisms.

However, the study's conclusions are often weakly supported by data, particularly given that this is not the first study to combine microscopy and single-cell transcriptomics of eukaryotic plankton using Smart-seq2.

Strengths:

A notable strength is the authors' generation of several single-cell transcriptomes for the diatom Chaetoceros, which could benefit from greater focus rather than broadly addressing eukaryotic single cells.

Weaknesses:

The study lacks comparison with other single-cell transcriptomics studies and it was presented as the first study that combines imaging and single-cell transcriptomics (smart-seq2) of eukaryotic plankton while in fact it is not. The sampling methodology is not replicable as the authors used a tea strainer instead of standard plankton collection equipment to filter larger cells. Terminology throughout the paper is unconventional, such as "public and private contigs," "single-organism genomics," "highly expressed contigs," and "optical methods." Additionally, the authors did not specify which database was used for taxonomic assignments. These issues may stem from the authors' limited background in microbial ecology. Overall, the study has many drawbacks and it could benefit from complete rewriting and focusing mainly on single-cell transcriptomics of diatoms.

Reviewer #2 (Public review):

Summary:

Fuqua et al investigated the relationship between prokaryotic box motifs and the activation of promoter activity using a mutagenesis sequencing approach. From generating thousands of mutant daughter sequences from both active and non-active promoter sequences they were able to produce a fantastic dataset to investigate potential mechanisms for promoter activation. From these large numbers of mutated sequences, they were able to generate mutual information with gene expression to identify key mutations relating to the activation of promoter island sequences.

Strengths:

The data generated from this paper is an important resource to address this question of promoter activation. Being able to link promoter modulated gene expression to mutational changes in previously nonactive promoter regions is exciting. This approach allows future large-scale studies to investigate evolutionary processes relating to changes in gene regulation in a statistically robust manner. Here there is a focus on the -10 and -35 boxes but other elements and interactions were explored including; H-NS binding, UP-element and TGn. Alongside this, the method of identifying key mutations using mutual information in this paper is well done and should be a standard in future studies for identifying regions of interest.

Weaknesses:

While the generation of the data is superb, as the authors have stated clearly themselves, there is a lot of scope for future studies to understand both causal relationships and utilise the data more effectively. The authors look at changes in regulatory expression based on a few observations that are treated independently but occur concurrently. While this study has backed up findings experimentally this may not always be possible. Previously this reviewer had suggested addressing this using complementary approaches such as analysis focusing on identifying important motifs, using something like a glm lasso regression to identify significant motifs, and then combining with mutational hotspot information would be more robust. The authors tried to implement such an approach in response to the review, but its complexity became beyond the scope. I look forward to the development of such methods that allow more complete exploration of similar datasets.

Comments on revised version:

The authors addressed all my previous comments. I believe the study is much improved and thank them for the time and effort they put into addressing the comments.

Reviewer #2 (Public review):

Summary:

In the manuscript "Decoding m6Am by simultaneous transcription-start mapping and methylation quantification" Liu and co-workers describe the development and application of CROWN-Seq, a new specialized library preparation and sequencing technique designed to detect the presence of cap-adjacent N6,2'-O-dimethyladenosine (m6Am) with single nucleotide resolution. Such a technique was a key need in the field since prior attempts to get accurate positional or quantitative measurements of m6Am positioning yielded starkly different results and failed to generate a consistent set of targets. As noted in the strengths section below the authors have developed a robust assay that moves the field forward.

Furthermore, their results show that most mRNAs whose transcription start nucleotide (TSN) is an 'A' are in fact m6Am (85%+ for most cell lines). They also show that snRNAs and snoRNAs have a substantially lower prevalence of m6Am TSNs.

Strengths:

Critically, the authors spent substantial time and effort to validate and benchmark the new technique with spike-in standards during development, cross-comparison with prior techniques, and validation of the technique's performance using a genetic PCIF1 knockout. Finally, they assayed nine different cell lines to cross-validate their results. The outcome of their work (a reliable and accurate method to catalog cap-adjacent m6Am) is a particularly notable achievement and is a needed advance for the field.

Weaknesses:

No major concerns were identified by this reviewer.

Mid-level Concerns:

(1) In Lines 625 and 626, the authors state that "our data suggest that mRNAs initate (mis-spelled by authors) with either Gm, Cm, Um, or m6Am." This reviewer took those words to mean that for A-initiated mRNAs, m6Am was the 'default' TSN. This contradicts their later premise that promoter sequences play a role in whether m6Am is deposited.

(2) Further, the following paragraph (lines 633-641) uses fairly definitive language that is unsupported by their data. For example in lines 637 and 638 they state "We found that these differences are often due to the specific TSS motif." Simply, using 'due to' implies a causative relationship between the promoter sequences and m6Am has been demonstrated. The authors do not show causation, rather they demonstrate a correlation between the promoter sequences and an m6Am TSN. Finally, despite claiming a causal relationship, the authors do not put forth any conceptual framework or possible mechanism to explain the link between the promoter sequences and transcripts initiating with an m6Am.

(3) The authors need to soften the language concerning these data and their interpretation to reflect the correlative nature of the data presented to link m6Am and transcription initiation.

Reviewer #2 (Public review):

Summary:

Kisspeptin neurons of the arcuate nucleus (ARC) are thought to be responsible for the pulsatile GnRH secretory pattern and to mediate feedback regulation of GnRH secretion by estradiol (E2). Evidence in the literature, including the work of the authors, indicates that ARC kisspeptin coordinate their activity through reciprocal synaptic interactions and the release of glutamate and of neuropeptide neurokinin B (NKB), which they co-express. The authors show here that E2 regulates the expression of genes encoding different voltage-dependent calcium channels, calcium-dependent potassium channels and canonical transient receptor potential (TRPC5) channels and of the corresponding ionic currents in ARC kisspeptin neurons. Using computer simulations of the electrical activity of ARC kisspeptin neurons, the authors also provide evidence of what these changes translate into in terms of these cells' firing patterns. The experiments reveal that E2 upregulates various voltage-gated calcium currents as well as 2 subtypes of calcium-dependent potassium currents, while decreasing TRPC5 expression (an ion channel downstream of NKB receptor activation), the slow excitatory synaptic potentials (slow EPSP) elicited in ARC kisspeptin neurons by NKB release and expression of the G protein-associated inward-rectifying potassium channel (GIRK). Based on these results, and on those of computer simulations, the authors propose that E2 promotes a functional transition of ARC kisspeptin neurons from neuropeptide-mediated sustained firing that supports coordinated activity for pulsatile GnRH secretion to a less intense burst-like firing pattern that could favor glutamate release from ARC kisspeptin. The authors suggest that the latter might be important for the generation of the preovulatory surge in females.

Strengths:

The authors combined multiple approaches in vitro and in silico to gain insights into the impact of E2 on the electrical activity of ARC kisspeptin neurons. These include patch-clamp electrophysiology combined with selective optogenetic stimulation of ARC kisspeptin neurons, reverse transcriptase quantitative PCR, pharmacology and CRISPR-Cas9-mediated knockdown of the Trpc5 gene. The addition of computer simulations for understanding the impact of E2 on the electrical activity of ARC kisspeptin cells is also a strength.

The authors add interesting information on the complement of ionic currents in ARC kisspeptin neurons and on their regulation by E2 to what was already known in the literature. Pharmacological and electrophysiological experiments appear of the highest standards and robust statistical analyses are provided throughout. The impact of E2 replacement on calcium and potassium currents is compelling. Likewise, the results of Trpc5 gene knockdown do provide good evidence that the TRPC5 channel plays a key role in mediating the NKB-mediated slow EPSP. Surprisingly, this also revealed an unsuspected role for this channel in regulating the membrane potential and excitability of ARC kisspeptin neurons.

Weaknesses:

The manuscript also has weaknesses that obscure some of the conclusions drawn by the authors.

One is that the authors compare here two conditions, OVX versus OVX replaced with high E2, that may not reflect the physiological conditions under which the proposed transition between neuropeptide-dependent sustained firing and less intense burst firing might take place (i.e. the diestrous [low E2] and proestrous [high E2] stages of the estrous cycle). This is an important caveat to keep in mind when interpreting the authors' findings. Indeed, that E2 alters certain ionic currents when added back to OVX females, does not mean that the magnitude of all of these ionic currents will vary during the estrous cycle.<br /> In addition, although the computational modeling indicates a role of the various E2-modulated conductances in causing a transition in ARC kisspeptin neuron firing pattern, their role is not directly tested in physiological recordings, weakening the link between these changes and the shift in firing patterns.

Overall, the manuscript provides interesting information about the effects of E2 on specific ionic currents in ARC kisspeptin neurons and some insights into the functional impact of these changes. However, some of the conclusions of the work, with regard, in particular, to the role of these changes in ion channels and to their implications for the LH surge, are not fully supported by the findings.

Reviewer #2 (Public review):

Summary:

In this article, Abssy, Osokin, Osborne, et al. aimed to demonstrate the effect of Peripheral Magnetic Stimulation (PMS) as a pain relief tool, studying its effects in an experimentally induced pain paradigm applied over healthy subjects. This is a relevant objective, as it will give a proxy indication of its utility as a clinical intervention to treat pain. Shockingly, in the first experiment, the authors found that this effect existed, not only in the active PMS groups but also in the sham PMS. With a clever second experiment, the authors used pink noise to mask the clicking sound and the PMS: this modification abolished the hypoalgesic effect of PMS.

Strengths:

This study presents an adequately calculated sample size (n = 100 for study 1 and n = 32 for study 2). This gives trustability to the results and allows for a correct disaggregated analysis to assess gender effects, which correct execution does not often occur. Nuisance variables are adequately addressed, figures and writing are clear, and I especially liked figures 4 and 5 for their easiness of interpretation. They explore two different stimulation protocols for the PMS, extending their results beyond parametrization. Secondary hyperalgesia is a particularly relevant measurement, as it is a common symptom in many relevant painful conditions. Pseudorandomization and counterbalanced design are also appreciated, as well as reinforcement of the results through Bayesian statistical approaches. Regarding the scientific content, the main result (auditory modulation of pain in PMS) is exciting and very interesting by itself and will be relevant for the pain community, granting further research, both from a fundamental and clinical perspective. Personally, I respect that they recognize that results did not match their a priori hypothesis, instead of committing HARKing. And it is a very thrilling mismatch for sure!

It will be especially interesting for those among us dedicated to neural stimulation for pain treatment.

Weaknesses:

Although the study presents solid results, some specific concerns make me reluctant to accept the interpretations that the authors take from said results. I list the most important here.

(1) My biggest concern in this paper is that the stimulation protocols are not applied after pain was induced in the subjects, but before. This is not bad in itself, but as the paper presents the stimulations as potential "treatments" it generates a severe mismatch between the objective, context (introduction), and impact (discussion) presented for the experiments, and how they are actually designed. This adds to the fact that healthy volunteers are used here to generate a study with low translational capability, that aims to be translational and provide an indication for clinics (maybe this is why the reduction in pain intensity caused by PMS when applied in patients, reported in references [29, 35 and 39], is not observed here).

(2) TENS treatment duration is simply too short (90s) to be considered a therapeutic TENS intervention. I get that this duration was chosen to match the one of PMS, but TENS is never applied like this in the clinics, in which the duration varies from 10 minutes to an hour (or more). This specific study comparing different durations recommends 40 minutes for knee osteoarthritis pain relief (PMID: 12691335). Under these conditions, this stimulation is more similar to a sham TENS than to a real TENS treatment: I would suggest interpreting it as such. As the paper is right now, it could give the impression that PMS could produce clinical effects not observed in TENS, but while the PMS application resembles a clinical one, the TENS application does not (due to its extremely short duration). As an example, giving paracetamol at a dose 10 times below its effective dose is a placebo, not a paracetamol treatment.

(3) This study measured pain, not central sensitization. Specifically, the effects refer to the area of secondary hyperalgesia. The IASP definition for central sensitization is "Increased responsiveness of nociceptive neurons in the central nervous system to their normal or subthreshold afferent input." (PMID: 32694387). No neuronal results are reported in this article. Therefore, central sensitization is not measured here, and we do not know if it is reduced by sound. This frontally clashes with the title of the article and with many interpretations of the results. For a deep review on this topic, I recommend PMID: 39278607 and the short article PMID: 30416715.

(4) There is no mention of blinding/masking/concealing in this manuscript. Was the therapist blind to whether they applied one protocol, another, or a placebo? Were the evaluators blind, as this can heavily influence their measurements? And the volunteers? Was allocation concealed? Was this blinding measured afterwards? Blinding is, together with randomization, the most important methodological feature for those interventional studies. For example, not introducing blinding and concealing directly makes a study lose 4 out of 10 points in the PEDro scale, failing to fulfill criteria 3, 5, 6, and 7 (https://pedro.org.au/english/resources/pedro-scale/). Continuing with methodological considerations, the dropout percentage is high (18% for the first and 25% for the second study), both above the 15% cutoff for criterion 8 of the PEDro, losing another point. It is not mentioned whether the statistical analysis was intention-to-treat or per-protocol. Assuming the second, criterion 9 is failed too. Also, although between-group comparisons are done for study 1, they are not for study 2. Criterion 10 depends on this, so I would recommend doing it to avoid failing it. As it is right now, the study will be a 3/10 on the PEDro scale, being therefore considered "low-quality level evidence". As some of these criteria can be fulfilled in this study, I will recommend doing so to increase its quality level to medium (more in "recommendations for authors").

(5) Data reporting and statistical treatment can be improved, as only differences are reported and regression to the mean is not accounted for in this study. Moreover, baseline levels for the dependent variables (control session) are not accessible for evaluation and they are not compared statistically, making it impossible to know if the groups were similar at baseline. This will imply failing criterion 3 of the PEDro, for a total of 2/10 points.

Reviewer #2 (Public review):

Summary:

The authors have developed a machine learning tool AIVE to predict the infectivity of SARS-CoV-2 variants and also a scoring metric to measure infectivity. A large number of virus sequences were used with very detailed analysis that incorporates hydrophoic, hydrophiclic, acid and alkaline characteristics. The protein structures were also considered to measure infectivity and search for core mutations. The study especially focused on the S protein of SARS-CoV-2. The contents of this study would be of interest to many researchers related to this area and the web-service would be helpful to easily analyze such data without indepth bioinformatics expertise.

Strengths:

- Analysis on large scale data<br /> - Experimental validation on a partial set of searched mutations<br /> - A user-friendly web-based analysis platform that is made public

Weaknesses:

- Complexity of the research

Comments on revisions:

The authors have addressed all my comments and is much more readable.

Reviewer #2 (Public review):

Summary:

The authors have attempted to modify and adapt the IH-KA model in mice to provide an improved approach to screening for new ASDs by partially mitigating the problem of randomly occurring seizures and relatively low seizure frequency in the IH-KA model. The authors used KA micro-injections to selectively kill the hippocampal CA3 area as a way to induce temporal lobe "epileptogenesis" (TLE), and then used optogenetics to activate CA1 pyramidal cells specifically. This approach allowed the authors to trigger generalized seizures where the tonic-clonic pattern of electrical activity was reminiscent of actual tonic-clonic behavioral convulsions. Administration of levitracetam (LEV) and diazepam (DZP), two widely used ASDs with different mechanisms, reduced the probability of optogenetically activated epileptic seizures in IH-KA mice, thus seeming to provide evidence for a new approach to screen ASDs. A variety of problems and issues with the approach and the results lead to confounds that raise serious concerns about the conclusions.

Major strengths and weaknesses of the Methods and Results:

Strengths:

The authors have designed a method for triggering seizures, and the figures show bona fide electrographic seizures with concomitant convulsive behavioral components. The optogenetically evoked seizures in IH-KA mice had the electrical properties of actual seizures and the tonic-clonic components were readily apparent. These seizures appeared different from seizures evoked in naïve mice, and the authors attribute this difference to the epileptogenic process, but this may not be correct.

The ASDs (i.e., LEV and DZP) reduced the success rate of the optogenetically evoked seizures in IH-KA mice, thus suggesting the potential usefulness of the model for testing ASDs. The paper discusses whether the Epilepsy Therapy Screening Program (ETSP) will be able to use this modification of the IH-KA model in place of (1) ASD screening with acute seizures in naïve animals, where the brain has not undergone "epileptogenesis", (2) testing ASDs on hippocampal paroxysmal discharges (HPDs) in the IH-KA model, which has undergone epileptogenesis, or (3) spontaneous epileptic seizures in animal models of TLE based on systemic treatments that lead to acute convulsive status epilepticus that have later undergone epileptogenesis. This proposed version of the IH-KA model aims to address the former problem (#1, above) by using a mouse model of TLE, and to address the latter problems (#2 and #3, above) of the seemingly random occurrence of epileptic seizures and the low seizure frequency by using optogenetically "triggered" seizures.

Weaknesses

Although the figures provide excellent examples of individual electrographic seizures and compare induced seizures in epileptic and naïve animals, it is unclear which criteria were used to identify an actual seizure induced by the optogenetic stimulus, versus a hippocampal paroxysmal discharge (HPD), an "afterdischarge", an "electrophysiological epileptiform event" (EEE, Ref #36, D'Ambrosio et al., 2010 Epilepsy Currents), or a so-called "spike-wave-discharge" (SWD). Were HPDs or these other non-seizure events ever induced using stimulation in animals with IH-KA? A critical issue is that these other electrical events are not actual seizures, and it is unclear whether they were included in the column showing data on "electrographic afterdischarges" in Figure 5 for the studies on ASDs. This seems to be a problem in other areas of the paper, also.

The differences between the optogenetically evoked seizures in IH-KA vs naïve mice are interpreted to be due to the "epileptogenesis" that had occurred, but the lesion from the KA-induced injury would be expected to cause differences in the electrically and behaviorally recorded seizures - even if epileptogenesis had not occurred. This is not adequately addressed.

The authors did not test whether an apparent "kindling" effect, apparently seen in naïve controls, also occurred in animals micro-injected with kainic acid (KA). This effect could cause model instability that might result in variability in response to ASDs. It is not clear whether the number of optogenetically induced seizures in epileptic animals would affect the response to drugs. It is also unclear how much of an improvement the animal model in the present work is over other similar models of TLE, where electrically triggered seizures could simply be applied to one of them.

The authors offer little mention of other research using animal models of TLE to screen ASDs, of which there are many published studies - many of them with other strengths and/or weaknesses. For example, although Grabenstatter and Dudek (2019, Epilepsia) used a version of the systemic KA model to obtain dose-response data on the effects of carbamazepine on spontaneous seizures, that work required use of KA-treated rats selected to have very high rates of spontaneous seizures, which requires careful and tedious selection of animals. The ETSP has published studies with an intra-amygdala kainic acid (IA-KA) model (West et al., 2022, Exp Neurol), where the authors claim that they can use spontaneous seizures to identify ASDs for DRE; however, their lack of a drug effect of carbamazepine may have been a false negative secondary to low seizure rates. The approach described in this paper may help with confounds caused by low or variable seizure rates. These types of issues should be discussed, along with others.

While the paper may be relevant for the ETSP and contract research organizations (CROs), the paper was not written to attract the interest of biological scientists, even those in this specific area of epilepsy research. It may be of low interest to other neuroscientists.

The outcome measure for testing LEV and DZP on seizures was essentially the fraction of unsuccessful or successful activations of seizures, where high ASD efficacy is based on showing that the optogenetic stimulation causes fewer seizures when the drug is present. The final outcome measure is thus a percentage, which would still lead to a large number of tests to be assured of adequate statistical power. Thus, there is a concern about whether this proposed approach will have high enough resolution to be more useful than conventional screening methods so that one can obtain actual dose-response data on ASDs.

The key issue the authors aim to address is the 30-40% of patients with DRE, but the real problem with DRE patients is not that these people have seizures with no effect of the ASDs; rather, although ASD may reduce seizure burden, these patients continue to have some remaining seizures even after high doses of ASDs, which often leads to adverse effects from the particular ASDs.

In several sections of the paper, the authors argue that two different groups are similar on the basis that no statistical difference was found between the two groups (i.e., p > 0.05); however, the failure to find a statistically significant difference, particularly with relatively small sample sizes, is not rigorous evidence that the two groups are actually similar - they are just "not significantly different."

It remains unclear that the optogenetically induced seizures in this model are better than similarly induced seizures in a naïve animal, and there is no evidence that the model will be useful for finding new ASDs to treat DRE.

Do the results support the conclusions?

Although the Results show examples of clear tonic-clonic seizures, it is not at all clear whether this approach is a significant improvement over previous methods used on animal models of TLE. The presented data from this method shows it provides an ability to detect the effect of widely used ASDs, but not that it will have the resolution to find better ASDs. The outcome measure of successful vs failed seizure inductions does not necessarily translate to a pathway for finding new ASDs for DRE, which often is a function of the side effects of the proposed new ASD. Although the recorded seizures in IH-KA rats differ in waveform from the ones in naïve mice, this could be due to the pattern of damage resulting from the micro-injection of KA or the density of expressed Chr2, which could be affected by sclerosis.

Impact and utility of methods and data.

The authors state that this approach should be used to test for and discover new ASDs for DRE, and also used for various open/closed loop protocols with deep-brain stimulation; however, the paper does not actually discuss rigorously or critically the background literature on other published studies in these areas or how this approach will improve future research for a broader audience than the ETSP and CROs. Thus, it is not clear whether the utility will apply more widely and how extensive a readership will be attracted to this work.

Final Conclusions:

Although this is an Interesting if not elegant new model for testing ASDs, it could be seen as a version of kindling (plus brain damage) in a rodent model, where some of the pathology of TLE is induced through focal injection of KA in the CA3 area of the hippocampus. Unfortunately, no evidence was presented that it will be any better than other models, although it could be faster and maybe easier than models based on spontaneous seizures. Although it has some similarities to the pathology of human TLE, the ablating part of the hippocampus does not account for the more widespread pathology that usually occurs elsewhere in the brain, as studied with imaging and with anatomy in surgical specimens from patients with DRE.

Although this approach with seizure induction via an optogenetic approach adds specificity to the type of cell that is stimulated (i.e., CA1 pyramidal cells), it is not apparent why this provides a better or more effective tool than simple electrical induction of seizures in any TLE model. Most important, it remains unclear how this addresses any aspect of drug resistance. To improve the ASD discovery process, an important new model must make a significant reduction in seizure burden, and would ideally improve the percentage of patients that become seizure-free. It is not clear how this model will do that.

In the end, the authors have created a model with some of the pathology of TLE, where they can then induce actual seizures via specific optogenetic stimulation. So, although it is potentially elegant work, it remains unclear what new information this model will tell us about epilepsy, and most importantly DRE - or how it will improve treatment outcomes.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

The primary weaknesses of this analysis is the relatively low sample size for analyzing disease subsets and moderate correlation values observed for putative biomarkers. Regardless, this data can be used to inform future studies aiming to understand the contribution of multifactorial dysbiosis to COVID-19 disease progression.

Reviewer #2 (Public review):

Schwintek et al. investigated whether a geological setting of a rock pore with water inflow on one end and gas passing over the opening of the pore on the other end could create a non-equilibrium system that sustains nucleic acid reactions under mild conditions. The evaporation of water as the gas passes over it concentrates the solutes at the boundary of evaporation, while the gas flux induces momentum transfer that creates currents in the water that push the concentrated molecules back into the bulk solution. This leads to the creation of steady state regions of differential salt and macromolecule concentrations that can be used to manipulate nucleic acids. First, the authors showed that fluorescent bead behavior in this system closely matched their fluid dynamic simulations. With that validation in hand, the authors next showed that fluorescently-labeled DNA behaved according to their theory as well. Using these insights, the authors performed a FRET experiment that clearly demonstrated hybridization of two DNA strands as they passed through the high Mg++ concentration zone, and, conversely, the dissociation of the strands as they passed through low Mg++ concentration zone. This isothermal hybridization and dissociation of DNA strands allowed the authors to perform an isothermal DNA amplification using a DNA polymerase enzyme. Crucially, the isothermal DNA amplification required the presence of the gas flux and could not be recapitulated using a system that was at equilibrium. These experiments advance our understanding of the geological settings that could support nucleic acid reactions that were key for the origin of life.

The presented data compellingly supports the conclusions made by the authors. In the revised submission, the authors have made convincing arguments supported by simulations that the present findings obtained with DNA would translate to RNA as well, thus making this work highly relevant for the field of origin of life.

A potential future experiment the authors could consider includes performing a prebiotically relevant reaction, such as non-enzymatic primer extension or ligation, in the described model of the rock pore geological setting.

Reviewer #2 (Public review):

Summary:

In the manuscript "Surprising Features of Nuclear Receptor Interaction Networks Revealed by Live Cell Single Molecule Imaging", Dahal et al combine fast single molecule tracking (SMT) with proximity-assisted photoactivation (PAPA) to study the interaction between RARa and RXRa. The prevalent model in the nuclear receptor field suggests that type II nuclear receptors compete for a limiting pool of their partner RXRa. Contrary to this, the authors find that over-expression of RARa but not RXRa increases the fraction of RXRa molecules bound to chromatin, which leads them to conclude that the limiting factor is the abundance of RARa and not RXRa. The authors also perform experiments with a known RARa agonist, all trans retinoic acid (atRA) which has little effect on the bound fraction. Using PAPA, they show that chromatin binding increases upon dimerization of RARa and RXRa.

The authors have done well to address my comments and specify limitations where they could not.

Reviewer #2 (Public review):

Summary:

To follow-up on recent reports of Xist-autosome interaction the authors examine female (and male transgenic) mESCs and MEFs by CHARTseq. Upon finding that only 10% of reads map to X, they sought to identify reproducible alternative sites of Xist-binding, and identify ~100 autosomal Xist-binding sites in active chromatin regions. They demonstrate a transient down-regulation of autosomal expression. They utilize published male transgenic inducible Xist mESC data to support their findings. In their system, inhibition of Xist reduces autosomal impact.

Strengths:

The authors address a topical and interesting question with a series of models including developmental timepoints and utilize unbiased approaches (CHARTseq, RNAseq). For the CHARTseq they have controls of both sense probes and male cells; and indeed do detect considerable background with their controls. The use of 'metagene' plots provides a visual summation of genic impact. They compare with published data.

Weaknesses:

The revised text and rebuttal clarified my confusion of the 'follow-up' analyses (Figure 4) compared to published datasets. Further, the figure legends have been improved.

While the controls were a strength, it appears that when focussed on bound regions, the background (from sense probes) is now also substantially higher than global background (compare 1E to 1A/B). Thus, why do these autosomal targets enrich for the sense probes, and how to distinguish from such background for the ∆B experiments? If male and sense are both controls, then why is sense lower for males than females, doesn't this suggest Xist impact? While authors note d0 might detect Tsix, the signal is only slightly reduced by d14 and never equivalent. Indeed, the new PCA (S1C) does show as noted that female Xist interactions are distinct from sense and male, but the male signal is even more distinct from sense probes.

It would have been preferable to see the dispersion of the Xist RNA cloud in these ∆B cells, rather than a reference.

Only 2 replicates were used, but there were multiple time-points: D0, D4, d7, d14; further, the correlation analysis showed good reproducibility, and in response to reviews they note that 2 replicates are standard of practice.

The conclusion that RepB is "required for localization to the ~100 genes" is based on density (panel 2E); however, these autosomal targets retain enrichment at TSSs (panel 2A) and indeed the text suggests they are the same sites, suggesting that in fact the choice of autosomal region binding is not RepB dependent. Thus, this remains unresolved for me.

The introduction is clear, and the senior author is a leader in the field; however, by this reviewer's count 19 of the 52 references include the senior author.

Better descriptors for the supplemental Excel files would be helpful.

Aim achievement: The authors do identify autosomal sites with enrichment of chromatin marks and evidence of silencing. Their revised text clarifies many issues, although this reviewer still remains unconvinced that the autosomal targeting is repB-dependent.

The impact of Xist on autosomes is important for consideration of impact of changes in Xist expression with disease (notably cancers). Knowing the targets (if consistent) would enable assessment of such impact.

Reviewer #2 (Public review):

Summary:

Longhurst et al. assessed cell cycle regulators using a chemogenetic CRISPR-Cas9 screen in the haploid human cell line HAP1. Besides known cell cycle regulators they identified the PRC2.1 subcomplex to be specifically involved in G1 progression, given that the absence of members of the complex makes the cells resistant to Palbociclib. They further showed that in HAP1 cells the PRC2.1, but not the PRC2.2 complex is important to repress the cyclins CCND1 and CCND2. This can explain the enhanced resistance to Palbociclib, a CDK4/6-Inhibitor, after PRC2.1 deletion.

Strengths:

The initial CRISPR screen is very interesting, because it uses three distinct chemicals that disturb the cell cycle at various stages. This screen mostly identified known cell cycle regulators, which demonstrates the validity of the approach. The results can be used as a resource for future research.

The most interesting outcome of the experiment is the finding that knockouts of the PRC2.1 complex make the cell resistant to Palbociclib. In further experiments, the authors focused on MTF2 and JARID2 as main components of PRC2.1 and PRC2.2, respectively. Via extensive analyses, including genome-wide experiments, they confirmed that MTF2 is particularly important to repress the cyclins CCND1 and CCND2. Absence of MTF2 therefore leads to increased expression of these genes, sufficient to make the cell resistant to Palbociclib. This result will likely be of wide interest to the community.

Weaknesses:

The work is limited to specific biological contexts, and the generality of the conclusions is uncertain.

Comments on revisions:

The revision offers new insights and is overall satisfying. I have no further recommendations that I consider essential.

Reviewer #2 (Public review):

Summary:

In this manuscript, Dr. Yao Li et al. documented the metabolomic profile of the aorta from OVX rats and that from OVX plus E2. These conditions mimic post-menopause hypertension and hormonal replacement therapy.

Strengths:

The authors state that this is probably the first study to examine the metabolic changes in the aorta of post-menopause hypertension.

Weaknesses:

There are several weaknesses, and a few of them are quite serious.

(1) The aorta is not a resistant artery and has little to do with hypertension. The authors should have used resistant arteries for this study. The expression of several adrenergic receptors and cholinergic receptors in the aorta and resistant arteries are different. It is unknown whether the aorta metabolomic profile has any relevance to BP and whether they are similar to that of the resistant arteries. I understand the logistics issue of obtaining enough tissues from resistant arteries. At least, once some leads are discovered in the aorta, the authors should validate it in resistant arteries. This should be feasible.

(2) The aorta and all the arteries have three layers. It is critically important to know whether the metabolic changes occur in the intima or in the media, while the adventitia probably has little to do with vasoconstriction and hypertension. If the authors want to use the aorta to conduct the preliminary study, they should completely remove the adventitia and then use samples with and without their endothelium stripped and then assess their metabolomic profiles. After the leads are obtained from this preliminary profiling, they should be validated in endothelium and smooth muscles of the resistant artery. The current experiments are not appropriately designed.

(3) The tail-cuff BP measurement is a technique of the last century. The current gold standard of BP measurement is by telemetry. The tail-cuff method is particularly problematic in this study because the 1-2 h restraining of the rats for more than 10 times BP measurement will cause significant stress in the animal, and their stress hormone secretion might cause biased metabolomic profiles in the OVX versus shames operated mice. The problem can be totally avoided by using telemetry.

(4) Although the L-AABA showed a high p-value (10^-4) of a decrease in the OVX rats, the fold change is small (2-3 folds). Such a small change should be validated using a different method to be convincing.

(5) The authors claim (or hypothesize) that the reduced AABA level in OVX can cause vascular remodeling. This can be easily validated by the histology of the OVX-resistant artery, and they should do that during the revision. The authors should also examine the M1 macrophage function from the OVX mice to validate their claimed link of AABA to M1.

(6) As mentioned above, the authors need to pinpoint the changes of AABA to target cells, i.e., endothelial cells, SMC, or M1, and then use in vitro or in vivo cell biology approaches to assess whether these cells in the OVX rat indeed have an abnormality in function and, indeed, such functional changes are responsible for the BP phenotype.

(7) The results of the current study can be condensed into 1 or 2 figures that can serve as a base or a starting point for a deeper scientific study.

Summary

The experimental design of this manuscript is inappropriate, and the methods are not up to the current standards. The whole study is descriptive and rudimentary. It lacks validation and mechanism. The data from this manuscript might be of some value and can serve as the first step for more investigation of the mechanism of post-menopause hypertension.

Reviewer #2 (Public review):

Summary:

Kan et al. presents the discovery of oxydifficidin as a potential antimicrobial against N. gonorrhoeae, including multi-drug resistant strains. The authors show the role of DedA flippase assisted uptake and the specificity of RplL in the mechanism of action for oxydifficidin. This mode of action could potentially offer a new therapeutic avenue, providing a critical addition to the limited arsenal of antibiotics effective against gonorrhea.

Strengths:

This study shows the potential of revisiting anti-bacterial agents/products for antibacterial activity against modern-day-concerning pathogens and highlights a new anti-gonoccoal mechanism of action. Indeed there is a recent growing body of research to revisit potential antimicrobial agents and metabolites from cultured bacterial species. The discovery of oxydifficidin interaction with RplL and its DedA-assisted uptake mechanism opens new research directions in understanding and combating antibiotic resistant N. gonorrhoeae. The antimicrobial activity of oxydifficidin is also active against N. meningitidis, a closely related species. Methodologically, the study is rigorous employing various experimental techniques including Tn-mutagenesis (TraDIS, Tn-Seq).

Weaknesses:

While the study demonstrates the in vitro effectiveness of oxydifficidin, there is a lack of in vivo validation (i.e., animal models) for assessing pre-clinical potential of oxydifficidin. However, I acknowledge that this would be a tremendous amount of work and likely outside the scope of this study. Potential SNPs within dedA or RplL raises concerns about how quickly resistance could emerge in clinical settings.

Reviewer #2 (Public Review):

In this paper, starting with unbiased CRISPRi screening, the authors found that perturbations in lipid homeostasis lead to proteostasis impairment. The screen and most follow-up experiments used the dye ProteoStat, which detects protein aggregates and the aggresome. Based upon their screen hits and subsequent analyses, the authors determined that increased levels of sphingolipids and cholesterol esters induce proteostasis defects, along with formation of protein aggregates that appear to be localized in the lysosome. The lysosome increases in content, but its function is not detectably perturbed.

Comments on revised version:

I am satisfied with the authors' actions in response to my public and specific suggestions, but not yet with the manuscript itself. I think that the paper would be improved if they showed the evidence arguing against an effect on proteasome activity but I can live with this omission. I think that the readability and ease of grasping the main points are improved by Figure 7. Inclusion of these simple but informative conceptual summaries is a must.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors re-analyze RNase-treated iMARGI data to systematically identify and analyze RNase-resistant RNA-chromatin interactions. In general

Strengths:

Analyses are well-thought-out and generally solid.

Weaknesses:

Conclusions are massively overstated, and though the analytical pipelines used are solid, the conclusions deriving from them lack the backing of solid computational and molecular controls.

Reviewer #2 (Public review):

Summary:

Crystallographic analysis revealed the asymmetric conformation of the dimer in the inhibitor-bound state. Based on this result, which is consistent with previous time-resolved analysis, authors verified the dynamics and distance between spin introduced label by DEER spectroscopy in solution and predicted possible patterns of asymmetric dimer.

Strengths:

Crystal structures with inhibitor bound provide detailed coordination in the binding pocket thus useful information for the PPase field and maybe for drug development.

Weaknesses:

The distance information measured by DEER is advantageous for verifying the dynamics and structure of membrane protein in solution. However, regarding T211 data, which, as the authors themselves stated, lacks measurement precision, it is unclear for readers how confident one can judge the conclusion leading from these data for the cytoplasmic side.

The distance information for the luminal site, which the authors claim is more accurate, does not indicate either the possibility or the basis for why it is the ensemble of two components and not simply a structure with a shorter distance than the crystal structure.

Reviewer #2 (Public review):

Summary:

Shapiro et al. set out to verify the American Beefalo Association's claim that Beefalo cattle possess 37.5% bison ancestry. They employ a comprehensive range of well-established population genomics methods to estimate ancestry in these hybrid populations, including PCA, ADMIXTURE, D and F statistics, and local ancestry inference. Their findings conclusively demonstrate that most Beefalo lack the claimed bison ancestry, with only 8 out of 47 samples showing any detectable bison ancestry, ranging from 2 - 18%.

Strengths:

The primary strength of this analysis lies in the comprehensive dataset available to the authors, which includes important foundational Beefalo individuals and various reference populations. The rigorous and multi-faceted methodological approach employs several well-established techniques in population genomics for detecting and measuring admixture. Each method used has a firm basis in the field, providing consistent and robust results. The authors' approach of using PCA to initially assess the data within a global context, followed by more specific analyses using ADMIXTURE and D-statistics, provides a clear and logical progression of evidence. The presentation of these results in figures is particularly effective, clearly illustrating the key findings of the study. Additionally, the examination of both autosomal and sex chromosome ancestry offers a more complete understanding of Beefalo genetic composition and the mechanics of bison-cattle hybridisation.

Weaknesses:

One limitation of this analysis is the relatively low coverage (~2x) of many Beefalo samples. However, the authors have taken steps to mitigate biases that may arise from this. Another weakness is the limited sampling of contemporary Beefalo populations, as the study focuses primarily on historical samples. This may limit our understanding of how Beefalo genetics may have changed over time.

Appraisal:

The authors have clearly achieved their primary aim using a rigorous and comprehensive methodology. Their extensive dataset and multi-faceted analytical approach provide strong support for their conclusions. The study not only addresses its main research question but also reveals unexpected insights into Beefalo genetics, particularly the presence of zebu ancestry.

Discussion:

This study is valuable for several reasons beyond its primary findings. First, it definitively addresses and refutes the claim of 37.5% bison ancestry in Beefalo, providing crucial information for those studying these interspecies hybrids and the viability of their offspring. Second, it reveals the unexpected presence of zebu ancestry in many Beefalo, raising intriguing questions about the breed's development and the potential role of zebu cattle in achieving desired traits. This finding suggests that the distinctive appearance of Beefalo may be due in part to zebu admixture rather than bison ancestry. Third, the study highlights the significant barriers to admixture between bison and cattle, both in controlled breeding programs and potentially in wild populations. This has important implications for conservation genetics and our understanding of gene flow between these species. Lastly, the study demonstrates the power of genomic analysis in verifying breed claims and understanding the complex history of domestic animal breeds. These findings open new avenues for research in bovine genomics, breed development, and the dynamics of interspecies hybridisation.

Reviewer #2 (Public review):

Summary:

This study explores the potential of inhibiting the p38-MK2 signaling pathway to enhance the efficacy of microtubule-targeting agents (MTAs) in breast cancer treatment using a dual-target inhibitor.

Strengths:

The study identifies the p38-MK2 pathway as a promising target to enhance the efficacy of microtubule-targeting agents (MTAs), offering a novel therapeutic strategy for breast cancer treatment. In addition, the study employs a wide range of techniques, especially live-cell imaging, to assess the microtubule dynamics in TNBC cells.

Weaknesses:

The study primarily uses RPE1 cells as the control for normal cells, which may not fully capture the response of normal mammary epithelial cells. While CMPD1 is shown to be effective in suppressing tumor growth in MDA-MB-231 xenograft, the study lacks detailed toxicity data to confirm its safety profile in vivo.

Reviewer #2 (Public review):

Summary:

This is an extremely interesting mouse study, trying to understand how sepsis is tolerated during obesity/NAFLD. The researchers combine a well-established model of NASH (Choline-deficiency with High Fat Diet) with a sepsis model (IP injection of 10mg/kg LPS), leading to dramatic mortality in mice. Using this model, they characterize the complex contributions of immune cells. Specifically, they find that NK-cells and Neutrophils contribute the most to mortality in this model due to IFNG and PD-L1+ Neutrophils.

Strengths:

The biggest strength of the manuscript is how clear the primary phenotypes/endpoints of their model are. Within 6 hours of LPS injection, there is a stark elevation of liver inflammation and damage, which is exacerbated by a High Fat/CholineDeficient diet (HFCD). And after 1 day, almost all of the mice die. Using these endpoints, the authors were able to identify which cells were critical for mortality in the model and the specific mediators involved.

Weaknesses:

A few key details regarding the experimental design and interpretation are missing.

Most important is the choice of a high-fat diet with choline deficiency. I believe this model was chosen because the experiments are shorter and typically result in a liver inflammatory phenotype with not as clear of an adipose/obesity phenotype. I actually think it is typically considered a NASH (Non-alcoholic Steatohepatitis) model. I don't think the manuscript includes any data regarding the physiology of these mice that you would expect in an obesity model: body weight, liver weight, blood glucose, etc.

You should include a description in the methods for how the survival studies were conducted. Were the mice just checked on once a day for death, or were there other endpoints for euthanasia, like severe weight loss?

The measurement of IFNG and TNF in tissue throughout the manuscript seemed inconsistent. For example, IFNG in Figure 3A is 0.05pg/g for Chow+LPS, and 0.15pg.g for HFCD+LPS. But in Figure 4H, Chow+LPS is 0.18pg/g and HFCD+LPS is 0.18pg/g, so there is no effect of HFCD in the IgG controls. Also, in Figure 4I and 4J, the TNF values are dramatically different for the controls (0.1 vs 1pg/g).

You can't conclude that CD4+ and CD8+ T cells or monocytes don't play a role in liver damage from your data, because you did not measure liver damage, only mortality. I understand using mortality as an endpoint, but without ALT/AST measurements or histology, it's hard to say what exactly happened in the livers.

I'm not sure the authors can conclude that neutrophils expressing PD-L1 live longer in the hepatic environment from an in vitro experiment. I think this is an interesting result in terms of crosstalk between these two cell types, but I'm not sure that in vivo the neutrophils would live longer.

Reviewer #2 (Public review):

Summary:

This study presents a valuable finding that the activation of TFEB by sulforaphane (SFN) could promote lysosomal exocytosis and biogenesis in NPC, suggesting a potential mechanism by SFN for the removal of cholesterol accumulation, which may contribute to the development of new therapeutic approaches for NPC treatment.

Strengths:

The cell-based assays are convincing, utilizing appropriate and validated methodologies to support the conclusion that SFN facilitates the removal of lysosomal cholesterol via TFEB activation.

Weaknesses:

(1) The in vivo experiments demonstrate the therapeutic potential of SFN for NPC. A clear dose-response analysis would further strengthen the proposed therapeutic mechanism of SFN. Additional data supporting the activation of TFEB by SFN for cholesterol clearance in vivo would strengthen the overall impact of the study

(2) In Figure 4, the authors demonstrate increased lysosomal exocytosis and biogenesis by SFN in NPC cells. Including a TFEB-KO/KD in this assay would provide additional validation of whether these effects are TFEB-dependent.

(3) For lysosomal pH measurement, the combination of pHrodo-dex and CF-dex enables ratiometric pH measurement. However, the pKa of pHrodo red-dex (according to Invitrogen) is ~6.8, while lysosomal pH is typically around 4.7. This discrepancy may account for the lack of observed lysosomal pH changes between WT and U18666A-treated cells. Notably, previous studies (PMID: 28742019) have reported an increase in lysosomal pH in U18666A-treated cells.

(4) The authors are also encouraged to perform colocalization studies between CF-dex and a lysosomal marker, as some researchers may be concerned that NPC1 deficiency could reduce or block the trafficking of dextran along endocytosis.

(5) In vivo data supporting the activation of TFEB by SFN for cholesterol clearance would significantly enhance the impact of the study. For example, measuring whole-animal or brain cholesterol levels would provide stronger evidence of SFN's therapeutic potential.

Reviewer #3 (Public review):

The manuscript by Goyal et al report substrate-bound and substrate-free structures of a tripartite ATP independent periplasmic (TRAP) transporter from a previously uncharacterized homolog, F. nucleatum. This is one of most mechanistically fascinating transporter families, by means of its QM domain (the domain reported in his manuscript) operating as a monomeric 'elevator', and its P domain functioning as a substrate-binding 'operator' that is required to deliver the substrate to the QM domain; together, this is termed an 'elevator with an operator' mechanism. Remarkably, previous structures had not demonstrated the substrate Neu5Ac bound. In addition, they confirm the previously reported Na+ binding sites, and report a new metal binding site in the transporter, which seems to be mechanistically relevant. Finally, they mutate the substrate binding site and use proteoliposomal uptake assays to show the mechanistic relevance of the proposed substrate binding residues.

Strengths:

The structures are of good quality, the presentation of the structural data has improved, the functional data is robust, the text is well-written, and the authors are appropriately careful with their interpretations. Determination of a substrate bound structure is an important achievement and fills an important gap in the 'elevator with an operator' mechanism.

Weaknesses:

Although the possibility of the third metal site is compelling, I do not feel it is appropriate to model in a publicly deposited PDB structure without directly confirming experimentally. The authors do not extensively test the binding sites due to technical limitations of producing relevant mutants; however, their model is consistent with genetic assays of previously characterized orthologs, which will be of benefit to the field. Finally, some clarifications of EM processing would be useful to readers, and it would be nice to have a figure visualizing the unmodeled lipid densities - this would be important to contextualize to their proposed mechanism.

Reviewer #2 (Public review):

Summary:

In this study, the authors aimed to identify if and how magnesium affects the ability of two particular bacteria species to resist the action of antibiotics. In my view, the authors succeeded in their goals and present a compelling study that will have important implications for the antibiotic resistance research community. Since metals like magnesium are present in all lab media compositions and are present in the host, the data presented in this study certainly will inspire additional research by the community. These could include research into whether other types of metals also induce multi-drug resistance, whether this phenomenon can be observed in other bacterial species, especially pathogenic species that cause clinical disease, and whether the underlying molecular determinants (i.e. enzymes) of metal-induced phenotypic resistance could be new antimicrobial drug targets themselves.

Strengths:

This study's strengths include that the authors used a variety of methodologies, all of which point to a clear effect of exogenous Mg2+ on drug resistance in the targeted species. I also comment the authors for carrying out a comprehensive study, spanning evaluation of whole cell phenotypes, metabolic pathways, genetic manipulation, to enzyme activity level evaluation. The fact that the authors uncovered a molecular mechanism underlying Mg2+-induced phenotypic resistance is particularly important as the key proteins should be studied further.

Weaknesses:

I thank the authors for improving their manuscript based on my previous suggestions. I still believe the Results section is long and bogs down at times.

In general, the conclusions drawn by the authors are justified by the data, except for the interpretation of some experiments. Importantly, this paper has discovered new antimicrobial resistance mechanisms and has also pointed to potential new targets for antimicrobials.

Comments on revisions:

I just wanted to thank the authors for addressing most of my previous comments.

Reviewer #2 (Public review):

Summary:

Homan et al. examined the effect of macrophage- or Kupffer cell-specific C3aR1 KO on MASLD/MASH-related metabolic or liver phenotypes.

Strengths:

Established macrophage- or Kupffer cell-specific C3aR1 KO mice, and showing comparable liver metabolic phenotypes between WT and macrophage-specific C3aR1KO mice in response to normal chow diet or MASH diet feeding.

Weaknesses:

Insufficient data showing the effects of C3aR1KO on liver macrophage phenotypes, such as hepatic macrophage profiles, macrophage activation status, etc, which are important for the development of liver steatosis and fibrosis.

Reviewer #3 (Public review):

This study explores the use of an adenine base editing strategy to knock down PTBP1 in astrocytes and neurons of a Parkinson's disease mouse model, as a potential AAV-BE therapy. The results indicate that editing Ptbp1 in neurons, but not astrocytes, leads to the formation of tyrosine hydroxylase (TH)+ cells, rescuing some motor symptoms.

Several aspects of the manuscript stand out positively. Firstly, the clarity of the presentation. The authors communicate their ideas and findings in a clear and understandable manner, making it easier for readers to follow.

The Materials and Methods section is well-elaborated, providing sufficient detail for reproducibility.

The logical flow of the manuscript makes sense, with each section building upon the previous one coherently.

The ABE strategy employed by the authors appears sound, and the manuscript presents a coherent and well-supported argument.

Positively, some of the data in this study effectively counteracts previous work in line with more recent publications, demonstrating the authors' ability to contribute to the ongoing conversation in the field.

Comments on revisions:

The authors have adequately addressed all the previous questions and suggestions by providing new data and/or adding necessary clarifications and deeper discussions. The newly presented data convincingly fills the gaps identified during the initial review process. The additional discussions and clarifications enhance both the clarity and transparency of the manuscript.

Reviewer #3 (Public review):

Summary:

Zhou and colleagues elegantly used pre-clinical mouse models to understand the nature of abnormally high GnRH/LH pulse secretion in polycystic ovary syndrome (PCOS), a major endocrine disorder affecting female fertility worldwide. This work brings a fundamental question of how altered gonadotropin secretion takes place upstream within the GnRH pulse generator core, which is defined by arcuate nucleus kisspeptin neurons.

Strengths:

Authors use state-of-the-art in vivo calcium imaging with fiber photometry and important physiological manipulations and measurements to dissect the possible neuronal mechanisms underlying such neuroendocrine derangements in PCOS. The additional use of unsupervised k-means clustering analysis for the evaluation of calcium synchronous events greatly enhances the quality of their evidence. The authors nicely propose that neuroendocrine dysfunction in PCOS might involve different setpoints through the hypothalamic-pituitary-gonadal (HPG) axis, and beyond kisspeptin neurons, which importantly pushes our field forward toward future investigations.

Weaknesses:

The reviewer agrees that the authors provide important evidence and have improved the quality of the manuscript following first-round revisions. However, they seem resistant to show frequency and amplitude averages in Figure 1 or as supplemental data. Whether the amplitude is dependent on fiber position and its influences on the analysis should be a point of discussion and not data omission. A more detailed analysis of frequency data would enhance the quality of their manuscript.

Comments on revised version:

This comment is related to Reviewer 3's comment # 2 (major) response:

The response does not justify why authors could simply show frequency and amplitude averages in Figure 1 or as supplemental data. Whether the amplitude is dependent on fiber position and its influences on the analysis should be a point of discussion and not data omission.

Reviewer #2 (Public Review):

Summary:

The goal of this paper is to present a new method, termed MINT, for decoding behavioral states from neural spiking data. MINT is a statistical method which, in addition to outputting a decoded behavioral state, also provides soft information regarding the likelihood of that behavioral state based on the neural data. The innovation in this approach is neural states are assumed to come from sparsely distributed neural trajectories with low tangling, meaning that neural trajectories (time sequences of neural states) are sparse in the high-dimensional space of neural spiking activity and that two dissimilar neural trajectories tend to correspond to dissimilar behavioral trajectories. The authors support these assumptions through analysis of previously collected data, and then validate the performance of their method by comparing it to a suite of alternative approaches. The authors attribute the typically improved decoding performance by MINT to its assumptions being more faithfully aligned to the properties of neural spiking data relative to assumptions made by the alternatives.

Strengths:

The paper did an excellent job critically evaluating common assumptions made by neural analytical methods, such as neural state being low-dimensional relative to the number of recorded neurons. The authors made strong arguments, supported by evidence and literature, for potentially high-dimensional neural states and thus the need for approaches that do not rely on an assumption of low dimensionality.

The paper was thorough in considering multiple datasets across a variety of behaviors, as well as existing decoding methods, to benchmark the MINT approach. This provided a valuable comparison to validate the method. The authors also provided nice intuition regarding why MINT may offer performance improvement in some cases and in which instances MINT may not perform as well.

In addition to providing a philosophical discussion as to the advantages of MINT and benchmarking against alternatives, the authors also provided a detailed description of practical considerations. This included training time, amount of training data, robustness to data loss or changes in the data, and interpretability. These considerations not only provided objective evaluation of practical aspects but also provided insights to the flexibility and robustness of the method as they relate back to the underlying assumptions and construction of the approach.

Impact:

This work is motivated by brain-computer interfaces applications, which it will surely impact in terms of neural decoder design. However, this work is also broadly impactful for neuroscientific analysis to relate neural spiking activity to observable behavioral features. Thus, MINT will likely impact neuroscience research generally. The methods are made publicly available, and the datasets used are all in public repositories, which facilitates adoption and validation of this method within the greater scientific community.

Reviewer #3 (Public review):

Summary:

Asabuki and Clopath study stochastic sequence learning in recurrent networks of Poisson spiking neurons that obey Dale's law. Inspired by previous modeling studies, they introduce two distinct learning rules, to adapt excitatory-to-excitatory and inhibitory-to-excitatory synaptic connections. Through a series of computer experiments, the authors demonstrate that their networks can learn to generate stochastic sequential patterns, where states correspond to non-overlapping sets of neurons (cell assemblies) and the state-transition conditional probabilities are first-order Markov, i.e., the transition to a given next state only depends on the current state. Finally, the authors use their model to reproduce certain experimental songbird data involving highly-predictable and highly-uncertain transitions between song syllables. While the findings are only moderately surprising, this is a well-written and welcome detailed study that may be of interest to experts of plasticity and learning in recurrent neural networks that respect Dale's law.

Strengths:

This is an easy-to-follow, well-written paper, whose results are likely easy to reproduce. The experiments are clear and well-explained. In particular, the study of the interplay between excitation and inhibition (and their different plasticity rules) is a highlight of the study. The study of songbird experimental data is another good feature of this paper; finches are classical model animals for understanding sequence learning in the brain. I also liked the study of rapid task-switching, it's a good-to-know type of result that is not very common in sequence learning papers.

Weaknesses:

One weakness I see in this paper is the derivation of the learning rules, which is semi-heuristic. The paper studies Poisson spiking neurons, for which learning rules can be derived from a statistical objective, typically maximum likelihood, as previously done in the cited literature. The authors provide a brief section connecting the learning rules to gradient descent on objective functions, but the link is only heuristic or at least not entirely presented. The reason is that the neural network state is not fully determined by (or "clamped to") the target during learning (for instance, inhibitory neurons do not even have a target assigned). So, the (total) gradient should take into account the recurrent contributions from other neurons, and equation 13 does not appear to be complete/correct to me. Moreover, the target firing rate is a mixture of external currents with currents arising from other neurons in the recurrent network. The authors ideally should start from an actual distribution matching objective (e.g., KL divergence, and not such a squared error), so that their main claims immediately follow from the mathematical derivations. Along the same line, it would be excellent to get some additional insights on the interaction of the two distinct plasticity rules, one of the highlights of the study. This could be naturally achieved by relating their distinct rules to a common principled objective.

The other major weakness (albeit one that is clearly discussed by the authors) is that the study assumes that every excitatory neuron is directly given its target state when learning. In machine learning language, there are no 'hidden' excitatory neurons. While this assumption greatly simplifies the derivation of efficient and biologically-plausible learning rules that can be mapped to synaptic plasticity, it also limits considerably the distributions that can be learned by the network, more precisely to those that satisfy the Markov property.

Reviewer #2 (Public review):

Summary:

The paper attempts to elucidate how feral (wild) pigs cause distortion of the environment in over 54 countries of the world, particularly Australia.

The paper displays proof that over $120 billion worth of facilities were destroyed annually in the United States of America.

The authors have tried to infer that the findings of their work were important and possess a convincing strength of evidence.

Strengths:

(1) Clearly stating feral (wild) pigs as a problem in the environment.

(2) Stating how 54 countries were affected by the feral pigs.

(3) Mentioning how $120 billion was lost in the US, annually, as a result of the activities of the feral pigs.

(4) Amplifying the fact that 14 species of animals were being driven into extinction by the feral pigs.

(5) Feral pigs possessing zoonotic abilities.

(6) Feral pigs acting as reservoirs for endemic diseases like brucellosis and leptospirosis.

(7) Understanding disease patterns by the social dynamics of feral pig interactions.

(8) The use of 146 GPS-monitored feral pigs to establish their social interaction among themselves.

Weaknesses:

(1) Unclear explanation of the association of either the female or male feral pigs with each other, seasonally.

(2) The "abstract paragraph" was not justified.

(3) Typographical errors in the abstract.

Reviewer #2 (Public review):

Summary

In this extensive comparative study, Moreno-Borrallo and colleagues examine the relationships between plasma glucose levels, albumin glycation levels, diet, and life-history traits across birds. Their results confirmed the expected positive relationship between plasma blood glucose level and albumin glycation rate but also provided findings that are somewhat surprising or contradicting findings of some previous studies (relationships with lifespan, clutch mass, or diet). This is the first extensive comparative analysis of glycation rates and their relationships to plasma glucose levels and life history traits in birds that are based on data collected in a single study and measured using unified analytical methods.

Strengths

This is an emerging topic gaining momentum in evolutionary physiology, which makes this study a timely, novel, and very important contribution. The study is based on a novel data set collected by the authors from 88 bird species (67 in captivity, 21 in the wild) of 22 orders, which itself greatly contributes to the pool of available data on avian glycemia, as previous comparative studies either extracted data from various studies or a database of veterinary records of zoo animals (therefore potentially containing much more noise due to different methodologies or other unstandardised factors), or only collected data from a single order, namely Passeriformes. The data further represents the first comparative avian data set on albumin glycation obtained using a unified methodology. The authors used LC-MS to determine glycation levels, which does not have problems with specificity and sensitivity that may occur with assays used in previous studies. The data analysis is thorough, and the conclusions are mostly well-supported (but see my comments below). Overall, this is a very important study representing a substantial contribution to the emerging field of evolutionary physiology focused on the ecology and evolution of blood/plasma glucose levels and resistance to glycation.

Weaknesses

My main concern is about the interpretation of the coefficient of the relationship between glycation rate and plasma glucose, which reads as follows: "Given that plasma glucose is logarithm transformed and the estimated slope of their relationship is lower than one, this implies that birds with higher glucose levels have relatively lower albumin glycation rates for their glucose, fact that we would be referring as higher glycation resistance" (lines 318-321) and "the logarithmic nature of the relationship, suggests that species with higher plasma glucose levels exhibit relatively greater resistance to glycation" (lines 386-388). First, only plasma glucose (predictor) but not glycation level (response) is logarithm transformed, and this semi-logarithmic relationship assumed by the model means that an increase in glycation always slows down when blood glucose goes up, irrespective of the coefficient. The coefficient thus does not carry information that could be interpreted as higher (when <1) or lower (when >1) resistance to glycation (this only can be done in a log-log model, see below) because the semi-log relationship means that glycation increases by a constant amount (expressed by the coefficient of plasma glucose) for every tenfold increase in plasma glucose (for example, with glucose values 10 and 100, the model would predict glycation values 2 and 4 if the coefficient is 2, or 0.5 and 1 if the coefficient is 0.5). Second, the semi-logarithmic relationship could indeed be interpreted such that glycation rates are relatively lower in species with high plasma glucose levels. However, the semi-log relationship is assumed here a priori and forced to the model by log-transforming only glucose level, while not being tested against alternative models, such as: (i) a model with a simple linear relationship (glycation ~ glucose); or (ii) a log-log model (log(glycation) ~ log(glucose)) assuming power function relationship (glycation = a * glucose^b). The latter model would allow for the interpretation of the coefficient (b) as higher (when <1) or lower (when >1) resistance in glycation in species with high glucose levels as suggested by the authors.

Besides, a clear explanation of why glucose is log-transformed when included as a predictor, but not when included as a response variable, is missing.

The models in the study do not control for the sampling time (i.e., time latency between capture and blood sampling), which may be an important source of noise because blood glucose increases because of stress following the capture. Although the authors claim that "this change in glucose levels with stress is mostly driven by an increase in variation instead of an increase in average values" (ESM6, line 46), their analysis of Tomasek et al.'s (2022) data set in ESM1 using Kruskal-Wallis rank sum test shows that, compared to baseline glucose levels, stress-induced glucose levels have higher median values, not only higher variation.

Although the authors calculated the variance inflation factor (VIF) for each model, it is not clear how these were interpreted and considered. In some models, GVIF^(1/(2*Df)) is higher than 1.6, which indicates potentially important collinearity; see for example https://www.bookdown.org/rwnahhas/RMPH/mlr-collinearity.html). This is often the case for body mass or clutch mass (e.g. models of glucose or glycation based on individual measurements).

It seems that the differences between diet groups other than omnivores (the reference category in the models) were not tested and only inferred using the credible intervals from the models. However, these credible intervals relate to the comparison of each group with the reference group (Omnivore) and cannot be used for pairwise comparisons between other groups. Statistics for these contrasts should be provided instead. Based on the plot in Figure 4B, it seems possible that terrestrial carnivores differed in glycation level not only from omnivores but also from herbivores and frugivores/nectarivores.

Given that blood glucose is related to maximum lifespan, it would be interesting to also see the results of the model from Table 2 while excluding blood glucose from the predictors. This would allow for assessing if the maximum lifespan is completely independent of glycation levels. Alternatively, there might be a positive correlation mediated by blood glucose levels (based on its positive correlations with both lifespan and glycation), which would be a very interesting finding suggesting that high glycation levels do not preclude the evolution of long lifespans.

Reviewer #2 (Public review):

The aim of this study is to investigate the role of Swi6 binding to RNA in heterochromatin assembly in fission yeast. Using in vitro protein-RNA binding assays (EMSA) they showed that Swi6/HP1 binds centromere-derived siRNA (identified by Reinhardt and Bartel in 2002) via the chromodomain and hinge domains. They demonstrate that this binding is regulated by a lysine triplet in the conserved region of the Swi6 hinge domain and that wild-type Swi6 favours binding to DNA-RNA hybrids and siRNA, which then facilitates, rather than competes with, binding to H3K9me2 and to a lesser extent H3K9me3.

However, the majority of the experiments are carried out in swi6 null cells overexpressing wild-type Swi6 or Swi63K-3A mutant from a very strong promoter (nmt1). Both swi6 null cells and overexpression of Swi6 are well known to exhibit phenotypes, some of which interfere with heterochromatin assembly. This is not made clear in the text. Whilst the RNA binding experiments show that Swi6 can indeed bind RNA and that binding is decreased by Swi63K-3A mutation in vitro (confusingly, they only much later in the text explained that these 3 bands represent differential binding and that II is likely an isotherm). The gels showing these data are of poor quality and it is unclear which bands are used to calculate the Kd. RNA-seq data shows that overall fewer siRNAs are produced from regions of heterochromatin in the Swi63K-3A mutant so it is unsurprising that analysis of siRNA-associated motifs also shows lower enrichment (or indeed that they share some similarities, given that they originate from repeat regions).

The experiments are seemingly linked yet fail to substantiate their overall conclusions. For instance, the authors show that the Swi63K-3A mutant displays reduced siRNA binding in vitro (Figure 1D) and that H3K9me2 levels at heterochromatin loci are reduced in vivo (Figure 3C-D). They conclude that Swi6 siRNA binding is important for Swi6 heterochromatin localization, whilst it remains entirely possible that heterochromatin integrity is impaired by the Swi63K-3A mutation and hence fewer siRNAs are produced and available to bind. Their interpretation of the data is really confusing.

The authors go on to show that Swi63K-3A cells have impaired silencing at all regions tested and the mutant protein itself has less association with regions of heterochromatin. They perform DNA-RNA hybrid IPs and show that Swi63K-3A cells which also overexpress RNAseH/rnh1 have reduced levels of dh DNA-RNA hybrids than wild-type Swi6 cells. They interpret this to mean that Swi6 binds and protects DNA-RNA hybrids, presumably to facilitate binding to H3K9me2. The final piece of data is an EMSA assay showing that "high-affinity binding of Swi6 to a dg-dh specific RNA/DNA hybrid facilitates the binding to Me2-K9-H3 rather than competing against it." This EMSA gel shown is of very poor quality, and this casts doubt on their overall conclusion.

Unfortunately, the manuscript is generally poorly written and difficult to comprehend. The experimental setups and interpretations of the data are not fully explained, or, are explained in the wrong order leading to a lack of clarity. An example of this is the reasoning behind the use of the cid14 mutant which is not explained until the discussion of Figure 5C, but it is utilised at the outset in Figure 5A.

Another example of this lack of clarity/confusion is that the abstract states "Here we provide evidence in support of RNAi-independent recruitment of Swi6". Yet it then states "We show that...Swi6/HP1 displays a hierarchy of increasing binding affinity through its chromodomain to the siRNAs corresponding to specific dg-dh repeats, and even stronger binding to the cognate siRNA-DNA hybrids than to the siRNA precursors or general RNAs." RNAi is required to produce siRNAs, so their message is very unclear. Moreover, an entire section is titled "Heterochromatin recruitment of Swi6-HP1 depends on siRNA generation" so what is the author's message?

The data presented, whilst sound in some parts is generally overinterpreted and does not fully support the author's confusing conclusions. The authors essentially characterise an overexpressed Swi6 mutant protein with a few other experiments on the side, that do not entirely support their conclusions. They make the point several times that the KD for their binding experiments is far higher than that previously reported (Keller et al Mol Cell 2012) but unfortunately the data provided here are of an inferior quality and thus their conclusions are neither fully supported nor convincing.

Reviewer #2 (Public review):

Summary:

In this manuscript entitled "p53 isoforms have a high aggregation propensity, interact with chaperones and lack 1 binding to p53 interaction partners", the authors suggest that the p53 isoforms have high aggregation propensity and that they can co-aggregate with canonical p53 (FLp53), p63 and p73 thus exerting a dominant-negative effect.

Strengths:

Overall, the paper is interesting as it provides some characterization of most p53 isoforms DNA binding (when expressed alone), folding structure, and interaction with chaperones. The data presented support their conclusion and bring interesting mechanistic insight into how p53 isoforms may exert some of their activity or how they may be regulated when they are expressed in excess.

Weaknesses:

The main limitation of this manuscript is that the isoforms are highly over-expressed throughout the manuscript, although the authors acknowledge that the level of expression is a major factor in the aggregation phenomenon and "that aggregation will only become a problem if the expression level surpasses a certain threshold level" (lines 273-274 and results shown in Figures S3D, 6E). The p53 isoforms are physiologically expressed in most normal human cell types at relatively low levels which makes me wonder about the physiological relevance of this phenomenon.

Furthermore, it was previously reported that some isoforms clearly induce transcription of target genes which are not observed here. For example, p53β induces p21 expression (Fujita K. et al. p53 isoforms Delta133p53 and p53beta are endogenous regulators of replicative cellular senescence. Nat Cell Biol. 2009 Sep;11(9):1135-42), and Δ133p53α induces RAD51, RAD52, LIG4, SENS1 and SOD1 expression (Gong, L. et al. p53 isoform D113p53/D133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage. Cell Res. 2015, 25, 351-369. / Gong, L. et al. p53 isoform D133p53 promotes the efficiency of induced pluripotent stem cells and ensures genomic integrity during reprogramming. Sci. Rep. 2016, 6, 37281. / Horikawa, I. et al. D133p53 represses p53-inducible senescence genes and enhances the generation of human induced pluripotent stem cells. Cell Death Differ. 2017, 24, 1017-1028. / Gong, L. p53 coordinates with D133p53 isoform to promote cell survival under low-level oxidative stress. J. Mol. Cell Biol. 2016, 8, 88-90. / Joruiz et al. Distinct functions of wild-type and R273H mutant Δ133p53α differentially regulate glioblastoma aggressiveness and therapy-induced senescence. Cell Death Dis. 2024 Jun 27;15(6):454.) which demonstrates that some isoforms can induce target genes transcription and have defined normal functions (e.g. Cellular senescence or DNA repair).

However, in this manuscript, the authors conclude that isoforms are "largely unfolded and not capable of fulfilling a normal cellular function" (line 438), that they do not have "well defined physiological roles" (line 456), and that they only "have the potential to inactivate members of the p53 protein family by forming inactive hetero complexes with wtp53" (line 457-458).

Therefore, I think it is essential that the authors better discuss this major discrepancy between their study and previously published research.

Reviewer #2 (Public review):

Summary:

In this manuscript, Wang et al analyze ~17,000 transcriptomes from 35 human tissues from the GTEx database and address transcriptomic variations due to age and sex. They identified both gene expression changes as well as alternative splicing events that differ among sexes. Using breakpoint analysis, the authors find sex dimorphic shifts begin with declining sex hormone levels with males being affected more than females. This is an important pan-tissue transcriptomic study exploring age and sex-dependent changes although not the first one.

Strengths:

(1) The authors use sophisticated modeling and statistics for differential, correlational, and predictive analysis.

(2) The authors consider important variables such as genetic background, ethnicity, sampling bias, sample sizes, detected genes, etc.

(3) This is likely the first study to evaluate alternative splicing changes with age and sex at a pan-tissue scale.

(4) Sex dimorphism with age is an important topic and is thoroughly analyzed in this study.

Weaknesses:

(1) The findings have not been independently validated in a separate cohort or through experiments. Only selective splicing factor regulation has been verified in other studies.

(2) It seems the authors have not considered PMI or manner of death as a variable in their analysis.

(3) The manuscript is very dense and sometimes difficult to follow due to many different types of analyses and correlations.

(4) Short-read data can detect and quantify alternative splicing events with only moderate confidence and therefore the generalizability of these findings remains to be experimentally validated.

Reviewer #2 (Public review):

Summary:

Sox9 is a transcription factor crucial for development and tissue homeostasis, and its expression continues in various adult eye cell types, including retinal pigmented epithelium cells, Müller glial cells, and limbal and corneal basal epithelia. To investigate its functional roles in the adult eye, this study employed inducible mouse mutagenesis. Adult-specific Sox9 depletion led to severe retinal degeneration, including the loss of Müller glial cells and photoreceptors. Further, lineage tracing revealed that Sox9 is expressed in a basal limbal stem cell population that supports stem cell maintenance and homeostasis. Mosaic analysis confirmed that Sox9 is essential for the differentiation of limbal stem cells. Overall, the study highlights that Sox9 is critical for both retinal integrity and the differentiation of limbal stem cells in the adult mouse eye.

Strengths:

In general, inducible genetic approaches in the adult mouse nervous system are rare and difficult to carry out. Here, the authors employ tamoxifen-inducible mouse mutagenesis to uncover the functional roles of Sox9 in the adult mouse eye.

Careful analysis suggests that two degeneration phenotypes (mild and severe) are detected in the adult mouse eye upon tamoxifen-dependent Sox9 depletion. Phenotype severity nicely correlates with the efficiency of Cre-mediated Sox9 depletion.

Molecular marker analysis provides strong evidence of Mueller cell loss and photoreceptor degeneration.

A clever genetic tracing strategy uncovers a critical role for Sox9 in limbal stem cell differentiation.

Weaknesses:

The Introduction can be improved by explaining clearly what was previously known about Sox9 in the eye. A lot of this info is mentioned in a single, 3-page long paragraph in the Discussion. However, the current study's significance and novelty would become clearer if the authors articulated in more detail in the Introduction what was already known about Sox9 in retina cell types (in vitro and in vivo).

Because a ubiquitous tamoxifen-inducible CreER line is employed, non-cell autonomous mechanisms possibly contribute to the observed retina degeneration. There is precedence for this in the literature. For example, RPE-specific ablation of Otx2 results in photoreceptor degeneration (PMID: 23761884). Have the authors considered the possibility of non-cell autonomous effects upon ubiquitous Sox9 deletion?

Given the similar phenotypes between animals lacking Otx2 and Sox9 in specific cell types of the eye, the authors are encouraged to evaluate Otx2 expression in the tamoxifen-induced Sox9 adult retina.

The most parsimonious explanation for the dual role of Sox9 in retinal cell types and limbal stem cells is that the cell context is different. For example, Sox9 may cooperate with TF1 in photoreceptors, TF2, in Mueller cells, and TF3 in limbal stem cells, and such cell type-specific cooperation may result in different outcomes (retinal integrity, stem cell differentiation). The authors are encouraged to add a paragraph to the discussion and share their thoughts on the dual role of Sox9.

One more molecular marker for Mueller glial cells would strengthen the conclusion that these cells are lost upon Sox9 deletion.

Using opsins as markers, the authors conclude that the photoreceptors are lost upon Sox9 deletion. However, an alternate possibility is that the photoreceptors are still present and that Sox9 is required for the transcription of opsin genes. In that case, Sox9 (like Otx2) may act as a terminal selector in photoreceptor cells. This point is particularly important because vertebrate terminal selectors (e.g., Nurr1, Otx2, Brn3a) initially affect neuron type identity and eventually lead to cell loss.

Quantification is needed for the TUNEL and GFAP analysis in Figure 3.

Line 269-320: The authors examined available scRNA-Seq data on adult retina. This data provides evidence for Sox9 expression in distinct cell types. However, the dataset does not inform about the functional role of Sox9 because Sox9 mutant cells were not analyzed with RNA-Seq. Hence, all the data that claim that this experiment provides insights into possible Sox9 functional roles must be removed. This includes panels F, G, and H in Figure 5. In general, this section of the paper (Lines 269-320) needs a major revision. Similarly, lines 442-446 in the Discussion should be removed.

Reviewer #2 (Public review):

This manuscript addresses an interesting and important question: the basic mechanisms underlying subthreshold intrinsic oscillations in the inferior olive. Instead of a direct investigation of the questions, the authors decide to study subthreshold oscillations in the C-elegance, where the connectivity pattern is known but does not exhibit sub-threshold oscillations. Furthermore, instead of the common description of gap-junction coupling by resistors, the authors decide to represent the system as a tight-binding Anderson Hamiltonian.

Weaknesses:

The authors study an architecture of the C-elegance instead of that of the inferior olive of mammals because the architecture of C-elegance is known.

No subthreshold oscillations were identified in the C-elegance.<br /> Instead of representing electrical coupling via resistors that connect neurons, the authors use a quantum formalism and introduce the tight-binding Anderson Hamiltonian. Why?

Equally spaced two virtual nodes were added between cells connected by a gap junction. Why?

Comments on revised version:

Last time, I recommended that the authors should represent electrical coupling via resistors that connect neurons instead of via the quantum formalism. The authors have not tested this direction.

Reviewer #2 (Public review):

Summary:

The authors did not find an increased representation of CS+ throughout reinforcement learning in the tuft dendrites of Rbp4-positive neurons from layer 5B of the barrel cortex, as previously reported for soma from layer 2/3 of the visual cortex.<br /> Alternatively, the authors observed an increased selectivity to both stimuli (CS+ and CS-) during reinforcement learning. This feature 1) was not present in repeated exposures (without reinforcement), 2) was not explained by animal's behaviour (choice, licking and whisking) and 3) was long-lasting, being present even when the mice disengaged from the task.<br /> Importantly, increased selectivity was correlated with learning (% correct choices), and neural discriminability between stimuli increased with learning.

In conclusion, the authors show that tuft dendrites from layer 5B of the barrel cortex increase the representation of conditioned (CS+) and unconditioned stimuli (CS-) applied to the whiskers, during reinforcement learning.

Strengths:<br /> The results presented are very consistent throughout the entire study, and therefore very convincing:

(1) The results observed are very similar using two different imaging techniques (using 2-photon -planar imaging- and SCAPE - volumetric imaging). Fig. 3 and Fig.4 respectively.<br /> (2) The results are similar using "different groups" of tuft dendrites for the analysis (e.g. initially unresponsive and responsive pre- and post-learning). Fig. 5.<br /> (3) The results are similar from a specific set of trials (with the same sensory input, but different choices). Fig.7.<br /> (4) Additionally, the selectivity of tuft dendrites from layer 5B of the barrel cortex was higher in the mice that exclusively used the whisker to respond to the stimuli (CS+ and CS-).

The results presented are controlled against a group of mice that received the same stimuli presentation, except the reinforcement (reward).

Additionally, the behaviour outputs, such as choice, whisking and licking could not account for the results observed.

Although there are no causal experiments, the correlation between selectivity and learning (% of correct choices), as well as the increased neural discriminability with learning, but not in repeated exposure, are very convincing.

Weaknesses:

The biggest weakness is the absence of causality experiments. Although inhibiting specifically tuft dendritic activity in layer 1 from layer 5 pyramidal neurons is very challenging, tuft dendritic activity in layer 1 could be silenced through optogenetic experiments as in Abs et al. 2018. By manipulating NDNF-positive neurons the authors could specifically modify tuft dendritic activity in the barrel cortex during CS presentations, and test if silencing tuft dendritic activity in layer 1 would lead to the lack of selectivity and an impairment of reinforcement learning. Additionally, this experiment will test if the selectivity observed during reinforcement learning is due to changes in the local network, namely changes in local synaptic connectivity, or solely due to changes in the long-range inputs.

Reviewer #2 (Public review):

Summary:

Bos and colleagues address the important question of how two major inhibitory interneuron classes in the neocortex differentially affect cortical dynamics. They address this question by studying Wilson-Cowan-type mathematical models. Using a linearized fixed point approach, they provide convincing evidence that the existence of multiple interneuron classes can explain the counterintuitive finding that inhibitory modulation can increase the gain of the excitatory cell population while also increasing the stability of the circuit's state to minor perturbations. This effect depends on the connection strengths within their circuit model, providing valuable guidance as to when and why it arises.

Overall, I find this study to have substantial merit. I have some suggestions on how to improve the clarity and completeness of the paper.

Strengths:

(1) The thorough investigation of how changes in the connectivity structure affect the gain-stability relationship is a major strength of this work. It provides an opportunity to understand when and why gain and stability will or will not both increase together. It also provides a nice bridge to the experimental literature, where different gain-stability relationships are reported from different studies.

(2) The simplified and abstracted mathematical model has the benefit of facilitating our understanding of this puzzling phenomenon. (I have some suggestions for how the authors could push this understanding further.) It is not easy to find the right balance between biologically-detailed models vs simple but mathematically tractable ones, and I think the authors struck an excellent balance in this study.

Weaknesses:

(1) The fixed-point analysis has potentially substantial limitations for understanding cortical computations away from the steady-state. I think the authors should have emphasized this limitation more strongly and possibly included some additional analyses to show that their conclusions extend to the chaotic dynamical regimes in which cortical circuits often live.

(2) The authors could have discussed -- even somewhat speculatively -- how VIP interneurons fit into this picture. Their absence from this modelling framework stands out as a missed opportunity.

(3) The analysis is limited to paths within this simple E, PV, SOM circuit. This misses more extended paths (like thalamocortical loops) that involve interactions between multiple brain areas. Including those paths in the expansion in Eqs. 11-14 (Fig. 1C) may be an important consideration.

Comments on revisions:

I think the authors have done a reasonable job of responding to my critiques, and the paper is in pretty good shape. (Also, thanks for correctly inferring that I meant VIP interneurons when I had written SST in my review! I have updated the public review accordingly.)

I still think this line of research would benefit substantially from considering dynamic regimes including chaotic ones. I strongly encourage the authors to consider such an extension in future work.

Reviewer #2 (Public review):

Summary:

Madrigal et al identified Tom1, a E3 ubiquitin ligase previously known to be involved in ribosome biogenesis, as a protein that binds to terminal tandem Src-homology 2 (tSH2) domain of Spt6. They mapped this interaction to the acid region of Tom1, which is also known to interact with histones. Cells with tom1 mutants that cannot bind Spt6 did not show temperature sensitive phenotypes displaced for tom1 null mutant. Using ChIP assays, they showed that Tom1 is enriched at gene bodies of highly transcribed genes, and a loss of tom1 leads to reduced nucleosomal changes at gene promoters. Finally, they also solved the structures of Tom1 lacking the acidic region and found that Tom1p can adopt a compact a-solenoidal "basket" similar to the previously described structure of HUWE1. Overall, this is an interesting study and I have the following suggestions to improve the manuscript.

Major concerns:

(1) Promoter regions are in general nucleosome free. How does Tom1 mutant affect nucleosome-sized fragments at the promoter regions?

(2) While Tom1 antibodies may not specific, could the author perform Tom1 ChIP-seq in wild type and tom 1 null cells? This dataset may be more informative than tagged Tom1 that may not be functional.

Reviewer #2 (Public review):

Summary:

In the present study, the authors investigated the nature of attentional templates during the preparatory period of goal-directed attention. By combining the use of 'pinging' the neural activity with a visual impulse and fMRI-based multivariate decoding, the authors found that the nature of the neural representations of the prospective feature target during the preparatory period was contingent on the presence of the 'pinging' impulse. While the preparatory representations contained highly similar information content as the perceptual representations when the pinging impulse was introduced, they fundamentally differed from perceptual representations in the absence of the pinging impulse. Based on these findings, the authors proposed a dual-format mechanism in which both a "non-sensory" template and a latent "sensory" template coexisted during attentional preparation. The former actively guides activity in the preparatory state, and the latter is utilized for future stimulus processing.

Strengths:

Overall, I think this is an interesting study that introduced a novel perspective concerning the nature of neural representations during attentional processing. Methodologically, the present study combines an innovative utilization of the pinging technique in working memory studies and fMRI-based multivariate pattern analysis. The method is sound and the results are convincing. While I appreciate the conceptual elegance of the dual-format idea proposed by the authors, there are several questions that need to be addressed more thoroughly to clarify some of the potential ambiguities of the results and to increase the plausibility of the author's theory.

Weaknesses:

(1) The origin of the latent sensory-like representation. By 'pinging' the neural activity with a high-contrast, task-irrelevant visual stimulus during the preparation period, the authors identified the representation of the attentional feature target that contains the same information as perceptual representations. The authors interpreted this finding as a 'sensory-like' template is inherently hosted in a latent form in the visual system, which is revealed by the pinging impulse. However, I am not sure whether such a sensory-like template is essentially created, rather than revealed, by the pinging impulses. First, unlike the classical employment of the pinging technique in working memory studies, the (latent) representation of the memoranda during the maintenance period is undisputed because participants could not have performed well in the subsequent memory test otherwise. However, this appears not to be the case in the present study. As shown in Figure 1C, there was no significant difference in behavioral performance between the ping and the no-ping sessions (see also lines 110-125, pg. 5-6). In other words, it seems to me that the subsequent attentional task performance does not necessarily rely on the generation of such sensory-like representations in the preparatory period and that the emergence of such sensory-like representations does not facilitate subsequent attentional performance either. In such a case, one might wonder whether such sensory-like templates are really created, hosted, and eventually utilized during the attentional process. Second, because the reference orientations (i.e. 45 degrees and 135 degrees) have remained unchanged throughout the experiment, it is highly possible that participants implicitly memorized these two orientations as they completed more and more trials. In such a case, one might wonder whether the 'sensory-like' templates are essentially latent working memory representations activated by the pinging as was reported in Wolff et al. (2017), rather than a functional signature of the attentional process.

(2) The coexistence of the two types of attentional templates. The authors interpreted their findings as the outcome of a dual-format mechanism in which 'a non-sensory template' and a latent 'sensory-like' template coexist (e.g. lines 103-106, pg. 5). While I find this interpretation interesting and conceptually elegant, I am not sure whether it is appropriate to term it 'coexistence'. First, it is theoretically possible that there is only one representation in either session (i.e. a non-sensory template in the no-ping session and a sensory-like template in the ping session) in any of the brain regions considered. Second, it seems that there is no direct evidence concerning the temporal relationship between these two types of templates, provided that they commonly emerge in both sessions. Besides, due to the sluggish nature of fMRI data, it is difficult to tell whether the two types of templates temporally overlap.

(3) The representational distance. The authors used Mahalanobis distance to quantify the similarity of neural representation between different conditions. According to the authors' hypothesis, one would expect greater pattern similarity between 'attend leftward' and 'perceived leftward' in the ping session in comparison to the no-ping session. However, this appears not to be the case. As shown in Figures 3B and C, there was no major difference in Mahalanobis distance between the two sessions in either ROI and the authors did not report a significant main effect of the session in any of the ANOVAs. Besides, in all the ANOVAs, the authors reported only the statistic term corresponding to the interaction effect without showing the descriptive statistics related to the interaction effect. It is strongly advised that these descriptive statistics related to the interaction effect should be included to facilitate a more effective and intuitive understanding of their data.

Reviewer #2 (Public review):

Summary:

This study investigates the molecular function of the N-glycan-dependent endoplasmic reticulum protein quality control system (ERQC) in Cryptococcus neoformans and correlates this pathway with key features of C. neoformans virulence, especially those mediated by extracellular vesicle transport. The findings provide valuable insights into the connection between this pathway and the biogenesis of C. neoformans extracellular vesicles.

Strengths:

The strength of this study lies primarily in the careful selection of appropriate and current methodologies, which provide a solid foundation for the authors' results and conclusions across all presented data. All experiments are supported by well-designed and established controls in the study of C. neoformans, further strengthening the validity of the results and conclusions drawn from them. The study presents novel data on this important pathway in C. neoformans, establishing its connection with C. neoformans virulence. Interestingly, the findings led the authors to understand the relationship between this pathway and the transport of key fungal virulence factors via extracellular vesicles. This was demonstrated in the study, paving the way for a deeper understanding of extracellular vesicle biogenesis-a field still filled with gaps but one to which this study contributes solid data, helping to clarify aspects of this process.

Weaknesses:

I do not see significant weaknesses in this study. The experiments are well-grounded, and the results are clearly presented. I believe the only weakness is that the paper could be condensed. Sections like the discussion, for instance, are extremely lengthy, which may make reading and, consequently, understanding more challenging for many readers. Regarding the presentation of the results, while clear, the figures contain a lot of information, and I believe that some of this content could be moved to supplementary figures.

Reviewer #2 (Public review):

Summary:

Wengert et al. generated and thoroughly characterized the developmental epileptic encephalopathy phenotype of Kcnc1A421V/+ knock-in mice. The Kcnc1 gene encodes the Kv3.1 channel subunit. Analogous to the role of BK channels in excitatory neurons, Kv3 channels are important for the recurrent high-frequency discharge in interneurons by accelerating the downward hyperpolarization of the individual action potential. Various Kcnc1 mutations are associated with developmental epileptic encephalopathy, but the effect of a recurrent A421V mutation was somewhat controversial and its influence on neuronal excitability has not been fully established. In order to determine the neurological deficits and underlying disease mechanisms, the authors generated cre-dependent KI mice and characterized them using neonatal neurological examination, high-quality in vitro electrophysiology, and in vivo imaging/electrophysiology analyses. These analyses revealed excitability defects in the PV+ inhibitory neurons associated with the emergence of epilepsy and premature death. Overall, the experimental data convincingly support the conclusion.

Strengths:

The study is well-designed and conducted at high quality. The use of the Cre-dependent KI mouse is effective for maintaining the mutant mouse line with premature death phenotype, and may also minimize the drift of phenotypes which can occur due to the use of mutant mice with minor phenotype for breeding. The neonatal behavior analysis is thoroughly conducted, and the in vitro electrophysiology studies are of high quality.

Weaknesses:

While not critically influencing the conclusion of the study, there are several concerns.

In some experiments, the age of the animal in each experiment is not clearly stated. For example, the experiments in Figure 2 demonstrate impaired K+ conductance and membrane localization, but it is not clear whether they correlated with the excitability and synaptic defects shown in subsequent figures. Similarly, it is unclear how old mice the authors conducted EEG recordings, and whether non-epileptic mice are younger than those with seizures.

The trafficking defect of mutant Kv3.1 proposed in this study is based only on the fluorescence density analysis which showed a minor change in membrane/cytosol ratio. It is not very clear how the membrane component was determined (any control staining?). In addition to fluorescence imaging, an addition of biochemical analysis will make the conclusion more convincing (while it might be challenging if the Kv3.1 is expressed only in PV+ cells).

While the study focused on the superficial layer because Kv3.1 is the major channel subunit, the PV+ cells in the deeper cortical layer also express Kv3.1 (Chow et al., 1999) and they may also contribute to the hyperexcitable phenotype via negative effect on Kv3.2; the mutant Kv3.1 may also block membrane trafficking of Kv3.1/Kv3.2 heteromers in the deeper layer PV cells and reduce their excitability. Such an additional effect on Kv3.2, if present, may explain why the heterozygous A421V KI mouse shows a more severe phenotype than the Kv3.1 KO mouse (and why they are more similar to Kv3.2 KO). Analyzing the membrane excitability differences in the deep-layer PV cells may address this possibility.

In Table 1, the A421V PV+ cells show a depolarized resting membrane potential than WT by ~5 mV which seems a robust change and would influence the circuit excitability. The authors measured firing frequency after adjusting the membrane voltage to -65mV, but are the excitability differences less significant if the resting potential is not adjusted? It is also interesting that such a membrane potential difference is not detected in young adult mice (Table 2). This loss of potential compensation may be important for developmental changes in the circuit excitability. These issues can be more explicitly discussed.

Reviewer #2 (Public review):

Summary:

Protein synthesis - translation - involves repeated recognition and incorporation of amino-acyl-tRNAs by the ribosome. This process is a trade-off between the rate and accuracy of selection (for review see (Johansson et al, 2008; Wohlgemuth et al, 2011)). The ribosome does not just maximise the rate or the accuracy, it balances the two. Therefore, it is possible to select mutants that translate faster than the wt (but are sloppy) or that are very accurate (more than the wt) but translate slower. Slow translation is detrimental as it limits the rate of protein synthesis (and, therefore, growth) and hyper-accurate mutants accumulate mis-translated proteins, which is detrimental for the cell.

Bi and colleagues employ genetics, MIC measurements, reporter assays, and structural biology to characterise the role of GidB rRNA methylase in translational accuracy in Mycobacterium smegmatis.

Strengths:

The genetics and phenotypic assays are convincing and establish the biological role of the methylase. The authors use a powerful set of complementary assays that convincingly demonstrate that the loss of GidB results in mistranslation.

Weaknesses:

(1) It would be essential to provide information regarding the growth rate and, ideally, translation rates in the gidB KO and the isogenic WT. As translation balances accuracy and speed, only characterising the speed is not sufficient to understand the phenomenon.

(2) Cryo-EM analysis of vacant 70S ribosomes is not sufficient for understanding the mechanisms underlying the accuracy defects in the gidB KO. One should assemble and solve structurally near-cognate and non-cognate complexes. I believe the authors are over-interpreting the scant structural data they have. Furthermore, current representation makes it impossible to assess the resolution of the structure, especially in the areas of interest.

References:

Johansson M, Lovmar M, Ehrenberg M (2008) Rate and accuracy of bacterial protein synthesis revisited. Curr Opin Microbiol 11: 141-147<br /> Wohlgemuth I, Pohl C, Mittelstaet J, Konevega AL, Rodnina MV (2011) Evolutionary optimization of speed and accuracy of decoding on the ribosome. Philos Trans R Soc Lond B Biol Sci 366: 2979-2986.

Reviewer #2 (Public review):

The authors demonstrate that a low parenteral glucose regimen can lead to improved bacterial clearance and survival from Staph epi sepsis in newborn pigs without inducing hypoglycemia, as compared to a high glucose regimen. Using RNA-seq, metabolomic, and proteomic data, the authors conclude that this is primarily mediated by altered hepatic metabolism.

The authors have addressed the concerns raised by the reviewers in their revised manuscript and have added additional information in the results and discussion part.<br /> Please address in Fig. 3- the genes PGM2 and GCK, which the authors mention, are downregulated in SE-Low compared to SE-high, but these are actually less downregulated in the SE group compared to Control group, where the the Con-low shows even more decrease in these genes compared to Con-high. So if anything, these genes are getting upregulated by infection.

Reviewer #2 (Public review):

Summary:

Chlamydial cell division is a peculiar event, whose mechanism was mysterious for many years. C. trachomatis division was shown to be polar and involve a minimal divisome machinery composed of both homologues of divisome and elongasome components, in the absence of an homologue of the classical division organizer FtsZ. In this paper, Harpring et al., show that FtsK is required at an early stage of the chlamydial divisome formation.

Strengths:

The manuscript is well-written and the results are convincing. Quantification of divisome component localization is well performed, number of replicas and number of cells assessed are sufficient to get convincing data. The use of a CRISPRi approach to knock down some divisome components is an asset and allows a mechanistic understanding of the hierarchy of divisome components.

Weaknesses:

The authors did not analyse the role of all potential chlamydial divisome components and did not show how FtsK may initiate the positioning of the divisome. Their conclusion that FtsK initiates the assembly of the divisome is an overinterpretation and is not backed by the data. However, data show convincingly that FtsK, if perhaps not the initiator of chlamydial division, is definitely an early and essential component of the chlamydial divisome.

Reviewer #2 (Public review):

This study uses all-atom MD simulation to explore the mechanics of channel opening for the NOMPC mechanosensitive channel. Previously the authors used MD to show that external forces directed along the long axis of the protein (normal to the membrane) result in AR domain compression and channel opening. This force causes two changes to the key TRP domains adjacent to the channel gate: 1) a compressive force pushes the TRP domain along the membrane normal, while 2) a twisting torque induces a clock-wise rotation on the TRP domain helix when viewing the bottom of the channel from the cytoplasm. Here, the authors wanted to understand which of those two changes is responsible for increasing the inner pore radius, and they show that it is the torque. The simulations in Figure 2 probe this question with different forces, and we can see the pore open with parallel forces in the membrane, but not with the membrane-normal forces. I believe this result as it is reproducible, the timescales are reaching 1 microsecond, and the gate is clearly increasing diameter to about 4 Å. This seems to be the most important finding in the paper, but the impact is limited since the authors already show how forces lead to channel opening, and this is further teasing apart the forces and motions that are actually the ones that cause the opening.

Reviewer #2 (Public review):

Summary:

This is an inspired study that merges the concept of individuality with evolutionary processes to uncover a new strategy that diversifies individual behavior that is also potentially evolutionarily adaptive.

The authors use a time-resolved measurement of spontaneous, innate behavior, namely handedness or turn bias in individual, isogenic flies, across several genetic backgrounds.

They find that an individual's behavior changes over time, or drifts. This has been observed before, but what is interesting here is that by looking at multiple genotypes, the authors find the amount of drift is consistent within genotype i.e., genetically regulated, and thus not entirely stochastic. This is not in line with what is known about innate, spontaneous behaviors. Normally, fluctuations in behavior would be ascribed to a response to environmental noise. However, here, the authors go on to find what is the pattern or rule that determines the rate of change of the behavior over time within individuals. Using modeling of behavior and environment in the context of evolutionarily important timeframes such as lifespan or reproductive age, they could show when drift is favored over bet-hedging and that there is an evolutionary purpose to behavioral drift. Namely, drift diversifies behaviors across individuals of the same genotype within the timescale of lifespan, so that the genotype's chance for expressing beneficial behavior is optimally matched with potential variation of environment experienced prior to reproduction. This ultimately increases the fitness of the genotype. Because they find that behavioral drift is genetically variable, they argue it can also evolve.

Strengths:

Unlike most studies of individuality, in this study, the authors consider the impact of individuality on evolution. This is enabled by the use of multiple natural genetic backgrounds and an appropriately large number of individuals to come to the conclusions presented in the study. I thought it was really creative to study how individual behavior evolves over multiple timescales. And indeed this approach yielded interesting and important insight into individuality. Unlike most studies so far, this one highlights that behavioral individuality is not a static property of an individual, but it dynamically changes. Also, placing these findings in the evolutionary context was beneficial. The conclusion that individual drift and bet-hedging are differently favored over different timescales is, I think, a significant and exciting finding.

Overall, I think this study highlights how little we know about the fundamental, general concepts behind individuality and why behavioral individuality is an important trait. They also show that with simple but elegant behavioral experiments and appropriate modeling, we could uncover fundamental rules underlying the emergence of individual behavior. These rules may not at all be apparent using classical approaches to studying individuality, using individual variation within a single genotype or within a single timeframe.

Weaknesses:

I am unconvinced by the claim that serotonin neuron circuits regulate behavioral drift, especially because of its bidirectional effect and lack of relative results for other neuromodulators. Without testing other neuromodulators, it will remain unclear if serotonin intervention increases behavioral noise within individuals, or if any other pharmacological or genetic intervention would do the same. Another issue is that the amount of drugs that the individuals ingested was not tracked. Variable amounts can result in variable changes in behavior that are more consistent with the interpretation of environmental plasticity, rather than behavioral drift. With the current evidence presented, individual behavior may change upon serotonin perturbation, but this does not necessarily mean that it changes or regulates drift.

However, I think for the scope of this study, finding out whether serotonin regulates drift or not is less important. I understand that today there is a strong push to find molecular and circuit mechanisms of any behavior, and other peers may have asked for such experiments, perhaps even simply out of habit. Fortunately, the main conclusions derived from behavioral data across multiple genetic backgrounds and the modeling are anyway novel, interesting, and in fact more fundamental than showing if it is serotonin that does it or not.

To this point, one thing that was unclear from the methods section is whether genotypes that were tested were raised in replicate vials and how was replication accounted for in the analyses. This is a crucial point - the conclusion that genotypes have different amounts of behavioral drift cannot be drawn without showing that the difference in behavioral drift does not stem from differences in developmental environment.

Reviewer #2 (Public review):

Summary:

The authors present a study of how modulatory activity from outside the classical receptive field (cRF) differs from cRF stimulation. They study neural activity across the different layers of V1 in two anesthetized monkeys using Neuropixels probes. The monkeys are presented with drifting gratings and border-ownership tuning stimuli. They find that border-ownership tuning is organized into columns within V1, which is unexpected and exciting, and that the flow of activity from cell-to-cell (as judged by cross-correlograms between single units) is influenced by the type of visual stimulus: border-ownership tuning stimuli vs. drifting-grating stimuli.

Strengths:

The questions addressed by the study are of high interest, and the use of Neuropixels probes yields extremely high numbers of single-units and cross-correlation histograms (CCHs) which makes the results robust. The study is well-described.

Weaknesses:

The weaknesses of the study are (a) the use of anesthetized animals, which raises questions about the nature of the modulatory signal being measured and the underlying logic of why a change in visual stimulus would produce a reversal in information flow through the cortical microcircuit and (b) the choice of visual stimuli, which do not uniquely isolate feedforward from feedback influences.

(1) The modulation latency seems quite short in Figure 2C. Have the authors measured the latency of the effect in the manuscript and how it compares to the onset of the visually driven response? It would be surprising if the latency was much shorter than 70ms given previous measurements of BO and figure-ground modulation latency in V2 and V1. On the same note, it might be revealing to make laminar profiles of the modulation (i.e. preferred - non-preferred border orientation) as it develops over time. Does the modulation start in feedback recipient layers?

(2) Can the authors show the average time course of the response elicited by preferred and non-preferred border ownership stimuli across all significant neurons?

(3) The logic of assuming that cRF stimulation should produce the opposite signal flow to border-ownership tuning stimuli is worth discussing. I suspect the key difference between stimuli is that they used drifting gratings as the cRF stimulus, the movement of the stimulus continually refreshes the retinal image, leading to continuous feedforward dominance of the signals in V1. Had they used a static grating, the spiking during the sustained portion of the response might also show more influence of feedback/horizontal connections. Do the initial spikes fired in response to the border-ownership tuning stimuli show the feedforward pattern of responses? The authors state that they did not look at cross-correlations during the initial response, but if they do, do they see the feedforward-dominated pattern? The jitter CCH analysis might suffice in correcting for the response transient.

(4) The term "nCRF stimulation" is not appropriate because the CRF is stimulated by the light/dark edge.

Reviewer #2 (Public review):

Summary:

This manuscript presents an important exploration of how intrinsic and synaptic conductances affect the robustness of neural circuits. This is a well-deserved question, and overall, the manuscript is written well and has a logical progression.

The focus on intrinsic plasticity as a potentially overlooked factor in network dynamics is valuable. However, while the stomatogastric ganglion (STG) serves as a well-characterized and valuable model for studying network dynamics, its simplified structure and specific dynamics limit the generalizability of these findings to more complex systems, such as mammalian cortical microcircuits.

Strengths:<br /> Clean and simple model. Simulations are carefully carried out and parameter space is searched exhaustively.

Weaknesses:

(1) Scope and Generalizability:<br /> The study's emphasis on intrinsic conductance is timely, but with its minimalistic and unique dynamics, the STG model poses challenges when attempting to generalize findings to other neural systems. This raises questions regarding the applicability of the results to more complex circuits, especially those found in mammalian brains and those where the dynamics are not necessarily oscillating. This is even more so (as the authors mention) because synaptic conductances in this study are inhibitory, and changes to their synaptic conductances are limited (as the driving force for the current is relatively low).

(2) Challenges in Comparison:<br /> A significant challenge in the study is the comparison method used to evaluate the robustness of intrinsic versus synaptic perturbations. Perturbations to intrinsic conductances often drastically affect individual neurons' dynamics, as seen in Figure 1, where such changes result in single spikes or even the absence of spikes instead of the expected bursting behavior. This affects the input to downstream neurons, leading to circuit breakdowns. For a fair comparison, it would be essential to constrain the intrinsic perturbations so that each neuron remains within a particular functional range (e.g., maintaining a set number of spikes). This could be done by setting minimal behavioral criteria for neurons and testing how different perturbation limits impact circuit function.

(3) Comparative Metrics for Perturbation:<br /> Another notable issue lies in the evaluation metrics for intrinsic and synaptic perturbations. Synaptic perturbations are straightforward to quantify in terms of conductance, but intrinsic perturbations involve more complexity, as changes in maximal conductance result in variable, nonlinear effects depending on the gating states of ion channels. Furthermore, synaptic perturbations focus on individual conductances, while intrinsic perturbations involve multiple conductance changes simultaneously. To improve fairness in comparison, the authors could, for example, adjust the x-axis to reflect actual changes in conductance or scale the data post hoc based on the real impact of each perturbation on conductance. For example, in Figure 6, the scale of the panels of the intrinsic (e.g., g_na-bar) is x500 larger than the synaptic conductance (a row below), but the maximal conductance for sodium hits maybe for a brief moment during every spike and than most of the time it is close to null. Moreover, changing the sodium conductance over the range of 0-250 for such a nonlinear current is, in many ways, unthinkable, did you ever measure two neurons with such a difference in the sodium conductance? So, how can we tell that the ranges of the perturbations make a meaningful comparison?