Reviewer #1 (Public Review):

In recent years, these investigators have been engaged in a debate regarding the classification of the sacral parasympathetic system as "sympathetic" rather than "parasympathetic," based on shared developmental ontogeny of spinal preganglionic neurons. In this current study, these investigators conducted single-cell RNAseq analyses of four groups of autonomic neurons: paravertebral sympathetic neurons (stellate and lumbar train ganglia), prevertebral sympathetic neurons (coeliac-mesenteric ganglia), rostral parasympathetic ganglia (sphenopalatine ganglia), and the caudal pelvic ganglia (containing traditionally recognized sacral "parasympathetic cholinergic neurons," which the investigators sought to challenge in terms of nomenclature). The authors argued that the pelvic ganglionic neurons shared the expression of more genes with sympathetic ganglia, as opposed to parasympathetic ganglia. Additionally, the pelvic neurons did not express a set of genes observed in the rostral parasympathetic sphenopalatine ganglia. Based on these findings, they claimed that the sacral autonomic system should be considered sympathetic rather than parasympathetic. However, these arguments face significant challenges.

Firstly, among the P1-4 clusters of pelvic neurons, the P3 cluster predominantly represents noradrenergic sympathetic neurons, known to be present in pelvic ganglia. These neurons share gene expression patterns typically found in sympathetic neurons and lack the key cholinergic features identified in the P1, P2, and P4 clusters. Consistently, the P3 cluster of neurons is located close to sympathetic neuron clusters on the map, echoing the conventional understanding that the pelvic ganglia are mixed, containing both sympathetic and parasympathetic neurons.

Secondly, as mentioned above, the P1, P2, and P4 clusters are cholinergic neurons, expressing ChAT (and VIP). The authors claimed that these neurons shared a large set of genes expressed in sympathetic neurons (class I genes shown in Figure 1B). A closer look at the expression showed that some genes are expressed at higher levels in sympathetic neurons and in P2 cluster neurons, but much weaker in P1, P2, and P4 neurons, such as Islet1 and GATA2, and the opposite is true for SST. Another set of genes is expressed weakly across clusters, like HoxC6, HoxD4, GM30648, SHISA9, and TBX20. Since the pelvic ganglia are in a caudal body part, it is not surprising to have genes expressed in pelvic ganglia, but not in rostral sphenopalatine ganglia, and vice versa (to have genes expressed in sphenopalatine ganglia, but not in pelvic ganglia), according to well recognized rostro-caudal body patterning, such as nested expression of hox genes.

Thirdly, noradrenergic sympathetic neurons and cholinergic neurons, by virtue of expressing different neurotransmitters, could have distinct roles. It is true that some cholinergic neurons reside in the sympathetic train ganglia as well, such as those innervating the sweat gland and some vascular systems; in this sense, the pelvic ganglia share some features with sympathetic ganglia, except that the pelvic ganglia contain a much higher percentage of cholinergic neurons compared with sympathetic ganglia. It is much simpler and easier to divide the autonomic nervous system into sympathetic neurons that release noradrenaline versus parasympathetic neurons that release acetylcholine, and these two systems often act in antagonistic manners, though in some cases, these two systems can work synergistically. It also does not matter whether or not pelvic cholinergic neurons could receive inputs from thoracic-lumbar preganglionic neurons (PGNs), not just sacral PGNs; such occurrence only represents a minor revision of the anatomy. In fact, it makes much more sense to call those cholinergic neurons located in the sympathetic chain ganglia parasympathetic. Thus, from the functionality point of view, it is not justified to claim that "pelvic organs receive no parasympathetic innervation".

Reviewer #1 (Public Review):

Summary: Seizure stimuli has long been recognized to exhibit potent effects on adult neurogenesis, from depletion of the NSC pool to promoting aberrant migration of adult-born neurons. However, the identity and source of extrinsic signals is still incompletely understood. The work by Noguchi et al., demonstrates that Shh from mossy cells is a major source of Shh signaling after KA-mediated acute seizures. This work is interesting because mossy cells undergo hyperactivation during seizures, so this study provides a mechanistic link between mossy cell neuronal activity control of neurogenesis through Shh signaling. Weaknesses are that only male mice were analyzed in the seizure induction experiments and several control groups are missing for seizure induction, tamoxifen induction, and the DREADD experiment.

Strengths:

1. The study uses rigorous and specific genetic approaches (e.g., GliLacZ/+ mice; ShhEGFP-Cre/+ mice; mossy cell selective conditional Shh knockout using Crlr-Cre mice) to demonstrate Shh signaling is activated by seizures in mossy cells and contributes to aberrant neurogenesis.

2. Use of DREADDs (Crlr-Cre; hM3Dq) to show mossy cells control adult neurogenesis through Shh in an activity-dependent manner.

3. Demonstration that Shh deletion in mossy cells leads to reduction of the NSC pool uses stringent methods and analysis, including BrdU pulse-chase and co-labeling with NSC markers.

Weaknesses:

1. The analysis of Shh deletion in mossy cells and influences of aging related NSC pool decline is not well connected with the rest of the study on the expression/requirement of Shh in mossy cells to regulate seizure-induced neurogenesis. To promote cohesion, the authors should examine/discuss what happens to mossy cells during aging - it is similar or different to what happens to mossy cell neuronal activity during seizures?

2. Only male mice were analyzed in the seizure induction experiments, leaving open the possibility of sex differences since previous reports suggest sex differences in adult neurogenesis.

3. Several control groups are missing:<br /> -For seizure induction: missing vehicle (instead of no KA treatment).<br /> -For TAM induction: missing corn oil only to check leakiness and specificity of transgene.<br /> -For DREADD experiment: missing vehicle (to control for hM3 non-specific effects)

Reviewer #1 (Public Review):

In this paper, the authors evaluate the utility of brain-age-derived metrics for predicting cognitive decline by performing a 'commonality' analysis in a downstream regression that enables the different contribution of different predictors to be assessed. The main conclusion is that brain-age-derived metrics do not explain much additional variation in cognition over and above what is already explained by age. The authors propose to use a regression model trained to predict cognition ("brain-cognition") as an alternative suited to applications of cognitive decline. While this is less accurate overall than brain age, it explains more unique variance in the downstream regression.

Comments on revised version:

I thank the authors for addressing many of my concerns with this revision. However, I do not feel they have addressed them all. In particular I think the authors could do more to address the concern I raised about the instability of the regression coefficients and about providing enough detail to determine that the stacked regression models do not overfit.

In considering my responses to the authors revision, I also must say that I agree with Reviewer 3 about the limitations of the brain age and brain cognition methods conceptually. In particular that the regression model used to predict fluid cognition will by construction explain more variance in cognition than a brain age model that is trained to predict age. To be fair, these conceptual problems are more widespread than this paper alone, so I do not believe the authors should be penalised for that. However, I would recommend to make these concerns more explicit in the manuscript.

Reviewer #1 (Public Review):

Referring to previous research findings, the authors explain the connection between NINJ1 and MVs. Additional experiments and clarifications will strengthen the conclusions of this study.

Below are some comments I feel could strengthen the manuscript:

1. The authors mentioned their choice of using heterozygous NINJ1+/- mice on page 4, because of lethality and hydrocephalus. Nonetheless, there is a substantial number of references that use homozygous NINJ1-/- mice. Could there be any other specific reasons for using heterozygous mice in this study?

2. Figure S2 clearly shows the method of pyroptosis induction by flagellin. It is also necessary as a prerequisite for this paper to show the changes in flagellin-induced pyroptosis in heterozygous NINJ1+/- mice.

3. IL-1ß levels controlled by GSDMD were not affected by NINJ1 expression according to previous studies (Ref 37, 29, Nature volume 618, pages 1065-1071 (2023)). GSDMD also plays an important role in TF release in pyroptosis. Are GSDMD levels not altered in heterozygous NINJ1 +/- mice?

4. In Fig 1 F, the authors used a fibrin-specific monoclonal antibody for staining fibrin, but it's not clearly defined. There may be some problem with the quality of antibody or technical issues. Considering this, exploring alternative methods to visualize fibrin might be beneficial. Fibrin is an acidophil material, so attempting H&E staining or Movat's pentachrome staining might help for identify fibrin areas.

Reviewer #1 (Public Review):

Summary:<br /> The study conducted on mice establishes a noteworthy connection between dietary protein intake and resistance exercise impact on metabolic health and muscle development. In sedentary mice, a diet rich in protein resulted in excessive fat accumulation and compromised blood sugar regulation in comparison to a diet low in protein. Intriguingly, when mice followed the high protein diet alongside progressive resistance training, they exhibited protection against surplus fat gain, though blood glucose regulation remained impaired. The research also revealed that resistance training notably enhanced muscle hypertrophy induced by exercise, particularly in mice on the high protein diet. Although the maximum strength achieved was similar across diets, this highlights the potential synergy between high protein consumption and resistance exercise in promoting skeletal muscle growth.

Strengths:<br /> The study possesses several significant strengths. Firstly, it combines controlled dietary manipulations with resistance exercise, providing a comprehensive understanding of their combined effects on metabolic health and muscle growth. The use of mouse models, while not directly translatable to humans, offers a controlled experimental environment, enabling precise measurements and observations. Moreover, the study reveals nuanced outcomes such as the differential impact of high protein intake on adiposity and muscle hypertrophy. The emphasis on both positive and negative findings lends balance to the conclusions, enhancing the overall credibility of the study. Additionally, the clear delineation of diet-exercise interactions contributes to the broader understanding of dietary and exercise recommendations for metabolic health and muscle development.

Weaknesses:<br /> Certain limitations warrant consideration. Firstly, the study's exclusive reliance on mice might limit the generalizability of the findings to humans due to inherent physiological differences. Additionally, the absence of direct investigation into the underlying molecular mechanisms responsible for the observed outcomes leaves room for speculation. Moreover, the research's concentration on male and young mice raises questions about the applicability of these findings to female and older subjects. Lastly, the study's duration and the specific resistance exercise protocol utilized might not fully reflect long-term human scenarios, underscoring the need for further research in more diverse populations and over extended timeframes.

Reviewer #1 (Public Review):

Building on previous work from the Tansey lab, here Howard et al. characterize transcriptional and translational changes upon WIN site inhibition of WDR5 in MLL-rearranged cancer cells. They first analyze whether C16, a newer generation compound, has the same cellular effects as C6, an early generation compound. Both compounds reduce the expression of WDR5-bound RPGs in addition to the unbound RPG RPL22L1. They then investigate differential translation by ribo-seq and observe that WIN site inhibition reduces the translational RPGs and other proteins related to biomass accumulation (spliceosome, proteasome, mitochondrial ribosome). Interestingly, this reduction adds to the transcriptional changes and is not limited to RPGs whose promoters are bound by WDR5. Quantitative proteomics at two-time points confirmed the downregulation of RPGs. Interestingly, the overall effects are modest, but RPL22LA is strongly affected. Unexpectedly, most differentially abundant proteins seem to be upregulated 24 h after C6 (see below). A genetic screen showed that loss of p53 rescues the effect of C6 and C16 and helped the authors to identify pathways that can be targeted by compounds together with WIN site inhibitors in a synergistic way. Finally, the authors elucidated the underlying mechanisms and analyzed the functional relevance of the RPL22, RPL22L1, p53, and MDM4 axis.

While this work is not conceptually new, it is an important extension of the observations of Aho et al. The results are clearly described and, in my view, very meaningful overall.

Major points:<br /> 1. The authors make statements about the globality/selectivity of the responses in RNA-seq, ribo-seq, and quantitative proteomics. However, as far as I can see, none of these analyses have spike-in controls. I recommend either repeating the experiments with a spike-in control or carefully measuring transcription and translation rates upon WIN site inhibition and normalizing the omics experiments with this factor.

2. Why are the majority of proteins upregulated in the proteomics experiment after 24 h in C6 (if really true after normalization with general protein amount per cell)? This is surprising and needs further explanation.

3. The description of the two CRISPR screens (GECKO and targeted) is a bit confusing. Do I understand correctly that in the GECKO screen, the treated cells are not compared with non-treated cells of the same time point, but with a time point 0? If so, this screen is not very meaningful and perhaps should be omitted. Also, it is unclear to me what the advantages of the targeted screen are since the targets were not covered with more sgRNAs (data contradictory: 4 or 10 sgRNAs per target?) than in Gecko. Also, genome-wide screens are feasible in culture for multiple conditions. Overall, I find the presentation of the screening results not favorable.

4. Can Re-expression of RPL22 rescue the growth arrest of C6?.

Reviewer #1 (Public Review):

Summary:<br /> In the manuscript titled "Disease modeling and pharmacological rescue of autosomal dominant Retinitis Pigmentosa associated with RHO copy number variation" the authors describe the use of patient iPSC-derived retinal organoids to evaluate the pathobiology of a RHO-CNV in a family with dominant retinitis pigmentosa (RP). They find significantly increased expression of rhodopsin, especially within the photoreceptor cell body, and defects in photoreceptor cell outer segment formation/maturation. In addition, they demonstrate how an inhibitor of NR2E3 (a rod transcription factor required for inducing rhodopsin expression), can be used to rescue the disease phenotype.

Strengths:<br /> The manuscript is very well written, the illustrations and data presented are compelling, and the authors' interpretation/discussion of their findings is logical.

Weaknesses:<br /> A weakness, which the authors have addressed in the discussion section, is the lack of an isogenic control, which would allow for direct analysis of the RHO-CNV in the absence of the other genetic sequence contained within the duplicated region. As the authors suggest, CRISPR correction of a large CNV in the absence of inducing unwanted on-target editing events in patient iPSCs is often very challenging. Given that they have used a no-disease iPSC line obtained from a family member, controlled for organoid differentiation kinetics/maturation state, and that no other complete disease-causing gene is contained within the duplicated region, it is unlikely that the addition of an isogenic control would yield significantly different results.

Aims and conclusions:<br /> This reviewer is of the opinion that the authors have achieved their aims and that their results support their conclusions.

Discussion:<br /> The authors have provided adequate discussion on the utility of the methods and data as well as the impact of their work on the field.

Reviewer #1 (Public Review):

Summary:<br /> Spinal cord injury (SCI) causes immediate and prolonged bladder dysfunction, for which there are poor treatments. Following up on evidence that AMPA glutamatergic receptors play a key role in bladder function, the authors induced spinal cord injury and its attendant bladder dysfunction and examined the effects of graded doses of allosteric AMPA receptor activators (ampakines). They show that ampakines ameliorate several prominent derangements in bladder function resulting from SCI, improving voiding intervals and pressure thresholds for voiding and sphincter function.

Strengths:<br /> Well-performed studies on a relevant model system. The authors induced SCI reproducibly and showed that they had achieved their model. The drugs revealed clear and striking effects. Notably, in some mice that had such bad SCI that they could not void, the drug appeared to restore voiding function.

Weaknesses:<br /> The studies are well conducted, but it would be helpful to include information on the kinetics of the drugs used, their half-life and how long they are present in rats after administration. What blood levels of the drugs are achieved after infusion? How do these compare with blood levels achieved when these drugs are used in humans?

Reviewer #1 (Public Review):

The process of EMT is a major contributor to metastasis and chemoresistance in breast cancer. By using a modified PyMT model that allows the identification of cells undergoing EMT and their decedents via S100A4-Cre mediated recombination of the mTmG allele, Ban et al. tackle a very important question of how tumor metastasis and therapy resistance by EMT can be blocked. They identified that pathways associated with ribosome biogenesis (RiBi) are activated during transition cell states. This finding represents a promising therapeutic target to block any transition from E to M (activated during cell dissemination and invasion) as well as from M to E (activated during metastatic colonization). Inhibition of RiBi-blocked EMT also reduced the establishment of chemoresistance that is associated with an EMT phenotype. Hence, RiBi blockage together with standard chemotherapy showed synergistic effects, resulting in impaired colonization/metastatic outgrowth in an animal model. The study is of great interest and of high clinical relevance as the authors show that blocking the transition from E to M or vice versa targets both aspects of metastasis, dissemination from the primary tumor, and colonization in distant organs.

The study is done with high skill using state-of-the-art technology and the conclusions are convincing and solid, but some aspects require some additional experimental support and clarification. It remains elusive whether blocking of EMT/MET is necessary for the synergistic effect of standard chemotherapy together with RiBi blockage or whether a general growth disadvantage of RiBi-treated cells independent of blocking transition is responsible. How can specific effects on state transition by RiBI block be separated from global effects attributed to overall reduced protein biosynthesis, proliferation etc.? Some other aspects are misleading or need extension.

Reviewer #1 (Public Review):

Summary: This study presents fundamental new insights into vesicular monoamine transport and the binding pose of the clinical drug tetrabenazine (TBZ) to the mammalian VMAT2 transporter. Specifically, this study reports the first structure for the mammalian VMAT (SLC18) family of vesicular monoamine transporters. It provides insights into the mechanism by which this inhibitor traps VMAT2 into a 'dead-end' conformation. The structure also provides some evidence for a novel gating mechanism within VMAT2, which may have wider implications for understanding the mechanism of transport in the wider SLC18 family.

Strengths: The structure is high quality, and the method used to determine the structure via fusing mVenus and the anti-GFP nanobody to the amino and carboxyl termini is novel. The binding and transport data are convincing, although limited. The binding position of TBZ is of high value, given its role in treating Huntington's chorea and for being a 'dead-end' inhibitor for VMAT2.

Weaknesses: The lack of additional mutational data and/or analyses on the impact of pH on ligand binding reduces the insights from these experiments. This reduces the strength of the conclusions that can be drawn about the mechanism of binding and transport or the novelty of the gating mechanism discussed above.

Joint Public Review:

Bull et al aimed to use data from observational studies and mendelian randomisation to explore if changes in circulating metabolites are associated with colorectal cancer development. As Mendelian randomisation uses information on genetic variations which are fixed at birth, it is less vulnerable to confounding than standard observational studies.

Overall, a major strength of the study is that it uses data from large cohort studies, one from childhood, adolescence, and early adulthood when the incidence of colorectal cancer is very low (reducing the likelihood of reverse causation) and before medication (such as statins which have the potential to affect metabolite levels) has been initiated.

This study has some weaknesses which have been acknowledged by the authors. Although the findings of this study indicate the potentially significant role that polyunsaturated fatty acids may have in colorectal cancer risk, the genes and therefore also the genetic variations (SNPs) associated with fatty acids often produce an effect for more than one fatty acid which may introduce bias. This together with the fact that there was limited information available on many specific fatty acids which are known causative metabolites for colorectal cancer, makes it difficult to establish with confidence which specific classes of fatty acids could potentially play a causative role in these associations. Also, the study populations are majority white European descent which may limit the generalizability of these findings to other populations.

The methodology used was largely acceptable to achieve the aims set out and the findings have shown an association between polyunsaturated fat levels and genetic liability to colorectal cancer.<br /> Overall, this is an important piece of work which has the potential to contribute to our understanding of the causal relationship between circulating metabolites at different stages of the life cycle and colorectal cancer risk as it would be extremely difficult to gather such evidence using other study designs. It opens the door for future research aiming to better understand the role that these metabolites could play in colorectal cancer risk prediction and in turn help identify groups of individuals who would benefit most from prevention and early detection interventions.

This work will be of interest not only to epidemiologists working in the area of GI tract cancers but also those interested in the different applications for mendelian randomisation within cancer epidemiology research.

Reviewer #1 (Public Review):

Summary: The authors seek to establish what aspects of nervous system structure and function may explain behavioral differences across individual fruit flies. The behavior in question is a preference for one odor or another in a choice assay. The variables related to neural function are odor responses in olfactory receptor neurons or in the second-order projection neurons, measured via calcium imaging. A different variable related to neural structure is the density of a presynaptic protein BRP. The authors measure these variables in the same fly along with the behavioral bias in the odor assays. Then they look for correlations across flies between the structure-function data and the behavior.

Strengths: Where behavioral biases originate is a question of fundamental interest in the field. In an earlier paper (Honegger 2019) this group showed that flies do vary with regard to odor preference, and that there exists neural variation in olfactory circuits, but did not connect the two in the same animal. Here they do, which is a categorical advance, and opens the door to establishing a correlation. The authors inspect many such possible correlations. The underlying experiments reflect a great deal of work, and appear to be done carefully. The reporting is clear and transparent: All the data underlying the conclusions are shown, and associated code is available online.

Weaknesses: The results are overstated. The correlations reported here are uniformly small, and don't inspire confidence that there is any causal connection. The main problems are<br /> 1. The target effect to be explained is itself very weak. Odor preference of a given fly varies considerably across time. The systematic bias distinguishing one fly from another is small compared to the variability. Because the neural measurements are by necessity separated in time from the behavior, this noise places serious limits on any correlation between the two.<br /> 2. The correlations reported here are uniformly weak and not robust. In several of the key figures, the elimination of one or two outlier flies completely abolishes the relationship. The confidence bounds on the claimed correlations are very broad. These uncertainties propagate to undermine the eventual claims for a correspondence between neural and behavioral measures.<br /> 3. Some aspects of the statistical treatment are unusual. Typically a model is proposed for the relationship between neuronal signals and behavior, and the model predictions are correlated with the actual behavioral data. The normal practice is to train the model on part of the data and test it on another part. But here the training set at times includes the testing set, which tends to give high correlations from overfitting. Other times the testing set gives much higher correlations than the training set, and then the results from the testing set are reported. Where the authors explored many possible relationships, it is unclear whether the significance tests account for the many tested hypotheses. The main text quotes the key results without confidence limits.

Reviewer #1 (Public Review):

Erbacher and colleagues provide further evidence for the function of epithelial cells as major contributors to the transduction of sensory stimuli. This technically advanced imaging study of human skin advances support for the anatomical and functional association of nerve fibers and skin keratinocytes. With combined high-resolution imaging and immunolabeling, the authors also advance the idea that gap junctions are at least one means by which direct neurochemical (e.g., ATP) communication from stimulated keratinocytes to nerve fibers can be achieved.

A major strength of the study is the combined use of super-resolution array tomography (srAT), expansion microscopy, structured illumination microscopy and immunolabeling to analyze human skin in situ as well as co-cultures of human neurons and keratinocytes. High resolution static and video imaging of skin clearly supports the ensheathment by keratinocytes of nerve fiber projections as they traverse layers of the epidermis. Another strength of this study is the srAT imaging combined with connexin Cx43 immunolabeling that focus on sites of nerve fiber-keratinocyte contact zones. Imaging of Cx43+ plaques support these sites as regions of direct epithelial-neural contact and as such, of communication.

Although imaging data support Cx43+/connexin plaques and neural ensheathment as regions of direct epithelial-neural communication, e.g., via keratinocyte release of ATP, this relationship remains correlative and lacking in quantification.

The conclusion of this paper regarding the anatomical relationship between nerves and keratinocytes is well supported. Data also support the proposal of connexin plaques as sites of communication, although analyses that validate this relationship, using experimental models and in human samples, remain for future studies.

Reviewer #1 (Public Review):

The authors provide compelling evidence that the activation of distinct populations of NTS neurons provides stronger decreases in eating/body weight when co-activated. Avoidance is not necessarily linked to the extent of the effects but seems to depend on specific neurons which when activated, not only reduce eating but also induce avoidance reactions. The results of this study provide strong data promoting multi-targeted approaches to reduce eating and body weight in obesity. Interestingly, none of the pathways identified is necessary for the weight-reducing effect of vertical sleeve gastrectomy. Future studies will hopefully shed light on the type of neurotransmitters released by these distinct populations of NTS neurons.

Reviewer #1 (Public Review):

Retinal ganglion cells are diverse. In recent years it was recognized that several subtypes are intrinsically photoresponsive (ipRGCs). In earlier work, it was suggested that hyperpolarization-activated channels (HCN) were the main responsive element contributing to generating the photocurrents that activate signaling by these cells. Other groups, including the authors, have shown evidence that other ionic mechanisms might be in play.

In the current manuscript, the authors present a thorough and careful characterization of the electrophysiology of two types of ipRGCs, M2, and M4. Both pharmacological and genetic ablation of specific ion channels in mice were employed along with posthoc identification of cell types. The authors identify an important experimental problem with one of the drugs employed previously to suggest the participation of HCN channels. This discovery leads the authors to suggest that in M4 ipRGCs, the depolarization induced by light is produced by activation of TRPC channels and inhibition of a leak of potassium channels. Importantly, prolonged application of the HCN channel blocker produced off-target (non-HCN related) effects that can explain previous results.

The authors go on to explore the responses of M2-type ganglion cells and also uncover the important participation of TRPC channels as well as a previously unrecognized role for T-type calcium channels. Since the authors also use pharmacological tools to uncover the participation of calcium channels in M2 cells, they make sure that the drugs employed do not produce off-target effects in cells where the ionic basis of the photocurrent is better established, namely the M1 type.<br /> The author's evidence as a whole is convincing, and should be a major contribution to understanding the physiology of ipRGCs, but should be confirmed by other groups with different experimental approaches.

Reviewer #1 (Public Review):

In this study, Chi et al. present a study on ctDNA profiling to predict the prognosis and treatment response of mTNBC patients. The authors report that ctDNA+ status and baseline ctDNA-related markers (MATH score and ctDNA%) are associated with the survival and treatment response. The data are well presented. However, some questions related to the association between ctDNA and clinical outcomes, and a lack of an external cohort to validate the predictive value of ctDNA need to be addressed. The Methods section also needs to be detailed.

Reviewer #1 (Public Review):

This study set out to test the causal involvement of the OFC in detecting auditory prediction errors at two levels of abstraction. The authors recorded EEG in patients with OFC damage and healthy age matched controls while they listened for deviations in sequences of tones in the Local-Global paradigm. This task can tease apart prediction errors at a local level (ie. within a sequence) and a global level (ie. between sequences). Focusing on the Mismatch Negativity (MMN) ERP component and the P3, which have both been previously linked to detecting violations in expectation and predictions, the study examined differences between neural responses elicited by patients and control subjects in four core conditions 1. standard sequences of tones (XXXXX XXXXX XXXXX XXXXX) that can be predicted both at local and global levels and should result in no prediction errors 2. Local deviations (XXXXY XXXXY XXXXY XXXXY) in which the final tone in the last sequence can only be predicted at the global level and which results in low-level prediction errors 3. Global deviations (XXXXY XXXXY XXXXY XXXXX) in which the final tone of the last sequence can be predicted only at the local level and results in higher level prediction errors. 4. Local+Global deviations (XXXXX XXXXX XXXXX XXXXY) in which the final tone of the last sequence is neither predicted at the global or local level and results in low and high prediction errors.

The timely and well designed study combines casual and correlative experimental methods. This unique strength allows the authors to identify differences in neural processing and link them directly to a specific region of the brain. The task is simple and intuitive, having been well characterized in previous literature. Its use here to investigate prediction errors beyond the typical reward-guided paradigms is particularly novel. As is the focus on the OFC which is often understudied relative to nearby ACC more commonly associated with prediction error coding. These strengths ensure the paper will likely have a wide impact across a number of fields.

The results suggest that OFC patients showed attenuated MMN to violations in local predictions as well as reduced and delayed MMN/P3a complex to combined violations in local and global predictions. By contrast, violations of prediction purely at the global level were preserved in the OFC group. No differences in processing local or global auditory prediction errors were observed in a brain damaged control group with lesions to the LPFC relative to controls. As these results stand they show a clear role of the OFC in the detection of prediction errors. This is particularly clear at the local level of processing.

However, as with many patient lesion studies, while the comparison directly against the healthy age matched controls is critical it would have strengthened the authors claims if they could show differences between the brain damaged control group. Given the previous literature that also links lateral PFC with prediction error detection, I understand that this region is potentially not the clearest brain damaged control group and therefore another lesion group might have strengthened claims of specificity. Furthermore, the authors do not offer an explanation for why no differences between lateral PFC and control groups were found when others have previously reported them. Identifying those differences would strengthen our understanding of the involvement of different structures in this task/function.

Furthermore, I believe it is important for the authors to clarify how the time frames to test for group differences of ERP components were defined. Were the components defined based on a grand average across lesions and controls or based or on the maximum range for both groups? As the paper is written currently this is unclear to me. It is also unclear why the group comparisons between controls and lateral PFC group were based only on the control group. To ensure no inadvertent biases towards the larger control group were introduced and ensure the studies findings were reliable, it would be appreciated if the authors could clarify this.

An additional potential weakness of the paper, and one that if addressed would increase our confidence that neural differences arise because of the specific lesion effect, is the lack of evidence that the lesion and control groups do not differ on measures that could inadvertently bias the neural data. For example, while the groups did not differ on demographics and a range of broad cognitive functions, were there any differences between the number or distribution of bad/noisy channels in each subject between the two groups? Were there differences in the number of blinks/saccades or distribution of blinks or saccades across the conditions in each subject across the two groups. On a similar note, while I appreciate this is a well established task could the authors clarify whether task difficulty is balanced across the different conditions? The authors appear to have used the counting task to ensure equal attention is paid across conditions although presumably the blocks differ in the number of deviant tones and therefore in the task difficulty. Typically, tasks to maintain attention are orthogonal to the main task and equally challenging across the different blocks. Is there a way to reassure readers that this has not affected the neural results.

Finally, one remaining weakness, which plagues all patient studies, is that of anatomical specificity. The authors have analysed what is, for the field, a large group of patients, and while the lesions appear to be relatively focused on the OFC the individuals vary in the degree to which different subregions within the OFC are damaged. This is increasingly important as evidence over the last 10 years has identified functional roles of these specific structures (Rushworth et al 2011, Neuron, Rudebeck et al 2017 Neuron). It would be important to ultimately know whether the detection of prediction errors was specific to a particular OFC subregion, a general mechanism across this area of cortex, or whether different subregions were more involved during different contexts or types of stimuli/contexts/tasks etc. Some comments on this would be appreciated.

In spite of the concerns raised above I believe that the authors have achieved their aims. I hope that by expanding and clarifying the sections outlined above the authors can be even more confident that their results support their conclusions.

As noted above, given the combination of methods and generalisability of the results the study will have a significant impact in a number of fields. I believe the use of an auditory paradigm will remind the community of the value of examining the generalisability of mechanisms across other sensory domains beyond vision. Unfortunately, though the data can not easily be shared (as is typical of patient data). However the authors explain in detail how permission could be sought by individual members of the community if needed.

Finally, while the authors have already cited widely across multiple fields, again speaking to the likely large impact the study will make, there does appear to be an unexplored conceptual link between the conclusions here that the OFC supports "the formation of predictions that define the current task by using context and temporal structure to allow old rules to be disregarded so that new ones can be rapidly acquired" and that lesions of the lateral portions of the OFC disrupt the assignment of credit or value to a stimuli that occurred temporally close to the outcome (Walton et al 2010, Noonan et al 2010, PNAS, Rudebeck et al 2017 Neuron, Noonan et al 2017, JON, Wittmann et al 2023 PlosB, note the wider imaging literature in line with this work Jocham et al 2014 Neuron and Wang et al bioRxiv). Without the OFC monkeys and humans appear to rely on an alternative, global learning mechanism that spreads the reinforcing properties of the outcome to stimuli that occurred further back in time. Could the authors speculate on how these two strains of evidence might converge? For example, does the OFC only assign credit in the event of a prediction error or does one mechanism subsume another?

Reviewer #1 (Public Review):

As central molecular scaffolds, Cullin ring ubiquitin ligases proteins play critical roles in the post-translational modification of cellular proteins. Since cyclin D1 is a pivotal regulator to form the CDK4/6 complex during cell cycle progression, understanding if additional cullin-associated E3 ligases participate in the regulation of cyclin D1 protein stability is interesting. The current study used an NIH3T3 cells-based siRNA library to screen 156 cullin-associated ubiquitin ligases genes. The results indicated that cullins are required for cyclin D1 degradation, and cullin-induced cyclin D1 degradation is ubiquitin-dependent and is mediated by multiple E3 ligases (Keap1, DDB2, WSB2, and Rbx1 subunits). Overall, this is a well-designed experimental study and the quality of the data collection and analysis are high and rigorous. The manuscript is well written. The conclusion stated by the authors is supported by their data logically.

Reviewer #1 (Public Review):

This relatively small-scale cohort trial has demonstrated ideal efficacy and safety of combinatory immunotherapy, radiotherapy and chemotherapy. The study design is straightforward and the major findings are held back by solid clinical data. However, the correlation between the primary endpoint selection and long term benefit is lacking, and the current adverse events are not yet comprehensively exhibited.

Reviewer #1 (Public Review):

This manuscript provides a comprehensive investigation of the effects of the genetic ablation of three different transcription factors (Srf, Mrtfa, and Mrtfb) in the inner ear hair cells. Based on the published data, the authors hypothesized that these transcription factors may be involved in the regulation of the genes essential for building the actin-rich structures at the apex of hair cells, the mechanosensory stereocilia and their mechanical support - the cuticular plate. Indeed, the authors found that two of these transcription factors (Srf and Mrtfb) are essential for the proper formation and/or maintenance of these structures in the auditory hair cells. Surprisingly, Srf- and Mrtfb- deficient hair cells exhibited somewhat similar abnormalities in the stereocilia and in the cuticular plates even though these transcription factors have very different effects on the hair cell transcriptome. Another interesting finding of this study is that the hair cell abnormalities in Srf-deficient mice could be rescued by AAV-mediated delivery of Cnn2, one of the downstream targets of Srf. However, despite a rather comprehensive assessment of the novel mouse models, the authors do not have yet any experimentally testable mechanistic model of how exactly Srf and Mrtfb contribute to the formation of actin cytoskeleton in the hair cells. The lack of any specific working model linking Srf and/or Mrtfb with stereocilia formation decreases the potential impact of this study.

Major comments:

Figures 1 & 3: The conclusion on abnormalities in the actin meshwork of the cuticular plate was based largely on the comparison of the intensities of phalloidin staining in separate samples from different groups. In general, any comparison of the intensity of fluorescence between different samples is unreliable, no matter how carefully one could try matching sample preparation and imaging conditions. In this case, two other techniques would be more convincing: 1) quantification of the volume of the cuticular plates from fluorescent images; and 2) direct examination of the cuticular plates by transmission electron microscopy (TEM).

In fact, the manuscript provides no single TEM image of the F-actin abnormalities either in the cuticular plate or in the stereocilia, even though these abnormalities seem to be the major focus of the study. Overall, it is still unclear what exactly Srf or Mrtfb deficiencies do with F-actin in the hair cells.

Figures 2 & 4 represent another example of how deceiving could be a simple comparison of the intensity of fluorescence between the genotypes. It is not clear whether the reduced immunofluorescence of the investigated molecules (ESPN1, EPS8, GNAI3, or FSCN2) results from their mis-localization or represents a simple consequence of the fact that a thinner stereocilium would always have a smaller signal of the protein of interest, even though the ratio of this protein to the number of actin filaments remains unchanged. According to my examination of the representative images of these figures, loss of Srf produces mis-localization of the investigated proteins and irregular labeling in different stereocilia of the same bundle, while loss of Mrtfb does not. Obviously, a simple quantification of the intensity of fluorescence conceals these important differences.

Reviewer #1 (Public Review):

The work by Yijun Zhang and Zhimin He at al. analyzes the role of HDAC3 within DC subsets. Using an inducible ERT2-cre mouse model they observe the dependency of pDCs but not cDCs on HDAC3. The requirement of this histone modifier appears to be early during development around the CLP stage. Tamoxifen treated mice lack almost all pDCs besides lymphoid progenitors. Through bulk RNA seq experiment the authors identify multiple DC specific target gens within the remaining pDCs and further using Cut and Tag technology they validate some of the identified targets of HDAC3.<br /> Collectively the study is well executed and shows the requirement of HDAC3 on pDCs but not cDCs, in line with the recent findings of a lymphoid origin of pDC.

While the authors provide extensive data on the requirement of HDAC3 within progenitors, the high expression of HDAC3 in mature pDCs may underly a functional requirement. Have you tested INF production in CD11c cre pDCs? Are there transcriptional differences between pDCs from HDAC CD11c cre and WT mice?

A more detailed characterization of the progenitor compartment that is compromised following depletion would be important, as also suggested in the specific points.

Reviewer #1 (Public Review):

This is a very exciting manuscript from Meng Wang's lab on lysosomal proteomics. They used several different protein tags to identify the lysosomal proteome. The exciting findings include A) specific lysosomal proteins exist in a tissue-specific manner B) lipl-4 overexpression and daf-2 extend life span using different mechanisms C) identification of novel lysosomal proteins D) demonstration of the function of several lysosomal proteins in regulation lysosome abundance and function.

Joint Public Review:

Summary:

This concise review provides a clear and instructive picture of the state-of-the-art understanding of protein kinases' activity and sets of approaches and tools to analyse and regulate it.

Strengths:

Three major parts of the work include: methods to map allosteric communications, tools to control allostery, and allosteric regulation of protein kinases. The work provides an important and timely view of the current status of our understanding of the function of protein kinases and state-of-the-art methods to study its allosteric regulation and to develop allosteric approaches to control it.

Weaknesses:

The authors may wish to consider first discussing the allosteric regulation of kinases, which can be further considered from the perspective of computational approaches to map and experimental methods to control it.

Reviewer #1 (Public Review):

Summary: The goal of this project is to test the hypothesis that individual differences in experience with multiple languages relate to differences in brain structure, specifically in the transverse temporal gyrus. The approach used here is to focus specifically on the phonological inventories of these languages, looking at the overall size of the phonological inventory as well as the acoustic and articulatory diversity of the cumulative phonological inventory in people who speak one or more languages. The authors find that the thickness of the transverse temporal gyrus (either the primary TTG, in those with one TTG, or in the second TTG, in people with multiple gyri) was related to language experience, and that accounting for the phonological diversity of those languages improved the model fit. Taken together, the evidence suggests that learning more phonemes (which is more likely if one speaks more than one language) leads to experience-related plasticity in brain regions implicated in early auditory processing.

Strengths: This project is rigorous in its approach--not only using a large sample, but replicating the primary finding in a smaller, independent sample. Language diversity is difficult to quantify, and likely to be qualitatively and quantitatively distinct across different populations, and the authors use a custom measure of multilingualism (accounting for both number of languages as well as age of acquisition) and three measures of phonological diversity. The team has been careful in discussion of these findings, and while it is possible that pre-existing differences in brain structure could lead to an aptitude difference which could drive one to learn more than one language, the fine-grained relationships with phonological diversity seem less likely to emerge from aptitude rather than experience.

Weaknesses: It is a bit unclear how the measures of phonological diversity relate to one another--they are partially separable, but rest on the same underlying data (the phonemes in each language). It would be helpful for the reader to understand how these measures are distributed (perhaps in a new figure), and the degree to which they are correlated with one another. Further, as the authors acknowledge, it is always possible that an unseen factor instead drives these findings--if typological lexical distance measures are available, it would be helpful to enter these into the model to confirm that phonological factors are the specific driver of TTG differences and not language diversity in a more general sense. That said, the relationship between phonological diversity and TTG structure is intuitive.

One curious aspect of this paper relates to the much higher prevalence of split or duplicate TTG in the sample. The authors do a good job speculating on how features of the TASH package might lead to this, but it is unclear where the ground truth lies--some discussion of validation of TASH against a gold standard would be useful.

Reviewer #1 (Public Review):

The authors demonstrate that reactivation of mild vs strong aversive contextual associations produces dissociable effects on fos expression across a wide network of relevant brain regions. Mild, 2-shock memory recruits a 'small-world' network in which amygdalar regions are functionally connected to other regions that modulate their activity and behavioral output, whereas strong, 10-shock memory isolates amygdalar nuclei from the rest of the network. These different patterns of correlated neural activity correspond with functional/behavioral differences - the authors confirm that weak, 2-shock memory is more effectively extinguished and is susceptible to reconsolidation relative to strong, 10-shock memory.

One major drawback of the manuscript is the fact that the data were collected from male subjects only. One might expect similar behavioral outcomes from male and female rats receiving 2-shock and 10-shock training. However, increasing attention to sex as a biological variable has revealed an interesting truth, namely that males and females can engage distinct neural pathways to arrive at the same behavioral destination. It should not be taken for granted that retrieval of aversive contextual associations would reproduce the same networks in females, and, as such, the manuscript does not give a complete accounting of the phenomenon under study.

Reviewer #1 (Public Review):

Bolumar et al. isolated and characterized EV subpopulations, apoptotic bodies (AB), Microvesicles (MV), and Exosomes (EXO), from endometrial fluid through the female menstrual cycle. By performing DNA sequencing, they found the MVs contain more specific DNA sequences than other EVs, and specifically, more mtDNA were encapsulated in MVs. They also found a reduction of mtDNA content in the human endometrium at the receptive and post-receptive period that is associated with an increase in mitophagy activity in the cells, and a higher mtDNA content in the secreted MVs was found at the same time. Last, they demonstrated that the endometrial Ishikawa cell-derived EVs could be taken by the mouse embryos and resulted in altered embryo metabolism.

This is a very interesting study and is the first one demonstrating the direct transmission of maternal mtDNA to embryos through EVs.

Reviewer #1 (Public Review):

Summary:<br /> Previously, researchers targeting certain brain areas in mice have relied on manual reconstruction of 3D trajectories based on published atlases of 2D sections in standardized anatomical planes. Over a decade ago, Leica's AngleTwo software provided an early proprietary software interface to rodent atlases based on 2D graphics. However, the more recent advent of open-source 3D gaming engines and CAD software (here the authors used Unity) and the adoption of a common 3D atlas framework (the Common Coordinate Framework, or CCF, from the Allen Institute) by the neuroscience community have enabled more advanced targeting based on 3D anatomy, as primate researchers and human clinicians have done previously with MRI data using bespoke and commercial software solutions. The Neuropixels Trajectory Explorer (https://github.com/petersaj/neuropixels_trajectory_explorer, by Andy Peters) pioneered a software interface to the 3D mouse atlas for electrode insertions, and here Birman et al. have built on the aforementioned previous efforts to provide the most comprehensive trajectory planning software in mice to date, which they call Pinpoint. The most critical improvement lies in the ability to model the experimental rig and instruments in the same 3D environment as the atlas, since previously researchers needed to iteratively guess and check whether instruments physically fit with each other and the other constraints imposed by the rig. Other key features include coordinate transforms to map the CCF to more accurate in vivo anatomical data, as well as an API and hardware interface to commonly used micromanipulators.

Strengths:<br /> The feature set in Pinpoint makes it the best available software for planning instrument trajectories given geometrical constraints. Additionally, the documentation and open-source nature of the software should allow many extensions and improvements in the future, and as the authors note, it can also be used as a powerful teaching tool. Especially as researchers continue to push the boundaries of concurrent electrodes and optical fibers or other instruments within a single brain, this software will be of great use for neuroscience.

Weaknesses:<br /> Although Pinpoint enables instrument insertion planning with geometrical constraints for the first time and has many other novel features, it remains to be quantified how useful it is in terms of time/efficiency gains and accuracy of planned trajectories. For instance, although using a coordinate transform to MRI anatomical data is more accurate than the CCF alone in principle, users will need to verify how much this improved planning ability translates to time saved and/or improved trajectories as reconstructed from histology of dyed electrode tracks. The utility of the hardware interface for automating experiments versus the risk of damaging instruments with such an approach also remains to be quantified. Researchers using experimental subjects other than adult mice will have to wait for future integration of their atlases of choice, although the open-source nature of the project invites others to try adding this and other desired features themselves.

Reviewer #1 (Public Review):

Summary:<br /> Mice can learn to associate sensory cues (sound and light) with a reward or activation of dopamine neurons in the ventral tegmental area (VTA), and then anticipate the reward from the sensory cue only. Using this paradigm, Harada et al. showed that after learning, the cue is able to induce dopamine release in the projection targets of the VTA, namely the nucleus accumbens and lateral hypothalamus (LH). Within the LH, dopamine release from VTA neurons (either by presentation of the cue or direct optical stimulation of VTA neurons) activates orexin neurons, measured as an increase in intracellular calcium levels.

Strengths:<br /> This study utilized genetically encoded optical tools to selectively stimulate dopamine neurons and to monitor dopamine release in target brain areas and the calcium response of orexin neurons. This allowed a direct assessment of the relationship between the behavioral response of the animals, the release of a key neurotransmitter in select brain areas, and its effect on target cells, with a precision previously not possible. The results shed light on the mechanism underlying reward-related learning and expectation.

Weaknesses:<br /> • The Ca increase in orexin neurons in response to optical stimulation of VTA DA neurons is convincing. However, there is an accumulated body of literature indicating that dopamine inhibits orexin neurons through D2 receptors, particularly at high concentrations both directly and indirectly (PMID 15634779, 16611835, 26036709, 30462527; but note that synaptic effects at low conc are excitatory - PMID 30462527, 26036709). There should be a clear acknowledgment of these previous studies and a discussion directly addressing the discrepancy. Furthermore, there are in-vivo studies that investigated the role of dopamine in the LH involving orexin neurons in different behavioral contexts (e.g. PMID 24236888). The statement found in the introduction "whether and how dopamine release modulates orexin neuronal activity has not been investigated vigorously" (3rd para of Introduction) is an understatement of these previous reports.

• Along these lines, previous reports of concentration-dependent bidirectional dopaminergic modulation of orexin neurons suggest that high and low levels of DA would affect orexin neurons differently. Is there any way to estimate the local concentration of DA released by the laser stimulation protocol used in this study? Could there be a dose dependency in the intensity of laser stimulation and orexin neuron response?

• The transient dip in DA signal during omission sessions in Fig2C (approx 1% decrease from baseline) is similar in amplitude compared to the decrease seen in non-laser trails shown in Fig 1C right panel (although the time course of the latter is unknown as the data is truncated). The authors should clarify whether those dips are a direct effect of the cue itself or indeed reward prediction error.

• There seem to be orexin-negative-GCaMP6 positive cells (Fig. 4B), suggesting that not all cells were phenotypically orexin+ at the time of imaging. The proportion of GCaMP6 cells that were ORX+ or negative and whether they responded differently to the stimuli should be indicated.

• Laser stimulation of DA neurons at the level of cell bodies (in VTA) induces an increase in DA release within the LH (Fig. 3C, D), however, there is no corresponding Ca signal in orexin neurons (Fig.4C). In contrast, stimulating DA terminals within the LH induces a robust, long-lasting Ca signal (> 30s) in orexin neurons (Fig. 5). The initial peak is blocked by raclopride but the majority of Ca signal is insensitive to DA antagonists (please add a positive control or cite references indicating that the dose of antagonists used was sufficient; also the timing of antagonist administration should be indicated). Taken together, these results seem to suggest that DA does not directly increase Ca signal in orexin neurons. What could be mediating the remaining component?

• Similarly, there is an elevation of Ca signal in orexin neurons that remains significantly higher after the cue/laser stimulation (Fig. 4F). It appears that it is this sustained component that is missing in omission trials. This can be analyzed further.

• Mice of both sexes were used in this study; it would be interesting to know whether sex differences were observed or not.

Reviewer #1 (Public Review):

Summary:<br /> Willems and colleagues test whether unexpected shock omissions are associated with reward-related prediction errors by using an axiomatic approach to investigate brain activation in response to unexpected shock omission. Using an elegant design that parametrically varies shock expectancy through verbal instructions, they see a variety of responses in reward-related networks, only some of which adhere to the axioms necessary for prediction error. In addition, there were associations between omission-related responses and subjective relief. They also use machine learning to predict relief-related pleasantness, and find that none of the a priori "reward" regions were predictive of relief, which is an interesting finding that can be validated and pursued in future work.

Strengths:<br /> The authors pre-registered their approach and the analyses are sound. In particular, the axiomatic approach tests whether a given region can truly be called a reward prediction error. Although several a priori regions of interest satisfied a subset of axioms, no ROI satisfied all three axioms, and the authors were candid about this. A second strength was their use of machine learning to identify a relief-related classifier. Interestingly, none of the ROIs that have been traditionally implicated in reward prediction error reliably predicted relief, which opens important questions for future research.

Weaknesses:<br /> To ensure that the number of omissions is similar across conditions, the task employs inaccurate verbal instructions; i.e. 25% of shocks are omitted, regardless of whether subjects are told that the probability is 100%, 75%, 50%, 25%, or 0%. Given previous findings on interactions between verbal instruction and experiential learning (Doll et al., 2009; Li et al., 2011; Atlas et al., 2016), it seems problematic a) to treat the instructions as veridical and b) average responses over time. Based on this prior work, it seems reasonable to assume that participants would learn to downweight the instructions over time through learning (particularly in the 100% and 0% cases); this would be the purpose of prediction errors as a teaching signal. The authors do recognize this and perform a subset analysis in the 21 participants who showed parametric increases in anticipatory SCR as a function of instructed shock probability, which strengthened findings in the VTA/SN; however given that one-third of participants (n=10) did not show parametric SCR in response to instructions, it seems like some learning did occur. As prediction error is so important to such learning, a weakness of the paper is that conclusions about prediction error might differ if dynamic learning were taken into account. Lastly, I think that findings in threat-sensitive regions such as the anterior insula and amygdala may not be adequately captured in the title or abstract which strictly refers to the "human reward system"; more nuance would also be warranted.

Reviewer #1 (Public Review):

Strengths:<br /> The authors introduced a new adapted paradigm from continuous flash suppression (CFS). The new CFS tracking paradigm (tCFS) allowed them to measure suppression depth in addition to breakthrough thresholds. This innovative approach provides a more comprehensive understanding of the mechanisms underlying continuous flash suppression. The observed uniform suppression depth across target types (e.g., faces and gratings) is novel and has new implications for how the visual system works. The experimental manipulation of the target contrast change rate, as well as the modeling, provided strong support for an early interocular suppression mechanism. The authors argue that the breakthrough threshold alone is not sufficient to infer about unconscious processing.

Weaknesses:<br /> A major finding in the current study is the null effect of the image categories on the suppression depth measured in the tCFS paradigm, from which the authors infer an early interocular mechanism underlying CFS suppression. This is not strictly logical as an inference based on the null effect. The authors may consider statistical evaluation of the null results, such as equivalence tests or Bayesian estimation.

More importantly, since limited types of image categories have been tested, there may be some exceptional cases. According to "Twofold advantages of face processing with or without visual awareness" by Zhou et al. (2021), pareidolia faces (face-like non-face objects) are likely to be an exceptional case. They measured bidirectional binocular rivalry in a blocked design, similar to the discrete condition used in the current study. They reported that the face-like non-face object could enter visual awareness in a similar fashion to genuine faces but remain in awareness in a similar fashion to common non-face objects. We could infer from their results that: when compared to genuine faces, the pareidolia faces would have a similar breakthrough threshold but a higher suppression threshold; when compared to common objects, the pareidolia faces would have a similar suppression threshold but a low breakthrough threshold. In this case, the difference between these two thresholds for pareidolia faces would be larger than either for genuine faces or common objects. Thus, it would be important for the authors to discuss the boundary between the findings and the inferences.

Reviewer #1 (Public Review):

This paper studies how amacrine cells influence retinal output signals. The approach taken is unusually direct. First, the amacrine light response is characterized. Second, the properties of signaling between the amacrine cell and ganglion cells is characterized by injecting current into the amacrine cell while measuring ganglion cell spiking. Third, the ganglion cell light response is analyzed in terms of components produced by signaling pathways that go through the amacrine cell and those that do not. Interpretation of the results relies on several important and largely untested assumptions. If some of the concerns that this dependence produces can be reduced the paper would be substantially stronger.

Linear vs. nonlinear and direct vs. indirect<br /> Influences of an amacrine cell on the ganglion cell response are separated into direct effects - in which the amacrine cell directly produces a component of the ganglion cell response - and indirect effects - in which the amacrine cell modulates component(s) of the ganglion cell response (e.g. lines 97-99). In various places direct and indirect are equated with linear and nonlinear. Importantly, this assumption forms the basis of the analysis in the paper. It is not clear why a direct pathway through the amacrine cell should be linear. For example, it seems entirely possible that nonlinear models would capture the amacrine cell light response better than linear models. Similarly, nonlinear models may better capture the transmission of signals from amacrine cells to ganglion cells. Clarity on this issue is essential to interpret the results in the paper. One example of this issue comes up in the sentence on line 233. The definition of modulation is precise but only in the context of the above assumptions.

Components of oSTA<br /> The set of pre-spike stimuli that are orthogonal to the "direct" STA is used to characterize the "indirect" pathways conveying signals to a ganglion cell. For the reasons noted above, it is not clear that this is accurate. In addition, the text describes the PCs of this orthogonal stimulus ensemble as features. This is introduced in the paragraph starting on line 177, and this paragraph has the disclaimer that these features do not correspond to neural pathways. That important caveat to interpretation could be reiterated in the following text - particularly in discussing the different forms of modulation.

Related to this point, the analysis of Figures 3 and 4 relies on the PCs of this orthogonal stimulus ensemble. Since the PCs themselves do not map onto pathways or mechanisms, it is not clear how to interpret some of the results. For example, when you see a polarity shift along one of the PCs, what happens along others (for example, could they also be shifting polarity such that the net effect is a change in kinetics but not a change in polarity)? This also comes up in the paragraph on line 236, as it is not clear how the separation works given the way the components used as the basis of the separation are defined.

Some of these issues are clarified in Figure 4D, and perhaps it would help to start with that description. I think this section would be much clearer if two types of modulation were noted and then it was laid out how that conclusion was reached.

Reviewer #1 (Public Review):

In this systematic and elegant structure-function analysis study, the authors delve into the intricate involvement of syntaxin 1 in various pivotal stages of synaptic vesicle priming and fusion. The authors use an original and fruitful approach based on the side-by-side comparison of the specific contributions of the two isoforms syntaxin 1 and syntaxin 2, and their respective SNARE domains, in priming, spontaneous, and Ca2+-dependent glutamate release. The experimental approach, mastered by the authors, offers an ideal means of unraveling the molecular roles played by syntaxins. Although it is not easy to come up with a model explaining all the observed phenotypes, the authors carefully restrict their conclusions to the role of the C-terminal half of the syntaxin1 C-terminal SNARE domain in the maintenance of the RRP and the clamping of neurotransmitter release. The study is carefully carried out, the conclusions are supported by high-quality data, and the manuscript is clearly written. In addition, the study clearly sets new questions that open new paths for future experimental work.

Reviewer #1 (Public Review):

Summary:<br /> In this paper, the effects of two sensory stimuli (visual and somatosensory) on fMRI responsiveness during absence seizures were investigated in GEARS rats with concurrent EEG recordings. SPM analysis of fMRI showed a significant reduction in whole-brain responsiveness during the ictal period compared to the interictal period under both stimuli, and this phenomenon was replicated in a structurally constrained whole-brain computational model of rat brains.

The conclusion of this paper is that whole-brain responsiveness to both sensory stimuli is inhibited and spatially impeded during seizures.

I also suggest the manuscript should be written in a way that is more accessible to readers who are less familiar with animal experiments. In addition, the implementation and interpretation of brain simulations need to be more careful and clear.

Strengths:<br /> 1. ZTE imaging sequence was selected over traditional EPI sequence as the optimal way to perform fMRI experiments during absence seizures.

2. A detailed classification of stimulation periods is achieved based on the relative position in time of the stimulation period with respect to the brain state.

3. A whole-brain model embedded with a realistic rat connectome is simulated on the TVB platform to replicate fMRI observations.

Weaknesses:<br /> 1. The analysis in this paper does not directly answer the scientific question posed by the authors, which is to explore the mechanisms of the reduced brain responsiveness to external stimuli during absence seizures (in terms of altered information processing), but merely characterizes the spatial involvement of such reduced responsiveness. The same holds for the use of mean-field modeling, which merely reproduces experimental results without explaining them mechanistically as what the authors have claimed at the head of the paper.

2. The implementations of brain simulations need to be more specific.

Contribution:<br /> The contribution of this paper is performing fMRI experiments under a rare condition that could provide fresh knowledge in the imaging field regarding the brain's responsiveness to environmental stimuli during absence seizures.

Reviewer #1 (Public Review):

Summary:<br /> Very systematic generation of phosphosite-specific antisera to monitor FFA2 phosphorylation in native cells and tissues. Provides evidence that FFA2 phosphorylation is tissue-specific.

Strengths:<br /> Technical tour de force, rigorous experimental approaches taking advantage of wt and DREADD versions of FFA2 to make sure that ligand-and receptor-dependent phosphorylations are indeed specific to FFA2.

Weaknesses:<br /> In this reviewer's opinion, the only shortcoming is that the implications of tissue-selective phosphorylation barcoding remain unexplored. However, I understand that tool development is required before tools are used to provide insight into the functional outcomes of receptor regulation by phosphorylation. The study is a technical tour de force to generate highly valuable tools. I have no major criticisms but suggest adding an additional aspect to the discussion as specified below.

Arrestins are highly flexible and dynamic phosphate sensors. If two arrestins have to recognize 800 different phosphorylated GPCRs, is it possible that any barcode serves the same purpose: arrestin recognition followed by signal arrest and internalization? Because phosphorylation barcoding is linked to G protein-independent signaling, which is claimed by some but is experimentally unsupported, and because arrestins don't transduce receptor signals on their own (they only scaffold signaling components and shuttle receptors within cellular compartments), I would also include this option in the discussion, i.e. that the different barcodes are a way nature may have chosen to regulate the location of 800 GPCRs by only 2 arrestins.

Reviewer #1 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

The protocol is not clear. I could not even find how many women were going to be enrolled, the timelines of the study, the statistical methods ("comparing" is not statistics) or the power calculations.

Tables 2 and 3 are too schematic - surely the authors must have an approximate idea of what the actual numbers are behind the green, red and yellow colors.

Figure 1 comparing screening and vaccination is somewhat misleading. They screen 20 birth cohorts but vaccinate only 5 birth cohorts. Furthermore, the theoretical gains of screening has not really been attained in any country in practice. Modelling can be a difficult task and the commentary does not provide any detail on how to evaluate what was done. It just seems unnecessary to attack vaccination as a motivation on why screening needs to be modernised.

Reviewer #1 (Public Review):

Summary:<br /> In this manuscript the authors use ATAC-seq to find regions of the genome of rat embryonic striatal neurons in culture that show changes in regulatory element accessibility following stimulation by KCl-mediated membrane depolarization. The authors compare 1hr and 4hr transcriptomes to see both rapid and late response genes. When they look at ATAC-seq data they see no changes in accessibility at 1hr but strong changes at 4hr. The differentially accessible sites were enriched for the AP-1 site, suggesting regulation by Fos-Jun family members, and consistent with the requirement for IEG expression, anisomycin blocked the increase in accessibility. To test the functional importance of this regulation the authors focus on a putative enhancer 45kb upstream of the activity-induced gene encoding the neuromodulator dynorphin (Pdyn). To test the function of this region, the authors recruited CRISPRi to the site, which blocked KCL-dependent Pdyn induction, or CRISPRa, which selectively increased Pdyn expression in the absence of KCl. Finally the authors reanalyze other human and rat datasets to show cell-type specific function of this enhancer correlated to Pdyn expression.

The idea that stimuli that induce expression of Fos in neurons can change accessibility of regulatory elements bound from Fos has been shown before, but almost all the data are from hippocampal neurons so it is nice to see the different cell type used here. The most interesting part of the study is the identification of the Pdyn enhancer because of the importance of this gene product in the function of striatal neurons. Overall the conclusions appear to be well supported by the data.

Joint Public Review:

In this study the authors confirm that one of the genes classified as essential in a Tn-mutagenesis study in A. baumannii is in fact an essential gene. It is also present in other closely related Gram negative bacteria and the authors designated it Aeg1.

The strength of the work is that it discovered that the depletion of Aeg1 leads to cell filamentation and that the requirement for Aeg1 can be suppressed by activation mutations in various cell division genes. These results suggest that Aeg1 plays an important role in cell division.

The weakness of the work is that it lacks convincing evidence to define Aeg1 place or role in the divisome assembly pathway. It is unclear what proteins are at the division site when Aeg1 is depleted and what proteins are required for Aeg1 to localize to the division site.

Reviewer #1 (Public Review):

In this manuscript the authors performed experiments and simulations which showed that substrate evaporation is the main driver of early construction in termites. Additionally, these experiments and simulations were designed taking into account several different works, so that the current results shine a light on how substrate evaporation is a sufficient descriptor of most of the results seen previously.

Through simulations and ingenious experiments the authors have shown how curvature is extremely correlated with evaporation, and therefore, how results coming from these 2 environmental factors can be explained through evaporation alone. The authors have continued to use their expertise of numerical simulations and a previously developed model for termite construction, to highlight and verify their findings. On my first pass of the manuscript I felt the authors were missing an experiment: an array of humidity probes to measure evaporation in the three spatial dimensions and over time. Technologically such an experiment is not out of reach, but the author's alternative (a substrate made with a saline solution and later measuring the salt deposits on the surface) was a very ingenious low tech solution to the problem.

The authors agree that future experiments should tackle finely controlled humidity levels and curvature in order to have a more quantitative measure termite behaviour, but the work done so far is more than sufficient to justify their current claims.

The results presented here are so far the best attempt on characterizing multiple cues that induce termite construction activity, and that possibly unifies the different hypothesis presented in the last 8 years into a single factor. More importantly, even if these results come from different species of termites than some of the previous works, they are relatable and seem to be mostly consistent, improving the strength of the author's claims.

Joint Public Review:

This concise review provides a clear and instructive picture of the state-of-the-art understanding of protein kinases' activity and sets of approaches and tools to analyse and regulate it.

Three major parts of the work include: methods to map allosteric communications, tools to control allostery, and allosteric regulation of protein kinases. The work provides an important and timely view of the current status of our understanding of the function of protein kinases and state-of-the-art methods to study its allosteric regulation and to develop allosteric approaches to control it.

Reviewer #1 (Public Review):

This work describes a structural analysis of the tripartite HipBST toxin-antitoxin (TA) system, which is related to the canonical two-component HipBA system composed of the HipA serine-threonine kinase toxin and the HipB antitoxin. The crystal structure of the kinase-inactive HipBST complex of the Enteropathogenic E. coli O127:H6 was solved and revealed that HipBST forms a hetero-hexameric complex composed of a dimer of HipBST heterotrimers that interact via the HipB subunit. The HipS antitoxin shows a structural resemblance to HipA N-terminal region and the HipT toxin represents to the core kinase domain of HipA, indicating that in HipBST the hipA toxin gene was likely split in two genes, namely hipS and hipT.<br /> -The structure also reveals a conserved and essential Trp residue within the HipS antitoxin, which likely prevents the conserved "Gly-rich loop" of HipT from adopting an inward conformation needed for ATP binding. This work also shows that the regulating Gly-rich loop of the HipT toxin contains conserved phosphoserine residues essential for HipT toxicity that are key players within the HipT active site interacting network and which likely control antitoxin binding and/or activity.

Strengths:

The manuscript is well written and the experimental work well executed. It shows that major features of the classical two-component HipAB TA system have somehow been rerouted in the case of the tripartite HipBST. This includes the N-terminal domain of the HipA toxin, which now functions as bona fide antitoxin, and the partly relegated HipB antitoxin, which could only function as a transcription regulator. In addition, this work shows a new mode of inhibition of a kinase toxin and highlights the impact of the phosphorylation state of key toxin residues in controlling the activity of the antitoxin.

Weaknesses:

The authors have convincingly addressed the previously raised weaknesses in their revised version of the manuscript.

Reviewer #1 (Public Review):

The authors develop reporter constructs in E. coli where gene expression, presumably translation, is repressed by MSI-1. This is a potentially useful tool for synthetic biologists, with the advantage over transcriptional regulation that one gene in an operon could be targeted. That being said, an important caveat of translational regulation that is not addressed in the manuscript is the potential for downstream effects on RNA stability and/or transcription termination. The authors' MSI-1-regulated reporter constructs could also be useful for mechanistic studies of MSI-1.

The author's initial construct design led to only weak regulation by MSI-1, presumably because the MSI-1 binding sites were not suitably positioned to repress translation initiation. A more rationally designed construct led to considerably greater repression. One weakness of the paper is that the authors did not use their redesigned construct that is more strongly repressed to demonstrate allosteric regulation by oleic acid using a comparable assay (e.g., flow cytometry) to that used in other experiments. The potential for allosteric regulation is a major strength of the MSI-1 system, so this is a significant gap. Similarly, the authors use the weakly regulated constructs to assess the effect of MSI-1 binding site mutations and for their mathematical modeling; these experiments would be better suited to the more strongly regulated construct.

1993 oslo akkoord: palestijns zelfbestuur

Reviewer #1 (Public Review):

Muscle models are important tools in the fields of biomechanics and physiology. Muscle models serve a wide variety of functions, including validating existing theories, testing new hypotheses, and predicting forces produced by humans and animals in health and disease. This paper attempts to provide an alternative to Hill-type muscle models that includes contributions of titin to force enhancement over multiple time scales. Due to the significant limitations of Hill-type models, alternative models are needed and therefore the work is important and timely.

The effort to include a role for titin in muscle models is a major strength of the methods and results. The results clearly demonstrate the weaknesses of Hill models and the advantages of incorporating titin into theoretical treatments of muscle mechanics. Another strength is to address muscle mechanics over a large range of time scales. Weaknesses include the decision to use a MTU model to simulate experiments from single muscle fibers, and failure to systematically address the limitations of the model, including equations for activation dynamics with no length dependence. It would also be useful for readers if the authors provided a discussion of the types of data that can be simulated using the model, along with potential pitfalls and how to determine model parameters.

The authors succeed in demonstrating the need to incorporate titin in muscle models. However, it remains unclear whether it will be practical for others to use this particular model for different types of data. Several ad hoc modifications were described in the paper, and the degree to which the model requires parameter optimization for different muscles, preparations and experiment types is also unclear.

Reviewer #1 (Public Review):

Aiming at the problem that Staphylococcus aureus can cause apoptosis of macrophages, the author found and verified that drug (R)-DI-87 can inhibit mammalian deoxycytidine kinase (dCK), weaken the killing effect of staphylococcus aureus on macrophages, and reduce the apoptosis of macrophages. And increase the infiltration of macrophages to the abscess, thus weakening the damage of Staphylococcus aureus to the host. This work provides new insights and ideas for understanding the effects of Staphylococcus aureus infection on host immunity and discovering corresponding therapeutic interventions.

The logic of the study is commendable, and the design is reasonable.

Some data related to the conclusion of the paper need to be supplemented, and some experimental details need to be described.

Reviewer #1 (Public Review):

Despite numerous studies on quinidine therapies for epilepsies associated with GOF mutant variants of Slack, there is no consensus on its utility due to contradictory results. In this study Yuan et al. investigated the role of different sodium selective ion channels on the sensitization of Slack to quinidine block. The study employed electrophysiological approaches, FRET studies, genetically modified proteins and biochemistry to demonstrate that Nav1.6 N- and C-tail interacts with Slack's C-terminus and significantly increases Slack sensitivity to quinidine blockade in vitro and in vivo. This finding inspired the authors to investigate whether they could rescue Slack GOF mutant variants by simply disrupting the interaction between Slack and Nav1.6. They find that the isolated C-terminus of Slack can reduce the current amplitude of Slack GOF mutant variants co-expressed with Nav1.6 in HEK cells and prevent Slack induced seizures in mouse models of epilepsy. This study adds to the growing list of channels that are modulated by protein-protein interactions, and is of great value for future therapeutic strategies.

Reviewer #1 (Public Review):

Summary:<br /> In this work the authors provide evidence that impairment of cell envelope protein homeostasis through blocking the machinery for disulfide bond formation restores the efficacy of antibiotics including beta-lactam drugs and colistin against AMR in Gram-negative bacteria.

Strengths:

The authors employ a thorough approach to showcase the restoration of antibiotic sensitivity through inhibition of the DSB machinery, including the evaluation of various antibiotics on both normal and Dsb-deficient pathogenic bacteria (i.e. Pseudomonas and Stenotrophomonas). The authors corroborate these findings by employing Dsb inhibitors in addition to delta dsbA strains. The methodology is appropriate and includes measuring MICs as well as validating their observations in vivo using the Galleria model.

Weaknesses:

The study would benefit from presenting raw data in some cases, such as MIC values and SDS-PAGE gels, by clarifying the number of independent experiments used, as well as further clarification on statistical significance for some of the data.

Reviewer #1 (Public Review):

Nitrogen metabolism is of fundamental importance to biology. However, the metabolism and biochemistry of guanidine and guanidine containing compounds, including arginine and homoarginine, have been understudied over the last few decades. Very few guanidine forming enzymes have been identified. Funck et al define a new type of guanidine forming enzyme. It was previously known that 2-oxogluturate oxygenase catalysis in bacteria can produce guanidine via oxidation of arginine. Interestingly, the same enzyme that produces guanidine from arginine also oxidises 2-oxogluturate to give the plant signalling molecule ethylene. Funck et al show that a mechanistically related oxygenase enzyme from plants can also produce guanidine, but instead of using arginine as a substrate, it uses homoarginine. The work will stimulate interest in the cellular roles of homoarginine, a metabolite present in plants and other organisms including humans and, more generally, in the biochemistry and metabolism of guanidines.

1. Significance<br /> Studies on the metabolism and biochemistry of the small nitrogen rich molecule guanidine and related compounds including arginine have been largely ignored over the last few decades. Very few guanidine forming enzymes have been identified. Funck et al define a new guanidine forming enzyme that works by oxidation of homoarginine, a metabolite present in organisms ranging from plants to humans. The new enzyme requires oxygen and 2-oxogluturate as cosubstrates and is related, but distinct from a known enzyme that oxidises arginine to produce guanidine, but which can also oxidise 2-oxogluturate to produce the plant signalling molecule ethylene.

Overall, I thought this was an exceptionally well written and interesting manuscript. Although a 2-oxogluturate dependent guanidine forming enzyme is known (EFE), the discovery that a related enzyme oxidises homoarginine is really interesting, especially given the presence of homoarginine in plant seeds. There is more work to be done in terms of functional assignment, but this can be the subject of future studies. I also fully endorse the authors' view that guanidine and related compounds have been massively understudied in recent times. I would like to see the possibility that the new enzyme makes ethylene explored. Congratulations to the authors on a very nice study.

Reviewer #1 (Public Review):

Summary:<br /> Cincotta et al set out to investigate the presence of glucocorticoid receptors in the male and female embryonic germline. They further investigate the impact of tissue-specific genetically induced receptor absence and/or systemic receptor activation on fertility and RNA regulation. They are motivated by several lines of research that report inter and transgenerational effects of stress and or glucocorticoid receptor activation and suggest that their findings provide an explanatory mechanism to mechanistically back parental stress hormone exposure-induced phenotypes in the offspring.

Strengths:<br /> - A chronological immunofluorescent assessment of GR in fetal and early life oocyte and sperm development.<br /> - RNA seq data that reveal novel cell type specific isoforms validated by q-RT PCR E15.5 in the oocyte.<br /> - 2 alternative approaches to knock out GR to study transcriptional outcomes. Oocytes: systemic GR KO (E17.5) with low input 3-tag seq and germline-specific GR KO (E15.5) on fetal oocyte expression via 10X single cell seq and 3-cap sequencing on sorted KO versus WT oocytes - both indicating little impact on polyadenylated RNAs<br /> - 2 alternative approaches to assess the effect of GR activation in vivo (systemic) and ex vivo (ovary culture): here the RNA seq did show again some changes in germ cells and many in the soma.<br /> - They exclude oocyte-specific GR signaling inhibition via beta isoforms.<br /> - Perinatal male germline shows differential splicing regulation in response to systemic Dex administration, results were backed up with q-PCR analysis of splicing factors.

Weaknesses:<br /> - The presence of a protein cannot be entirely excluded based on IF data (staining of spermatids is referred to but not shown).<br /> - The authors do not consider post-transcriptional level a) modifications also trigged by GR activation b) non-coding RNAs (not assessed by seq).<br /> - Sequencing techniques used are not total RNA but either are focused on all polyA transcripts (10x) or only assess the 3' prime end and hence are not ideal to study splicing, The number of replicates in the low input seq is very low and hence this might be underpowered. Since Dex treatment showed some (modest) changes in oocyte RNA - effects of GR depletion might only become apparent upon Dex treatment as an interaction.<br /> - Effects in oocytes following systemic Dex might be indirect due to GR activation in the soma.<br /> - Even though ex vivo culture of ovaries shows GR translocation to the nucleus it is not sure whether the in vivo systemic administration does the same.

The conclusion that fetal oocytes are "intrinsically buffered to GR signalling" is very strong, given that "only" poly A sequencing and few replicates of 3-prime sequencing have been analyzed and information is lacking on whether GR is activated in germ cells in the systemically dex-injected animals.

This work is a good reference point for researchers interested in glucocorticoid hormone signaling fertility and RNA splicing. It might spark further studies on germline-specific GR functions and the impact of GR activation on alternative splicing.

While the study provides a characterization of GR and some aspects of GR perturbation, and the negative findings in this study do help to rule out a range of specific roles of GR in the germline, there is still a range of other potential unexplored options. The introduction of the study eludes to implications for intergenerational effects via epigenetic modifications in the germline, however, it does not mention that the indirect effects of reproductive tissue GR signaling on the germline have indeed already been described in the context of intergenerational effects of stress. Also, the study does not assess epigenetic modifications.

The conclusion that the persistence of a phenotype for up to three generations suggests that stress can induce lasting epigenetic changes in the germline is misleading. For the reader who is unfamiliar with the field, it is important to define much more precisely what is referred to as "a phenotype". Furthermore, this statement evokes the impression that the very same epigenetic changes in the germline have been observed across multiple generations.

The evidence of the presence of GR in the germline is also somewhat limited - since other studies using sequencing have detected GR in the mature oocyte and sperm.

The discussion ends again on the implications of sex-specific differences of GR signaling in the context of stress-induced epigenetic inheritance. It states that the observed differences might relate to the fact that there is more evidence for paternal lineage findings, without considering that maternal lineage studies in epigenetic inheritance are generally less prevalent due to some practical factors - such as more laborious study design making use of cross-fostering or embryo transfer. Since the authors comment on RNA-mediated inheritance it seems inevitable to again consider indirect effects.

Reviewer #1 (Public Review):

This is a well-designed study that explores the BEF relationships in fragmented landscapes. Although there are massive studies on BEF relationships, most of them were conducted at local scales, few considered the impacts of landscape variables. This study used a large dataset to specifically address this question and found that habitat loss weakened the BEF relationships. Overall, this manuscript is clearly written and has important implications for BEF studies as well as for ecosystem restoration.

My only concern is that the authors should clearly define habitat loss and fragmentation. Habitat loss and fragmentation are often associated, but they are different terms. The authors consider habitat loss a component of habitat fragmentation, which is not reasonable. Please see my specific comments below.

Joint Public Review:

This work by Liu CSC et al. is an extension of the author's previous work on the role of Piezo1 mechano-sensor in human T cell activation. In this study, the authors address whether Piezo1 plays a role in T-cell chemotactic migration.

The authors used CD4+ T cells or Jurkat T cells to test the effects of siRNA-mediated depletion of Piezo1 on chemotactic migration. They establish that Piezo1 is implicated in chemotactic migration, although the effects of depletion are relatively moderate.

They show that Piezo1 is redistributed to the leading edge of T-cells.

They identify that relocation of Piezo1 to the leading edge follows an increase in membrane tension.

In Piezo-1 depleted cells, they observe a moderate reduction of LFA-1 polarity. With the use of specific inhibitors, they propose Piezo1 activation to be downstream of focal adhesion formation and upstream of calpain-mediated LFA-1, integrin alpha L beta 2, or CD11a/CD18 recruitment at the leading edge.

Strengths:<br /> Together with their 2018 paper, this study presents Pieszo1 as a regulator of T-cell activation, implicating it as a player in the coordination of the chemotactic immune response.

Weaknesses:<br /> Most of the effects observed are relatively modest. The authors did not challenge the cells with various physico-mechanical conditions to see when Piezo-1 might become really important. For instance, there are no experiments that expose T cells to varying counter-acting forces to see how piezo1 might affect migration.

Technical weaknesses:<br /> The authors state that "these high tension edges are usually further emphasized at later time points", but after ten minutes the median tension and tension (Figure 2C and Supplementary Figure 2C respectively) reduce down to the pretreatment time point. It would be clearer if the author stated within which timeframe the tension edges are "further emphasised".

Figures 3 and 4 - The author states the number of cells quantified from the images, but it is not clear whether the data is actually from 3 biological replicates.

Some of the data has no representative images or videos included. there is no video in the supplementary for Figures 1 A and B. There are no representative images of transwell migration assay in Figures 1 D and E.

Reviewer #1 (Public Review):

In 'Systems analysis of miR-199a/b-5p and multiple miR-199a/b-5p targets during chondrogenesis', Patel et al. present a variety of analyses using different methodologies to investigate the importance of two miRNAs in regulating gene expression in a cellular model of cartilage development. They first re-analysed existing data to identify these miRNAs as one of the most dynamic across a chondrogenesis development time course. Next, they manipulated the expression of these miRNAs and showed that this affected the expression of various marker genes as expected. An RNA-seq experiment on these manipulations identified putative mRNA targets of the miRNAs which were also supported by bioinformatics predictions. These top hits were validated experimentally and, finally, a kinetic model was developed to demonstrate the relationship between the miRNAs and mRNAs studied throughout the paper.

I am convinced that the novel relationships reported here between miR-199a/b-5p and target genes FZD6, ITGA3, and CAV1 are likely to be genuine. It is important for researchers working on this system and related diseases to know all the miRNA/mRNA relationships but, as the authors have already published work studying the most dynamic miRNA (miR-140-5p) in this biological system I was not convinced that this study of the second miRNA in their list provided a conceptual advance on their previous work.

I was also concerned with the lack of reporting of details of the manipulation experiments. The authors state that they have over-expressed miR-199a-5p (Figure 2A) and knocked down miR-199b-5p (Figure 2B) but they should have reported their proof that these experiments had worked as predicted, e.g. showing the qRT-PCR change in miRNA expression. Similarly, I was concerned that one miRNA was over-expressed while the other was knocked down - why did the authors not attempt to manipulate both miRNAs in both directions? Were they unable to achieve a significant change in miRNA expression or did these experiments not confirm the results reported in the manuscript?

I had a number of issues with the way in which some of the data was presented. Table 1 only reported whether a specific pathway was significant or not for a given differential expression analysis but this concealed the extent of this enrichment or the level of statistical significance reported. Could it be redrawn to more similarly match the format of Figure 3A? The various shades of grey in Figure 2 and Figure 4 made it impossible to discriminate between treatments and therefore identify whether these data supported the conclusions made in the text. It also appeared that the same results were reported in Figure 3B and 3C and, indeed, Figure 3B was not referred to in the main text. Perhaps this figure could be made more concise by removing one of these two sets of panels.

Overall, while I think that this is an interesting and valuable paper, I think its findings are relatively limited to those interested in the role of miRNAs in this specific biomedical context.

Reviewer #1 (Public Review):

Summary:

In this work, Frank, Bergamasco, Mlodzianoski et al study two microcephaly-associated patient variants in TRABID to identify and characterize a previously unrecognized role of this deubiquitylation enzyme during neurodevelopment. The authors generate TRABID p.R438W and p.A451V knock in mice, which exhibit smaller neuronal and glial cell densities as well as motor deficits, phenotypes that are consistent with the congenital defects observed in the patients. Through in vitro and cellular immunoprecipitation assays, the authors demonstrate that the p.R438W variant impairs the K29- and K63-chain cleavage activity of TRABID, while the p.A451V variant reduces binding to the STRIPAK complex, a previously identified TRABID interactor with established functions in cytoskeletal organization and neural development. Ubiquitylation assays performed in HEK293T cells further reveal that the hypomorphic patient variants are deficient in deubiquitylating APC, a previously identified substrate of TRABID that has been shown to control the neuronal cortical cytoskeleton during neurite outgrowth. Ex vivo experiments provide evidence that axonal APC trafficking and neurite outgrowth is disturbed in differentiating neural progenitors isolated from mouse embryos carrying Trabid patient alleles. From these experiments the authors propose a model in which TRABID- and STRIPAK-dependent APC deubiquitylation regulates its axonal trafficking to ensure faithful neurite outgrowth and misregulation of this function leads to neurodevelopmental phenotypes in TRABID/ZRANB1 patients.

Strengths:

This study describes a previously unrecognized function of TRABID in neurodevelopment and establishes knock in mice as model to study congenital defects of TRABID/ZRANB1 patients. In addition, the authors identify control of axonal trafficking of APC by deubiquitylation as a potential mechanism through which TRABID regulates neurite outgrowth and whose dysregulation could be the molecular basis of the neurodevelopmental phenotypes observed in TRABID/ZRANB1 patients.

Weaknesses:

While the proposed underlying mechanism of how hypomorphic TRABID mutations lead to the patient phenotypes is conceivable and supported by the author's data, there is no functional evidence provided that the mouse phenotypes (reduced neuron/glia densities or motor deficits) are indeed due to aberrant APC deubiquitylation and trafficking. In addition, some aspects of the proposed mechanism, i.e. the claim that APC deubiquitylation is STRIPAK-dependent, should be strengthened by orthogonal approaches.

Reviewer #1 (Public Review):

Summary:

This paper shows that E. coli exhibits a chemotactic response to potassium by measuring both the motor response (using a bead assay) and the intracellular signaling response (CheY phosporylation level via FRET) to step changes in potassium concentration. They find increase in potassium concentration induces a considerable attractant response, with an amplitude larger than aspartate, and cells can quickly adapt (but possibly imperfectly). The authors propose that the mechanism for potassium response is through modifying intracellular pH; they find both that potassium modifies pH and other pH modifiers induce similar attractant responses. It is also shown, using Tar- and Tsr-only mutants, that these two chemoreceptors respond to potassium differently. Tsr has a standard attractant response, while Tar has a biphasic response (repellent-like then attractant-like). Finally, the authors use computer simulations to study the swimming response of cells to a periodic potassium signal secreted from a biofilm and find a phase delay that depends on the period of oscillation.

Strengths:

The finding that E. coli can sense and adapt to potassium signals and the connection to intracellular pH is quite interesting and this work should stimulate future experimental and theoretical studies regarding the microscopic mechanisms governing this response. The evidence (from both the bead assay and FRET) that potassium induces an attractant response is convincing, as is the proposed mechanism involving modification of intracellular pH.

Weaknesses:

The authors show that changes in pH impact fluorescent protein brightness and modify the FRET signal; this measurement explains the apparent imprecise adaptation they measured. However, this effect reduces confidence in the quantitative accuracy of the FRET measurements. For example, part of the potassium response curve (Fig. 4B) can be attributed to chemotactic response and part comes from the pH modifying the FRET signal. Measuring the full potassium response curve of the no-receptor mutants as a control would help quantify the true magnitude of the chemotactic response and the adaptation precision to potassium.

The measured response may also be impacted by adaptation. For other strong attractant stimuli, the response typically shows a low plateau before it recovers (adapts). However, in the case of Potassium, the FRET signal does not have an obvious plateau following the stimuli. Do the authors have an explanation for that? One possibility is that the cells may have already partially adapted when the response reaches its minimum, which could indicate a different response and/or adaptation dynamics from that of a regular chemo-attractant? In any case, directly measuring the response to potassium in mutants without adaptation enzymes (CheR, CheB) and with the receptors in different methylation levels would shed more light on the problem.

There seems to be an inconsistency between the FRET and bead assay measurements, the CW bias shows over-adaptation, while the FRET measurement does not. The small hill coefficient of the potassium response curve and the biphasic response of the Tar-only strain, while both very interesting, require further explanation since these are quite different than responses to more conventional chemoattractants.

Reviewer #1 (Public Review):

This study addresses the temporal patterning of a specific Drosophila CNS neuroblast lineage, focusing on its larval development. They find that a temporal cascade, involving the Imp and Syb genes changes the fate of one daughter cell/branch, from glioblast (GB) to programmed cell death (PCD), as well as gates the decommissioning of the NB at the end of neurogenesis.

Reviewer #1 (Public Review):

Summary:

A novel serine protease and an inhibitor pair regulate cell migration in the neural crest. This is a very important study that describes a novel pathway controlling neural crest migratory behavior through a pair of protease and inhibitor regulators that act in the extracellular space. Using very high technical standards in Xenopus embryos they show that knockdown of the inhibitor SerpinE2 prevents cell migration and that this is restored by simultaneous knockdown of the serine protease HtrA1.

Strengths:

The reproduction of classical cranial neural crest extirpations and their phenocopy by SerpinE2 morpholino is remarkable. The experiments provided must represent many years of work, and the paper is written in a very scholarly fashion. The data is of the highest quality.

Weaknesses:

The paper is very long and contains many years of experiments, making it at times difficult to read. The paper contains so much data that it would help the readership if the present version were revised in order to make it more digestible.

Reviewer #1 (Public Review):

Pathogenic mutations of mTOR pathway genes have been identified in patients with malformation of cortical development and intractable epilepsy. Nguyen et al., established an in vivo rodent model to investigate the impact of different mTOR pathway gene dysfunction on neuronal intrinsic membrane excitability and cortical network activity. The results demonstrate that activation of mTORC1 activators or inactivation of mTORC1 repressors leads to convergent mTOR pathway activation and alterations of neuronal morphology, the key pathological feature of human FCD and hemimegalencephaly. However, different mTOR pathway gene mutations also exhibited variations in modulating Ih current and synaptic activity in rodent cortical neurons. These findings provide novel insights into the mechanism of seizure generation associated with cortical malformation.

1. The authors found differences in the initial spike doublet of action potentials between cortical neurons in experimental and control conditions (Figure 2e). The action potential firing frequency of the first two APs (instant firing frequency) of recorded neurons shall be quantified to investigate whether there are statistical differences between the action potential firing frequency in cortical neurons in different experimental groups versus control conditions.

2. The mTORS12215Y induced the largest changes in Ih current amplitudes in cortical neurons compared with other experimental conditions. Whether the HCN4 channel expression is regulated by mTOR pathway activation, or could there be possible interactions between the HCN channel and mTORS12215Y mutant protein?

3. A comparison of the electrophysiological characteristics of cortical neurons in different experimental conditions in the present study and pathological neurons in human FCD reported in previous literature could be interesting. Inducing pathological gene mutations or knocking out key genes in mTOR pathway in the rodent cortex - which approach could better model human FCD?

Reviewer #1 (Public Review):

Summary:

The authors study the appearance of oscillations in motifs of linear threshold systems, coupled in specific topologies. They derive analytical conditions for the appearance of oscillations, in the context of excitatory and inhibitory links. They also emphasize the higher importance of the topology, compared to the strength of the links. Finally, the results are confirmed with WC oscillators, which are also linear. The findings are to some extent confirmed with spiking neurons, though here results are less clear, and they are not even mentioned in the Discussion.

Overall, the results are sound from a theoretical perspective, but I still find it hard to believe that they are of significant relevance for biological networks, or in particular for the oscillations of BG-thalamus-cortex loop in PD. I find motifs in general to be too simplistic for multiscale and generally large networks as is the case in the brain. Moreover, the division of regions is more or less arbitrary by definition, and having such a strong dependence on an odd/even number of inhibitory links is far from reality. Another limitation is the fact that the cortex is considered a single node. Similarly, decomposing even such a coarse network in all possible (238 in this case) motifs doesn't seem of much relevance, when I assume that the emergence of pathological rhythms is more of an emergent phenomenon.

Strengths:

From the point of view of nonlinear dynamics, the results are solid, and the intuition behind the proofs of the theorems is well explained.

Weaknesses:

As stated in the summary, I find the work to be too theoretical without a real application in biological systems or the brain, where the networks are generally very large. It is not the problem in the simplicity of the model or of the topology, it is often the case that the phenomena are explained by very reduced systems, but the problem is that the applicability of the finding cannot be extended. E.g. the Kuramoto model uses all-to-all coupling, or similar with QIF neurons which also need to follow a Lorentzian distribution in order to derive a mean field. But in those cases, relaxing the strict conditions that were necessary for the derivations, still conserves the main findings of the analysis, which I don't see being the case here. The odd/even number rule is too strict, and talking about a fixed and definite number of cycles in the actual brain seems too simplistic.

Being linear is another strong assumption, and it is not clear how much of the results are preserved for spiking neurons, even though there is such an analysis, or maybe for other nonlinear types of neuronal masses.

Delays are also mentioned, and their impact on the oscillatory networks is as expected: it reduces the amplitude, but there is no link to the literature, where this is an established phenomenon during synchronization. Finally, the authors should also discuss the time-delays as a known phenomenon to cause or amplify oscillations at different frequencies in a network of coupled oscillators, e.g Petkoski & Jirsa Network Neuroscience 2022, Tewarie et al. NeuroImage 2019, Davis et al. Nat Commun 2021.

Reviewer #1 (Public Review):

Summary:<br /> The Drosophila wing disc is an epithelial tissue, the study of which has provided many insights into the genetic regulation of organ patterning and growth. One fundamental aspect of wing development is the positioning of the wing primordia, which occurs at the confluence of two developmental boundaries, the anterior-posterior and the dorsal-ventral. The dorsal-ventral boundary is determined by the domain of expression of the gene apterous, which is set early in the development of the wing disc. For this reason, the regulation of apterous expression is a fundamental aspect of wing formation.

In this manuscript, the authors used state-of-the-art genomic engineering and a bottom-up approach to analyze the contribution of a 463 base pair fragment of apterous regulatory DNA. They find compelling evidence about the inner structure of this regulatory DNA and the upstream transcription factors that likely bind to this DNA to regulate apterous early expression in the Drosophila wing disc.

Strengths:<br /> This manuscript has several strengths concerning both the experimental techniques used to address the problem of gene regulation and the relevance of the subject. To identify the mode of operation of the 463 bp enhancer, the authors use a balanced combination of different experimental approaches. First, they use bioinformatic analysis (sequence conservation and identification of transcription factors binding sites) to identify individual modules within the 463 bp enhancer. Second, they identify the functional modules through genetic analysis by generating Drosophila strains with individual deletions. Each deletion is characterized by looking at the resulting adult phenotype and also by monitoring apterous expression in the mutant wing discs. They then use a clever method to interfere in a more dynamic manner with the function of the enhancer, by directing the expression of catalytically inactive Cas9 to specific regions of this DNA. Finally, they recur to a more classical genetic approach to uncover the relevance of candidate transcription factors, some of them previously known and others suggested by the bioinformatic analysis of the 463 bp sequence. This workflow is clearly reflected in the manuscript, and constitutes a great example of how to proceed experimentally in the analysis of regulatory DNA.

Weaknesses:<br /> There are several caveats with the data that might be constructed as weaknesses, some of them are intrinsic to this detailed analysis or to the experimental difficulties of dealing with the wing disc in its earliest stages, and others are more conceptual and are offered here in case the authors may wish to consider them.

1) The primordium of the wing region of the wing imaginal disc is defined by the expression of the gen vestigial, which is regulated by inputs coming from the dorsal-ventral boundary (Notch and wg) and from the anterior-posterior boundary (Dpp). Having such a principal role in wing primordium specification and expansion, I am surprised that this manuscript does not mention this gene in the main text and only contains indirect references to it. I consider that the manuscript would have benefited a lot by including vestigial in the analysis, at least as a marker of early wing primordium. This might allow us to visualize directly the positioning of the primordium in the apterous mutants generated in this study, adding more verisimilitude to the interpretations that place this domain based on indirect evidence.

2) The authors place some emphasis on the idea that their work addresses possible coordination between setting the D/V boundary and the A/P boundary:

Abstract: "Thus, the correct establishment of ap expression pattern with respect to en must be tightly controlled", "...challenging the mechanism by which apE miss-regulation leads to AP defects." "Detailed mutational analyses using CRISPR/Cas revealed a role of apE in positioning the DV boundary with respect to the AP boundary"<br /> Introduction: "However, little is known about how the expression pattern of ap is set up with respect that of en. In other words, how is the DV boundary positioned with respect to the AP boundary?"<br /> "How such interaction between ap and the AP specification program arises is unknown."<br /> Results: "Some of these phenotypes are reminiscent of those reported for apBlot (Whittle, 1979) and point towards a yet undescribed crosstalk between ap early expression and the AP specification program."

At the same time, they express the notion, with which this reviewer agrees, that all defects observed in A/P patterning arising as a result of apterous miss-regulation are due to the fact that in their mutants, apterous expression is lost mainly in the posterior dorsal compartment, bringing novel confrontations between the A/P and the D/V boundaries.

To me, the key point is why the expression of apterous in different mutants of the OR463 enhancer affects only the posterior compartment. This should be discussed because it is far from obvious that apterous expression has different regulatory requirements in the anterior and posterior compartments.

3) The description of gene expression in the wing disc of novel apterous mutants is only carried out in late third instar discs (Figs. 2, 3, 5, and 7). This is understandable given the technical difficulties of dealing with early discs, as those shown in the analysis of candidate apterous regulatory transcription factors (Fig. 4F, Fig. 6 C-D). However, because the effects of the mutants on apterous expression are expected to occur much earlier than the time of expression analysis, this fact should be discussed.

Joint Public Review:

In this work Wu, J., et al., highlight the importance of a previously overlooked region on kinases: the αC-β4 loop. Using PKA as a model system, the authors extensively describe the conserved regulatory elements within a kinase and how the αC-β4 loop region integrates with these important regulatory elements. Previous biochemical work on a mutation within the αC-β4 loop region, F100A showed that this region is important for the synergistic high affinity binding of ATP and the pseudo substrate inhibitor PKI. In the current manuscript, the authors assess the importance of the αC-β4 loop region using computational methods such as Local Spatial Pattern Alignment (LSP) and MD simulations. LSP analysis of the F100A mutant showed decreased values for degree centrality and betweenness centrality for several key regulatory elements within the kinase which suggests a loss in stability/connectivity in the mutant protein as compared to the WT. Additionally, based on MD simulation data, the side chain of K105, another residue within the αC-β4 loop region had altered dynamics in the F100A mutant as compared to the WT protein. While these changes in the αC-β4 loop region seem to be consistent with the previous biochemical data, the results are preliminary and the manuscript can be strengthened (as the authors themselves acknowledge) with additional experiments. Specific comments/concerns are listed below.

1. MD simulations were carried out using a binary complex of the catalytic subunit of PKA and ATP/Mg and not the ternary complex of PKA, ATP/Mg and PKI. MD simulations carried out using the ternary complex instead of the binary complex would be more informative, especially on the role of the αC-β3 loop region in the synergistic binding of ATP/Mg and PKI.

2. The LSP analysis shows a decrease in degree centrality for the αC-β4 loop region in the F100A mutant compared to the WT protein which suggests a gain in stability in this region for the F100A mutant (Fig. 8A). These results seem to be contradictory to the MD simulation data which shows the side chain dynamics of K105 destabilizes the αC-β4 loop region in the F100A mutant (Fig. 10B). It would be helpful if the authors could clarify this apparent discrepancy.

3. The foundation for the experiments carried out in this paper are based on previous NMR and computational data for the F100A mutant. However, the specific results and conclusions from these previous experiments are not clearly described.

Reviewer #1 (Public Review):

Summary:<br /> This work seeks to isolate the specific effects of phosphoinositide 3-kinase (PI3K) on the trafficking of the ion channel TRPV1, distinct from other receptor tyrosine kinase-activated effectors. It builds on earlier studies by the same group (Stein et al. 2006; Stratiievska et al. 2018), which described the regulatory relationship between PI3K, nerve growth factor (NGF), and TRPV1 trafficking. A central theme of this study is the development of methods that precisely measure the influence of PI3K on TRPV1 trafficking and vice versa. The authors employ a range of innovative methodologies to explore the dynamics between TRPV1 and PI3K trafficking.

Strengths:<br /> A major strength of this study is the application of innovative methods to understand the interaction between PI3K and TRPV1 trafficking. The key techniques presented include:

1) The optogenetic trafficking system based on phytochrome B, introduced in this research. Its interaction mechanism, dependent on reversible light activation, is comprehensively explained in Figures 1 and 2, with the system's efficacy demonstrated in Figure 3.

2) An extracellular labeling method using click chemistry, which although not exclusive to this study, introduces specific reagents engineered for membrane impermeability.

The central biological insight presented here is the sufficiency of PI3K activation to guide TRPV1 trafficking to the plasma membrane. An additional notable discovery is the potential regulation of insulin receptors via this mechanism.

The paper's strengths are anchored in its innovative methodologies and the valuable collaboration between groups specializing in distinct areas of research.

Weaknesses:<br /> The paper might benefit from a more streamlined structure and a clearer emphasis on its findings. A possible way to enhance its impact might be to focus more on its methodological aspects. The methodological facets stand out as both innovative and impactful. These experiments are well-executed and align with biological expectations. It's evident how these techniques could be tailored for many protein trafficking studies, a sentiment echoed in the manuscript (lines 287-288). When seen through a purely biological lens, some findings, like those concerning the PI3K-TRPV1 interaction, are very similar to previous work (Stratiievska et al. 2018). A biological focus demands further characterization of this interaction through mutagenesis. Also, the incorporation of insights on the insulin receptor feels somewhat tangential. A cohesive approach could be to reshape the manuscript with a primary focus on methodology, using TRPV1 and InsR as illustrative examples.

Reviewer #1 (Public Review):

• A summary of what the authors were trying to achieve.

The authors cultured pre- and Post-vaccine PBMCs with overlapping peptides encoding S protein in the presence of IL-2, IL-7, and IL-15 for 10 days, and extensively analyzed the T cells expanded during the culture; by including scRNAseq, scTCRseq, and examination of reporter cell lines expressing the dominant TCRs. They were able to identify 78 S epitopes with HLA restrictions (by itself represents a major achievement) together with their subset, based on their transcriptional profiling. By comparing T cell clonotypes between pre- and post-vaccination samples, they showed that a majority of pre-existing S-reactive CD4+ T cell clones did not expand by vaccinations. Thus, the authors concluded that highly-responding S-reactive T cells were established by vaccination from rare clonotypes.

• An account of the major strengths and weaknesses of the methods and results.

Strengths<br /> • Selection of 4 "Ab sustainers" and 4 "Ab decliners" from 43 subjects who received two shots of mRNA vaccinations.<br /> • Identification of S epitopes of T cells together with their transcriptional profiling. This allowed the authors to compare the dominant subsets between sustainers and decliners.

Weaknesses<br /> • Fig. 3 provides the epitopes, and the type of T cells, yet the composition of subsets per subject was not provided. It is possible that only one subject out of 4 sustainers expressed many Tfh clonotypes and explained the majority of Tfh clonotypes in the sustainer group. To exclude this possibility, the data on the composition of the T cell subset per subject (all 8 subjects) should be provided.<br /> • S-specific T cells were obtained after a 10-day culture with peptides in the presence of multiple cytokines. This strategy tends to increase a background unrelated to S protein. Another shortcoming of this strategy is the selection of only T cells amenable to cell proliferation. This strategy will miss anergic or less-responsive T cells and thus create a bias in the assessment of S-reactive T cell subsets. This limitation should be described in the Discussion.<br /> • Fig. 5 shows the epitopes and the type of T cells present at baseline. Do they react to HCoV-derived peptides? I guess not, as it is not clearly described. If the authors have the data, it should be provided.<br /> • As the authors discussed (L172), pre-existing S-reactive T cells were of low affinity. The raw flow data, as shown in Fig. S3, for pre-existing T cells may help discuss this aspect.

Reviewer #1 (Public Review):

Drawing on insights from preceding studies, the researchers pinpointed mutations within the spag7 gene that correlate with metabolic aberrations in mice. The precise function of spag7 has not been fully described yet, thereby the primary objective of this investigation is to unravel its pivotal role in the development of obesity and metabolic disease in mice. First, they generated a mice model lacking spag7 and observed that KO mice exhibited diminished birth size, which subsequently progressed to manifest obesity and impaired glucose tolerance upon reaching adulthood. This behaviour was primarily attributed to a reduction in energy expenditure. In fact, KO animals demonstrated compromised exercise endurance and muscle functionality, stemming from a deterioration in mitochondrial activity. Intriguingly, none of these effects was observed when using a tamoxifen-induced KO mouse model, implying that Spag7's influence is predominantly confined to the embryonic developmental phase. Explorations within placental tissue unveiled that mice afflicted by Spag7 deficiency experienced placental insufficiency, likely due to aberrant development of the placental junctional zone, a phenomenon that could impede optimal nutrient conveyance to the developing fetus. Overall, the authors assert that Spag7 emerges as a crucial determinant orchestrating accurate embryogenesis and subsequent energy balance in the later stages of life.

The study boasts several noteworthy strengths. Notably, it employs a combination of animal models and a thorough analysis of metabolic and exercise parameters, underscoring a meticulous approach. Furthermore, the investigation encompasses a comprehensive evaluation of fetal loss across distinct pregnancy stages, alongside a transcriptomic analysis of skeletal muscle, thereby imparting substantial value. However, a pivotal weakness of the study centres on its translational applicability. While the authors claim that "SPAG7 is well-conserved with 97% of the amino acid sequence being identical in humans and mice", the precise role of spag7 in the human context remains enigmatic. This limitation hampers a direct extrapolation of findings to human scenarios. Additionally, the study's elucidation of the molecular underpinnings behind the spag7-mediated anomalous development of the placental junction zone remains incomplete. Finally, the hypothesis positing a reduction in nutrient availability to the fetus, though intriguing, requires further substantiation, leaving an aspect of the mechanism unexplored.

Hence, in order to fortify the solidity of their conclusions, these concerns necessitate meticulous attention and resolution in the forthcoming version of the manuscript. Upon the comprehensive addressing of these aspects, the study is poised to exert a substantial influence on the field, its significance reverberating significantly. The methodologies and data presented undoubtedly hold the potential to facilitate the community's deeper understanding of the ramifications stemming from disruptions during pregnancy, shedding light on their enduring impact on the metabolic well-being of subsequent generations.

both being aware of ones tendency towards a wandering mind, and the ability to refocus towards the here and now.

Reviewer #1 (Public Review):

The authors start from the premise that neural circuits exhibit "representational drift" -- i.e., slow and spontaneous changes in neural tuning despite constant network performance. While the extent to which biological systems exhibit drift is an active area of study and debate (as the authors acknowledge), there is enough interest in this topic to justify the development of theoretical models of drift.

The contribution of this paper is to claim that drift can reflect a mixture of "directed random motion" as well as "steady state null drift." Thus far, most work within the computational neuroscience literature has focused on the latter. That is, drift is often viewed to be a harmless byproduct of continual learning under noise. In this view, drift does not affect the performance of the circuit nor does it change the nature of the network's solution or representation of the environment. The authors aim to challenge the latter viewpoint by showing that the statistics of neural representations can change (e.g. increase in sparsity) during early stages of drift. Further, they interpret this directed form of drift as "implicit regularization" on the network.

The evidence presented in favor of these claims is concise. Nevertheless, on balance, I find their evidence persuasive on a theoretical level -- i.e., I am convinced that implicit regularization of noisy learning rules is a feature of most artificial network models. This paper does not seem to make strong claims about real biological systems. The authors do cite circumstantial experimental evidence in line with the expectations of their model (Khatib et al. 2022), but those experimental data are not carefully and quantitatively related to the authors' model.

To establish the possibility of implicit regularization in artificial networks, the authors cite convincing work from the machine-learning community (Blanc et al. 2020, Li et al., 2021). Here the authors make an important contribution by translating these findings into more biologically plausible models and showing that their core assumptions remain plausible. The authors also develop helpful intuition in Figure 4 by showing a minimal model that captures the essence of their result.

In Figure 2, the authors show a convincing example of the gradual sparsification of tuning curves during the early stages of drift in a model of 1D navigation. However, the evidence presented in Figure 3 could be improved. In particular, 3A shows a histogram displaying the fraction of active units over 1117 simulations. Although there is a spike near zero, a sizeable portion of simulations have greater than 60% active units at the end of the training, and critically the authors do not characterize the time course of the active fraction for every network, so it is difficult to evaluate their claim that "all [networks] demonstrated... [a] phase of directed random motion with the low-loss space." It would be useful to revise the manuscript to unpack these results more carefully. For example, a histogram of log(tau) computed in panel B on a subset of simulations may be more informative than the current histogram in panel A.

Reviewer #1 (Public Review):

C. elegans is a pre-eminent model for developmental genetics, and its invariant lineage makes it possible in theory to define molecular features such as gene expression comprehensively and at single cell resolution across the organism.

Previously published single-cell RNA-seq studies have mapped gene expression across the lineage through the 16-cell stage (Tintori et al 2017, Hashimshony et al 2016), and at later stages (Packer et al 2019, with good coverage starting at the 100-cell stage and some coverage at the ~50-cell stage). This left the critical period around gastrulation (~28-cell and ~50-cell) without comprehensive transcriptome data. This study covers this gap with a heroic effort involving the manual isolation and analysis of over 800 cells from embryos of known stage, combined with painstaking curation using known markers from small scale studies and larger imaging-based expression atlases. Importantly, the dataset overlaps at early and late stages with data prior studies.

The data quality and overlap with Tintori and Packer datasets both appear high, but to make this inference required additional analysis from Supplemental Table 6 by this reviewer as it is not explored or described in the manuscript. Analyses demonstrating continuity with these datasets would greatly increase the value of the resource.

The authors show that specific lineages and stages preferentially express TFs with different classes of DNA binding domains. This extends previous work implicating homeodomains as preferentially involved in nervous system patterning and as enriched in neural and muscle progenitors in mid-stage embryos.

They also show that C. elegans homologs of Drosophila early embryonic regulators (which function based on spatial position in that system) tend to also be patterned in early C. elegans embryos, but with lineage-specific patterns. This conserved use of regulators would be fairly remarkable given the dramatically different developmental modes in these two species, although this observation is not backed up by quantitative analyses.

Finally, there is an argument that combinations of TFs expressed in lineage-specific patterns give rise to "stripe" patterns. This section is also not based on statistical analyses but suggests the possibility that lineage and positional regulation may be more convoluted than was previously thought.

Reviewer #1 (Public Review):

In this manuscript, the authors have performed extensive imaging analysis of six human histone H1 variants, their enrichment and localization, their differential dynamics during interphase and mitosis, and their association with lamina-associated domains (LADs) or nucleolus-associated domains. The manuscript is well-written with high-quality confocal and super-resolution images. Various interesting observations are made on distribution patterns of H1 variants. H1.2, H1.3, and H1.5 are shown to be universally enriched at the nuclear periphery whereas H1.4 and H1X are found to be distributed throughout the nucleus. Interestingly, H1X was the only H1 variant found to be abundant in nucleoli. Depletion of H1 variants has been shown to affect chromatin structure in a variant-specific manner, with H1.2 knock-down resulting in global chromatin decompaction. Overall, the study presents several interesting insights on H1 variants conducted in a large number of cell lines.

Major Comments:<br /> 1) Though the co-immunostaining of a nucleolar marker (NPM1) is performed with H1X, it would be interesting to explore the localization of H1 variants with respect to some of the proteins critically involved in chromatin organization such as PC4 or HP1alpha. Since the phosphorylated form of PC4 has been shown to interact with H1, which variants specifically interact with PC4 and how their dynamic changes in interphase and mitosis would be worth exploring.

2) The manuscript would be a complete study if any physiological significance with the H1 variant distribution could be shown.

Reviewer #1 Public Review

Summary<br /> This paper presents a new, but simple and low-cost technique for multimodal EM imaging that combines the strengths of both volume scanning electron microscopy (SEM) and electron microscopic tomography. The novel ATUM-Tomo approach enables the consecutive inspection of selected areas of interest by correlated serial SEM and TEM, optionally in combination with CLEM, as demonstrated here. The most important feature of ATUM-Tomo, particularly of correlative ATUM-Tomo, is that it can bridge scales, from the cellular to the high-resolution subcellular scale, from micrometer to low nanometer resolution. This is particularly important for ultrastructural analyses of biological regions of interest, which is demonstrated here for focal pathologies or rare cellular and subcellular events. Both imaging modalities are non-destructive, thus allowing re-imaging and hierarchical imaging at the SEM and TEM levels. This is particularly important for precious samples, including human biopsies and samples from complex CLEM experiments. Beyond the demonstrated neuropathology-related application, further use in investigating normal and pathologically altered brains, including human brain tissue samples that require high-resolution SEM and TEM in combination with immunohistochemistry, and virus or tracer injections, would be possible. Thus, ATUM-Tomo provides new possibilities in multimodal volume EM imaging for diverse areas of biological research.

Strengths<br /> This paper is a very nice piece of work, bringing together modern high-end state-of-the-art technology that will allow us to investigate diverse biological questions in different areas of interest and at different scales. The paper is clear and well-written, although some additions are necessary to the methods section and the scientific results as exemplified by investigations of the blood-brain barrier. The discussion would benefit from an expansion of the part dealing with the scientific results. The paper is accompanied by excellent figures, supplemental information, and colored 3D-reconstructions, which makes it easy for the reader to follow the experimental procedure and the scientific context. The authors may consider moving the supplemental figures into the main body of the paper, which would then still contain 'only' eight figures.

Weaknesses<br /> There is some imbalance between the description of the state-of-the-art methodology and the scientific context.

Reviewer #1 (Public Review):

Sun and co-authors have determined the crystal structures of EHEP with/without phlorotannin analog, TNA, and akuBGL. Using the akuBGL apo structure, they also constructed model structures of akuBGL with phlorotannins (inhibitor) and laminarins (substrate) by docking calculation. They clearly showed the effects of TNA on akuBGL activity with/without EHEP and resolubilization of the EHEP-phlorotannin (eckol) precipitate under alkaline conditions (pH >8). Based on this knowledge, they propose the molecular mechanism of the akuBGL-phlorotannin/laminarin-EHEP system at the atomic level. Their proposed mechanism is useful for further understanding of the defensive-offensive association between algae and herbivores.

Reviewer #1 (Public Review):

Gambelli et al. provide a structural study of the SlaA/SlaB S-layer of the archaeon Sulfolobus acidocaldarius. S-layers form an essential component of most archaeal cell envelopes, where their self-assembling properties and activity as cell envelope support structures have raised substantial interest, both from researchers seeking to understand the fundamental biology of archaea, as well as researchers seeking to exploit the biomaterial properties of S-layers in biotechnological applications. Both interests are hampered by the paucity of structural information on archaeal S-layer assembly, structure, and function to date, in large part due to technical difficulties in their study.

In this study, Gambelli and coworkers overcome these difficulties and report the high-resolution 3D cryoEM structures of the purified SlaA monomers at three different pH, as well as the medium resolution 3D cryoET structures of the SlaA/SlyB lattices determined from S-layer fragments isolated from the Sulfolobus cells.

The structural work is generally well executed, although lacks in detail in places to allow a proper review, particularly in the cryoET. A further drawback of the current manuscript is that the structural work remains rather descriptive and speculative, with little validation of the proposed models.

The authors run a plethora of representation, analyses, prediction, and simulation software on their structures resulting in an abundance of Figures that risk overloading the reader and in several cases bring little new insight beyond unsubstantiated speculation.

The structural description of the S. acidocaldarius S-layer will be of high general interest and the authors have made a substantial leap forward, but the current manuscript would benefit from a better validation and basic atomic description of the SlaA/SlaB S-layer.

Specific points.

- It is not possible to review the quality of the SlaA and SlaA/SlaB models in the cryoET reconstruction. No detailed fits of the map and model are shown, and no correlation statistics are given (the latter is also true for the higher resolution 3D reconstructions at pH4, 7, and 10). To be of use to the community, the S-layer model and cryoET maps should also be deposited in PDB and EMDB, and an autodep report and ideally the cryoET maps should be available.

- The authors spend a great deal on the MD simulation of the SlaA glycans and the description of the 'glycan shield' and its possible role in subunit electrostatics and intersubunit contacts. This does not result in testable hypotheses, however, and does not bring much more than vague speculation on the role of the glycans or the subunits contacts in S-layer assembly and stability. For the primary description of the SlaA/B S-layer, more important would be a detailed atomic description and validation of the intermolecular contacts in the proposed lattice model. Given the low resolution of the cryoET, this would require MD simulation of the contacts. Lattice stability during MD simulation and/or the confirmation of lattice contacts by cross-linking mass spectrometry would go a great way in validating the proposed lattice model.

- The discussion of the subunit electrostatics and the role they could play in subunit assembly/disassembly remains superficial and speculative. No real model or hypothesis is put forward, let alone validated.

- The authors solve the cryoEM structure of SlaA released and purified form S. acidocaldarius S-layers by an alkaline pH shift. When shifted back to acidic pH, does this native material self-assemble in vitro? If not, do the authors have an explanation for this? Are components missing or could the solved structures represent SlaA conformations that are no longer assembly competent?

Reviewer #1 (Public Review):

The manuscript by Royall et al. builds on previous work in the mouse that indicates that neural progenitor cells (NPCs) undergo asymmetric inheritance of centrosomes and provides evidence that a similar process occurs in human NPCs, which was previously unknown.

The authors use hESC-derived forebrain organoids and develop a novel recombination tag-induced genetic tool to birthdate and track the segregation of centrosomes in NPCs over multiple divisions. The thoughtful experiments yield data that are concise and well-controlled, and the data support the asymmetric segregation of centrosomes in NPCs. These data indicate that at least apical NPCs in humans undergo asymmetric centrosome inheritance. The authors attempt to disrupt the process and present some data that there may be differences in cell fate, but this conclusion would be better supported by a better assessment of the fate of these different NPCs (e.g. NPCs versus new neurons) and would support the conclusion that younger centriole is inherited by new neurons.

Reviewer #1 (Public Review):

Myelodysplastic syndrome (MDS) represents as a rather complex and serious hematologic malignancy that affects the production of normal blood cells in the bone marrow. Some types of MDS could stay mild for years and other types of MDS could be more serious and progressed into AML. Tremendous efforts have been made to investigate the pathogenesis and treatment of MDS. For instance, a pile of papers has found that iron chelation therapy could benefit the overall survival in low risk MDS patients. Yet, the risk and benefit of this therapy remain in much debate. The authors demonstrated that erythrocyte precursors could re-gain EPO responsiveness after DFP chelation therapy. In addition, the authors investigated iron trafficking in erythroblasts using the MDS mouse model. The paper is rather interesting as it discussed the biological effects and underlying mechanisms of DFP for the treatment of low-risk MDS. More importantly, the paper adds practical values and theoretical evidences for chelation therapy towards low-risk MDS. The paper is overall well-written.

Reviewer #1 (Public Review):

In this manuscript, Kim et al. investigate the molecular basis for hindbrain segmentation by performing combined single cell nucleus RNAseq and ATACseq (scMultiome) on zebrafish embryonic hindbrain tissue. Hindbrain segmentation is fundamental to head development in vertebrate species. Decades of research have provided many insights into the gene regulatory cascades that control the progressive subdivision of the hindbrain territory into segments (rhombomeres). These studies have enabled the formulation of gene regulatory network (GRN) models that depict these regulatory interactions. However, many aspects of the GRN need further clarification, including the early steps of pre-rhombomeric patterning, and the factors that respond to axial signaling pathways such as RA and FGF. The dataset in this study provides a comprehensive view of gene expression and chromatin states during hindbrain segmentation, thus it is a valuable resource for characterizing the underlying GRN. The authors demonstrate the utility of this data by comparing the molecular profiles between different rhombomeres and tracing when and how these profiles arise during development.

Four main findings are presented:

1. Each rhombomere has a unique molecular profile.<br /> 2. There is no clear molecular signature for odd versus even rhombomeres, nor any overt repeating two-segment molecular identities.<br /> 3. The mature rhombomeres emerge through the subdivision of three mixed-identity 'primary hindbrain progenitor domains' (PHPDs) that correspond to r2/r3, r4, and r5/r6, respectively.<br /> 4. RA and FGF signaling control formation of the primary hindbrain progenitor domains.

These findings are well supported by the data but in my opinion they mainly confirm what was already known and do not significantly advance our mechanistic understanding of rhombomere formation, which is the aim of the paper.

Strengths:<br /> This comprehensive dataset will be very valuable to researchers in the field. The authors successfully demonstrate its utility by resolving unique molecular profiles for each rhombomere and identifying some novel markers.

The authors make excellent use of HCR to validate their findings, such as the co-expression of vgll3 and egr2b in r2/r3 cells at 10hpf, which implies mixed identities of PHPD cells.

The performance of scMultiome analysis on tissue from DEAB-treated embryos (depleted RA signaling) is exciting and holds much promise for identifying RA-dependent gene regulatory cascades that govern caudal hindbrain patterning. Assessing the contribution of control versus DEAB-treated cells to the various UMAP clusters is a very nice way to identify the altered cell states in the RA-depleted hindbrain. This confirms a complete absence of r5 and r6 in the DEAB-treated embryos at this developmental stage, as was inferred from in-situ approaches in earlier studies.

Weaknesses:<br /> The major weakness of this work is that it only provides an incremental mechanistic advance to our current understanding of the molecular basis for rhombomere formation. The descriptions of gene expression are useful but for the most part they are rather shallow lines of enquiry that confirm what was already known from previous, less comprehensive studies of gene expression. For example, regarding the identification of PHPDs, it has long been known that r5/r6 share a progenitor domain that is demarcated by mafba expression. Similarly, RA and Fgf signaling have already been shown to be required for anterior-posterior patterning in the pre-rhombomeric hindbrain. The identification of mixed-identity progenitors in PHPDs, and the characterisation of the changes in transcription and chromatin state in response to RA signaling perturbation are really exciting starting points for deeper analysis of the underlying GRN. However, it is a shame that no effort is made to glean mechanistic insights from this dataset by computational GRN inference.

Reviewer #1 (Public Review):

This manuscript by Proskurin, Manakov, and Karpova, posits a unique role for the anterior cingulate cortex (ACC) in the flexible control of learned sequences of motor actions. The authors marshall evidence from behavioral-electrophysiological analyses in support of two major claims: 1) that action encoding by ACC ensembles tracks 1) the current "context", i.e., which behavioral sequence is rewarded, and 2) the "prevalence", i.e., number of repetitions of one specific sequence. An important aspect of this later point is that the authors propose prevalence encoding is not strictly dependent on trial-by-trial reward receipt.

In this work, the authors wish to focus on self-initiated behavior when the correct behavioral sequence, out of four or fewer (mostly two it appears), changes across blocks in an unsignaled manner. Rats learn to enter a left and right nose poke in a sequence of three responses, with a required entry into a central port prior to each intra-sequence response, with correct sequence completion reinforced by a sucrose delivery in the relevant side nose poke port. Extracellular spike activity is acquired from well-trained rats performing this task. The authors' analyses of the behavior of well-trained rats show rats adjust their behavior when the block switches to a different one of the sequences in the known 'library'. The rats also perform non-reinforced responses/sequences within a given block which the authors suggest is exploration likely not triggered by changes in reinforcement in contrast to behavior change after a block switch.

The authors next provide a very rich set of analyses to examine the encoding of responses and sequences by ACC neural activity. Overall, these data provide intriguing support for ACC's integral contributions to flexible behavioral control. However, some of the individual analyses are a bit difficult to follow and could be clarified with greater detail within the results section of the paper, permitting an easier evaluation of the quality of the supporting data. Second, there are some proposals that could be strengthened by fuller analysis, in particular the authors' suggestion that "prevalence" encoding is distinct from reward encoding and/or is not impacted by reward presence or omission. Given the likely rich data set in hand, the authors could do more to demonstrate how "prevalence" encoding interacts with reinforcement parameters or perhaps be more specific in their word choice. More importantly, I was left unclear on how "prevalence" encoding intersects with the decision to repeat the same behavioral sequence on the next trial or not. These issues aside, this work provides further information on the physiology of ACC during flexible behavior and will add importance to this field.

Below are specific issues:

1. Some greater attention to the behavioral parameters could be helpful, especially regarding the impact of reward rate on behavior. For example, looking at some of the figures of individual rat behavior, exploratory sequences seemed triggered by reward omission. Is this just a chance for the examples chosen or is there something systematic here? Upon block switch, how exactly does the switch in sequences emitted by the rat track with reinforcement history? The authors mention that reinforcement probability differed across sessions, and one would thus expect switching behavior would as well. Because of the interesting existence of sometimes quite long 'tails' of performance of the original sequence after a block switch, I am wondering how the length of such tails relates to reinforcement rate parameters.

2. The authors provide strong data indicating that a given L or R response is associated with distinct ACC activity depending on which sequence that response is embedded within, a finding reminiscent of other reports in multiple brain regions. While not a criticism per se, I was interested in the center port responses, also embedded within unique sequences, yet never preceding reward. A key difference in the performance of a given R or L response is that it is sometimes the terminal response, and thus the rat knows a given R or L response to be sometimes reinforced in one of the contexts, but not the other, in each of these comparisons. I wonder if there was an opportunity to cleanly demonstrate the context dependence of a given individual action by comparing center port responses across distinct sequences.

3. In analyzing neural activity accompanying the behavioral persistence of the dominant sequence after a block change, the authors find that the ACC ensemble firing pattern is closer to the original dominant sequence pattern during reinforcement and less like this pattern during exploration. This makes sense and must be the case, as, in the example shown in the figure, the rat does not "know" the block has switched since no reward has yet been delivered that would signal that switch. (As an aside, it would be interesting to know, given a specific reward schedule in a given session, what would be the maximum number of unrewarded trials within the block, and how might that impact the performance/reward expectation during the tails?)<br /> As time, and trials, progress the rat is approaching the point at which it explores another strategy. The authors find strengthened "prevalence" encoding with increasing sequence repetition, but if this parameter is related to behavioral change/flexibility, this was not clear to me. Might there be something unique about the last trials in a tail "predicting" an upcoming switch? Can the authors please expand?<br /> Relatedly, if the prediction of upcoming behavioral change is not observed in the neural activity from sequence steps 2-6, it is notable that these are the steps 'within' the sequence, that leaves out the initiation (first center poke) and termination (reward/reward omission). Thus one could imagine this information is "missed" in the current analysis given that both the reward period and the initiation of a trial at the center are not analyzed. This does lead me to suggest a softening of some claims made of identifying "unifying principles" of ACC function, as the authors state, based on the analyses included in the current report, since the neural activity related to the full unit of behavior is not considered. (I appreciate the motivation behind this focus on within-sequence behavior - the wish to compare time periods with similar movement parameters .)

4. The variance in neural activity explained by the prevalence models is on average quite low. However, the authors find that the variance explained differs quite dramatically by anatomical coordinate within ACC. Would it make sense to focus the control analyses (vigor, reward history, and so on) on those sessions/ensembles with greater variance explained, ie, perhaps there might be greater sensitivity to detecting interactions among variables within ensembles recorded more rostrally?

5. A very intriguing aspect of this work is the position that (from the abstract): "Prevalence encoding in the ACC is ...independent of reward delivery." This is a novel aspect of the current work. However, I am wondering if the authors can refine and expand upon this. I find it difficult to disentangle prevalence encoding and impacts of reward in the way the data and interpretation are presented in some areas of the text. While neural encoding may not reflect trial-by-trial reward receipt, clearly the rat's decision to repeat a given sequence or initiate a new sequence is impacted by reinforcement parameters and reward expectation. Thus being very exact in the interpretation would be helpful.

Reviewer #1 (Public Review):

Summary:

Fox, Dan, and Loewenstein investigated how people explored six maze-like environments. They show that roughly one-third of their participants make choices now that increase the potential for future information gain and also temporally discount potential information gain based on how far in the future potential gains might be. The authors argue that rather than valuing exploration in its own right, participant behavior is most consistent with using exploration as a way to reduce uncertainty. They then propose a reinforcement learning (RL) model in which agents estimate an "exploration value" (the expected cumulative information gained by taking a given action in a given state) using standard RL techniques for estimating value (expected cumulative reward). They find that this model exhibits several qualitative similarities with human behavior and that it best captures the temporal dynamics of human exploration when propagating information through the entire history of a behavioral episode (as opposed to merely propagating it in a single step as some of the simplest RL models do).

While the core insight and basic method of the paper are compelling, the way in which both the behavioral experiment and computational modeling were conducted raise concerns that mean that, in their present form, the results do not fully justify the conclusions. After resolving these issues, the work would demonstrate how human exploration is sensitive to long-range dependencies in information gain, as well as valuable insights about how best to characterize this behavior computationally. I am not particularly well-versed in the literature on exploration so cannot comment on novelty here.

Strengths:<br /> The entire paper is logically well-motivated. It builds on a valuable basic insight, namely that while bandit tasks are an ideally minimal platform for testing certain questions about decision-making and exploration, richer paradigms are needed to capture the long-range informational dependencies distinguishing between various approaches to exploration.

Even so, the maze navigation paradigm explored here remains simple. Participants navigate a maze with two main branches which are identical save for minimal, theoretically motivated differences. Moreover, the tested differences are designed to clearly and explicitly test well-identified questions. The task, and really the entire paper, is clearly organized, and each component is logically connected to a larger argument.

The proposed model is also simple, clearly presented, and a clever way of applying ideas typically used to reason about reward-motivated behavior to reason here about information-motivated behavior.

One other strength of this work is that it combines behavioral experiments with computational modeling. This approach pairs a detailed and objectively specified theory (i.e. the model) with novel data specifically designed to test that theory and thus in principle presents a particularly strong test of the authors' hypotheses.

Weaknesses:<br /> Despite many strengths in the underlying logic of the paper, the presented evidence does not provide compelling support for the conclusions. In particular:

- The main claims are based on the behavior of 452 participants classed as good explorers, out of 1,052 participants included in the analyses and 1,336 participants who completed the study. That is, the authors' broad claims about human exploration are based on a third of their total sample; the other two-thirds displayed very different behavior, including 20% who performed at or below chance levels. That is, while a significant sub-population may demonstrate the claimed abilities, it is far from clear that they are universal.

- While the experimental manipulations are elegant, the behavioral study seems underpowered. In each of the primary manipulations, key theoretical predictions are not statistically validated. For example, in Experiment 1, the preference for the right door increases from the 4:3 condition to the 5:2 condition, but not when moving from the 5:2 condition to the 6:1 condition, as predicted (Figure 1c). Similar results can be seen for other analyses in Figures 3b and 4b. Relatedly, the experiments comprised just 20 episodes, and it is unclear whether that was sufficiently long for participants to demonstrate asymptotic behavior (e.g. Figure 5b). Either more participants or greater differences between conditions (e.g. testing 9:8, 12:5, and 15:2 conditions in a revised Experiment 1), as well as running a greater number of total episodes, would be needed to resolve this concern.

- The model is presented after the behavioral results, giving the impression that it was perhaps constructed to fit the data. No attempt is made to fit the model to a subset of the data and then validate the rest or give any clear indication as to how the model parameters were set. Moreover, as noted, even where the model is successful, it only explains the behavior of a minority of the total participants. No modeling work is done to explain the behavior of the other two-thirds of the participants.

- The authors helpfully discuss several meaningful alternative models of exploration, such as visit-counting and incorporating an objective function sensitive to information gain. They do not, however, compare their model against these or any other meaningful baselines. Moreover, the comparison between model and human participants is qualitative rather than quantitative. These issues could be resolved by introducing a more rigorous analysis quantitatively comparing a variety of theoretically relevant models as quantitative explanations of the human data.

Reviewer #1 (Public Review):

In this study, the authors utilise different chemical inhibitors and celular markers to examine the roles of macropinocytosis in WNT signalling activation in development (Xenopus), cell culture (3T3 cells) and cancer (CRC sections). Furthermore, they investigate the effect of the inflammation inducer Phorbol-12-myristate-13-acetate (PMA) in WNT signalling activation through macropinocytosis. The authors show 1) that PMA induces macropinocytosis-dependent WNT signalling activation, and 2) that CRC development correlates with increased levels and co-localisation of macropinocytosis components and b-catenin.

I found the analyses and conclusions compelling. Additional epistatic analyses could be done in the future to further disentangle the roles of macropinocytosis during WNT signalling activation, especially upon oncogenic alterations (e.g. in APC). The studies on CRC samples open interesting questions for specialists in tumour progression.

Reviewer #1 (Public Review):

Summary:<br /> Ngoune et al. present compelling evidence that Slender cells are challenged to infect tsetse flies. They explore the experimental context of a recent important paper in the field, Schuster et al., that presents evidence suggesting the proliferative Slender bloodstream T.brucei can infect juvenile tsetse flies. Schuster et al. were disruptive to the widely accepted paradigm that the Stumpy bloodstream-form is solely responsible for tsetse infection and T.brucei transmission potential.

Evidence presented here shows that in all cases, Stumpy form parasites are exponentially more capable of infecting tsetse flies. They further show that Slender cells do not infect mature flies.

However, they raise questions of immature tsetse immunological potential and field transmission potential that their experiments do not address. Specifically, they do not show that teneral tsetse flies are immunocompromised, that tsetse flies must be immunocompromised for Slender infection nor that younger teneral tsetse infection is not pertinent to field transmission.

Strengths:<br /> Experimental Design is precise and elegant, outcomes are convincing. Discussion is compelling and important to the field. This is a timely piece that adds important data to a critical discussion of host: parasite interactions, of relevance to all parasite transmission.

Weaknesses:<br /> As above, the authors dispute the biological relevance of teneral tsetse infection in the wild, without offering evidence to the contrary. Statements need to be softened for claims regarding immunological competence or relevance to field transmission.

Reviewer #1 (Public Review):

DeKraker et al. propose a new method for hippocampal registration using a novel surface-based approach that preserves the topology of the curvature of the hippocampus and boundaries of hippocampal subfields. The surface-based registration method proved to be more precise and resulted in better alignment compared to traditional volumetric-based registration. Moreover, the authors demonstrated that this method can be performed across image modalities by testing the method with seven different histological samples. This work has the potential to be a powerful new registration technique that can enable precise hippocampal registration and alignment across subjects, datasets, and image modalities.

Reviewer #1 (Public Review):

Summary:<br /> Van der Heijden et al perform an ambitious analysis of single-unit activity in the interposed nuclei of multiple mouse models of cerebellar dysfunction. Based on these recordings, they develop a classifier to predict the behavioral phenotype (ataxic, dystonic, or tremor) of each model, suggesting that highly regular spiking is associated with ataxia, irregular spiking is associated with dystonia and rhythmic spiking is associated with tremor. After developing this classifier, they show that activating Purkinje neurons in different patterns that evoke interposed nuclear activity similar to their "ataxic", "dystonic", and "tremor" firing patterns induce similar behaviors in healthy mice. These results show convincingly that specific patterns of cerebellar output are sufficient to cause specific movement abnormalities. The extent to which cerebellar nuclear firing patterns are solely responsible for phenotypes in human disease remains to be established, however.

Strengths:<br /> Major strengths are the recordings across multiple phenotypic models including genetic and pharmacologic manipulations, and the robust phenotypes elicited by Purkinje neuron stimulation.

Weaknesses:<br /> The number of units recorded was small for each model (on the order of 20), limiting the conclusions that can be drawn from the recording/classifier experiments.

Joint Public Review:

Summary<br /> This is a very meticulous and precise anatomical description of the external sensory organs (sensillia) in Drosophila larvae. Extending on their previous study (Rist and Thum 2017) that analyzed the anatomy of the terminal organ, a major external taste organ of fruit fly larva, the authors examined the anatomy of the remaining head sensory organs - the dorsal organ, the ventral organ, and the labial organ-also described the sensory organs of the thoracic and abdominal segments. Improved serial electron microscopy and digital modeling are used to the fullest to provide a definitive and clear picture of the sensory organs, the sensillia, and adjacent ganglia, providing an integral and accurate map, which is dearly needed in the field. The authors revise all the data for the abdominal and thoracic segments and describe in detail, for the first time, the head and tail segments and construct a complete structural and neuronal map of the external larval sensilla.

Strengths<br /> It is a very thorough anatomical description of the external sensory organs of the genetically amenable fruitfly. This study represents a very useful tool for the research community that will definitely use it as a reference paper. In addition to the classification and nomenclature of the different types of sensilla throughout the larval body, the wealth of data presented here will be valuable to the scientific community. It will allow for investigating sensory processing in depth. Serial electron microscopy and digital modeling are used to the fullest to provide a comprehensive, definitive, and clear picture of the sensory organs. The discussion places the anatomical data into a functional and developmental frame. The study offers fundamental anatomical insights, which will be helpful for future functional studies and to understand the sensory strategies of Drosophila larvae in response to the external environment. By analyzing different larval stages (L1 and L3), this work offers some insights into the developmental aspects of the larval sense organs and their corresponding sensory cells.

Weaknesses<br /> There are no apparent weaknesses, although it is not a complete novel anatomical study. It revisits many data that already existed, adding new information. However, the repetitiveness of some data and prior studies may be avoided for easy readability.

Reviewer #1 (Public Review):

Summary:<br /> The authors aimed to infer the trajectories of long range and local neuronal synchrony across the Alzheimer's disease continuum, relative to neurodegeneration and cognitive decline. The trajectories are inferred using event-based models, which infer a set of data-driven disease stages from a given dataset. The authors develop an adapted event-based modelling approach, in which they characterise each stage as a particular biomarker increasing by a particular z-score deviation from controls. Fitting infers the optimal set of z-scores to use for each biomarker and the order in which each biomarker reaches each z-score. The authors apply this approach to data from 148 individuals (70 cognitively unimpaired older adults and 78 individual with mild cognitive impairment or Alzheimer's disease), identifying trajectories in which long-range (amplitude-envolope correlation) and local (regional spectral power) neuronal synchrony in the alpha and beta bands becomes abnormal prior to neurodegeneration (measured as the volume of the parahippocampal gyrus) and cognitive decline (measured using the mini-mental state examination).

Strengths:<br /> - The main strength is that the authors assess two models. In the first they derive a staging system based only on the volume of the parahippocampal gyrus and mini-mental state examination score. They then investigate how neuronal synchrony metrics change compared to this staging system. In the second they derive a staging system that also includes an average (combined long-range and local) neuronal synchrony metric and investigate how long-range and local synchrony metrics change relative to this staging system. This is a strength as the first model provides confidence that there is not overfitting to the neuronal synchrony data, and the second provides more detailed insights into the dynamics of the early neuronal synchrony changes.<br /> - Another strength is that the authors automatically infer the optimal z-scores to choose, rather than having to pre-select them manually, as in previous approaches.

Weaknesses:<br /> - The dataset is small and no external validation is performed.<br /> - A high proportion of the data is from controls (nearly 50%) with no biomarker evidence of Alzheimer's disease, and so the changes may be driven by aging or other non-Alzheimer's effects.<br /> - Inferring the optimal z-scores is a strength, however as different sets of z-scores are allowed per biomarker, there is a concern that the changes reflected are mainly driven by the choice of z-score, rather than the markers themselves (e.g. if lower z-scores are selected for one marker than another, then changes in that marker will appear to be detected earlier, even if both markers change at the same time).<br /> - In equation 2 it is unclear why the gaussian is measured based on a sum over I. The more obvious choice would be to use a multivariate gaussian with no covariance, which would mean taking the product rather than the sum over I.<br /> - In the original event-based model, k is a hidden variable. Presumably that is also the case here, however the notation k=stage(j) makes it seem like each subject is assigned a stage during the sequence optimisation.<br /> - Typically for event-based modelling, positional variance diagrams are created from the markov chain monte carlo samples of the event sequence, enabling visualisation of the uncertainty in the sequence, but these are not included in the study.<br /> - Many of the figures in the manuscript (e.g. Figure 1E/G, Figure 2A/B, Figure 3A/B/E/F/I/J, Figure 4 A/B/E/F/I/J) are based on averages in both the x and the y axis. In the x dimension, individuals have a weighted contribution to the value on the y axis, depending on their stage probability. In the y dimension, the values are averages across those individuals, and the error bars represent the standard error rather than the standard deviation. Whilst the trajectories themselves are interesting, they may not be discriminative at the individual level and may be more heterogeneous than it appears.<br /> - The bootstrapped statistical analyses comparing metrics between the stages do not consider the variability in the sequence.

Reviewer #1 (Public Review):

In this manuscript, Lee et al. compared encoding of odor identity and value by calcium signaling from neurons in the ventral pallidum (VP) in comparison to D1 and D2 neurons in the olfactory tubercle (OT).

Strengths: They utilize a strong comparative approach, which allows the comparison of signals in two directly connected regions. First, they demonstrate that both D1 and D2 OT neurons project strongly to the VP, but not the VTA or other examined regions, in contrast to accumbal D1 neurons which project strongly to the VTA as well as the VP. They examine single unit calcium activity in a robust olfactory cue conditioning paradigm that allows them to differentiate encoding of olfactory identity versus value, by incorporating two different sucrose, neutral and air puff cues with different chemical characteristics. They then use multiple analytical approaches to demonstrate strong, low-dimensional encoding of cue value in the VP, and more robust, high-dimensional encoding of odor identity by both D1 and D2 OT neurons, though D1 OT neurons are still somewhat modulated by reward contingency/value. Finally, they utilize a modified conditioning paradigm that dissociates reward probability and lick vigor to demonstrate that VP encoding of cue value is not dependent on encoding of lick vigor during sucrose cues, and that separable populations of VP neurons encode cue value/sucrose probability and lick vigor.

Weaknesses: The conclusions of the data are mostly well supported by the analyses, but the statistical analysis is somewhat limited and needs to be clarified and extended.

1) The manuscript includes limited direct statistical comparison of the neural populations, and many of the comparisons between the subregions are descriptive, including descriptions of the percentage of neurons having specific response types, or differences in effect sizes or differing "levels" of significance. An additional direct comparison of data from each subpopulation would help to confirm whether the differences reported are statistically meaningful.

2) When hypothesis tests are conducted between the neural populations, it is not clear whether the authors have accounted for the random effect of the subject, or whether individual units were treated as fully independent. For instance, pairwise differences are reported in Figures 4I, 5G/I/L, and others, but the statistical methods are unclear. Assessment of the statistics is further limited by the lack of reporting of degrees of freedom.

Reviewer #1 (Public Review):

Summary:<br /> Ever-improving techniques allow the detailed capture of brain morphology and function to the point where individual brain anatomy becomes an important factor. This study investigated detailed sulcal morphology in the parieto-occipital junction. Using cutting-edge methods, it provides important insights into local anatomy, individual variability, and local brain function. The presented work advances the field and will stimulate future research into this important area.

Strengths:<br /> Detailed, very thorough methodology. Multiple raters mapped detailed sulci in a large cohort. The identified sulcal features and their functional and behavioural relevance are then studied using various complementary methods. The results provide compelling evidence for the importance of the described sulcal features and their proposed relationship to cortical brain function.

Weaknesses:<br /> A detailed description/depiction of the various sulcal patterns is missing. A possible relationship between sucal morphology and individual demographics might provide more insight into anatomical variability. The unique dataset offers to opportunity to provide insights into laterality effects that should be explored.

Reviewer #1 (Public Review):

Summary:<br /> The authors provide very compelling evidence that the lateral septum (LS) engages in theta cycle skipping.

Strengths:<br /> The data and analysis are highly compelling regarding the existence of cycle skipping.

Weaknesses:<br /> The manuscript falls short on describing the behavioral or physiological importance of the witnessed theta cycle skipping, and there is a lack of attention to detail with some of the findings and figures:

More/any description is needed in the article text to explain the switching task and the behavioral paradigm generally. This should be moved from only being in methods as it is essential for understanding the study.

An explanation is needed as to how a cell can be theta skipping if it is not theta rhythmic.

The most interesting result, in my opinion, is the last paragraph of the entire results section, where there is more switching in the alternation task, but the reader is kind of left hanging as to how this relates to other findings. How does this relate to differences in decoding of relative arms (the correct or incorrect arm) during those theta cycles or to the animal's actual choice? Similarly, how does it relate to the animal's actual choice? Is this phenomenon actually behaviorally or physiologically meaningful at all? Does it contribute at all to any sort of planning or decision-making?

The authors state that there is more cycle skipping in the alternation task than in the switching task, and that this switching occurs in the lead-up to the choice point. Then they say there is a higher peak at ~125 in the alternation task, which is consistent. However, in the final sentence, the authors note that "This result indicates that the representations of the goal arms alternate more strongly ahead of the choice point when animals performed a task in which either goal arm potentially leads to reward." Doesn't either arm potentially lead to a reward (but different amounts) in the switching task, not the alternation task? Yet switching is stronger in the alternation task, which is not constant and contradicts this last sentence.

Additionally, regarding the same sentence - "representations of the goal arms alternate more strongly ahead of the choice point when the animals performed a task in which either goal arm potentially leads to reward." - is this actually what is going on? Is there any reason at all to think this has anything to do with reward versus just a navigational choice?

Similarly, the authors mention several times that the LS links the HPC to 'reward' regions in the brain, and it has been found that the LS represents rewarded locations comparatively more than the hippocampus. How does this relate to their finding?

Reviewer #1 (Public Review):

Summary:

This manuscript describes the development of an oral THC consumption model in mice where THC is added to a chocolate flavored gelatin. The authors compared the effects of THC consumed in this highly palatable gelatin (termed E-gel) to THC dissolved in a less palatable gelatin (CTR-gel), and to i.p. injections of multiple doses of THC, on the classic triad of CB1R dependent behaviors (hypolocomotion, antinociception, and body temperature).

The authors found that they could achieve consumption of higher concentrations of THC in the E-gel than the CTR-gel, and that this led to larger total dose exposure and decreases in locomotor activity, antinociception, and body temperature reductions similar to 3-4 mg/kg THC when tested after 2 hour consumption and roughly 10 mg/kg if tested immediately after 1 hour consumption. The majority of THC E-gel consumption was found to occur in the first hour on the first exposure day. THC E-gel consumption was lower than VEH E-gel consumption and this persisted on a subsequent consumption day, suggesting that the animals may form a taste aversion and that THC at the dose consumed likely has aversive properties, consistent with the literature on i.p. dosing. The authors also report the pharmacokinetics in brain and plasma of THC and metabolites after 1 or 2 hour consumption, finding high levels of THC in the brain that begins to dissipate at 2.5 hours is gone 24 hours later. Finally, the authors tested THC effects on the acoustic startle response and found an inverted dose response that was more pronounced in males than females after i.p. dosing and a greater startle response in males after E-gel dosing.

Overall, the authors find that voluntary oral consumption of THC can achieve levels of intake that are consistent with the present and prior reported literature on i.p. dosing.

Strengths:

The strengths of the article include a direct comparison of voluntary oral THC consumption to noncontingent i.p. administration, the use of multiple THC doses and oral THC formulations, the inclusion of multiple assays of cannabinoid agonist effects, and the inclusion of males and females. Additional strengths include monitoring intake over 10 minute intervals and validating that effects are CB1R dependent via antagonist studies.

Weaknesses:

1. The abstract does not discuss the reduction of E-gel consumption that occurs after multiple days of exposure to the THC formulation, but rather implies that a new model for chronic oral self-administration has been developed. Given that only two days of consumption was assessed, it is not clear if the model will be useful to determine THC effects beyond the acute measures presented here. The abstract should clarify that there was evidence of reduced consumption/aversive effects with repeated exposures.<br /> 2. In the results section, the authors sometimes describe effects in terms of the concentration of gel as opposed to the dose consumed in mg/kg, which can make interpretation difficult. For example, the text describing Figure 1i states that significant effects on body temperature were achieved at 4 mg CTR-gel and 5 mg THC-gel, but were essentially equivalent doses consumed? It would be helpful to describe what average dose of THC produced effects given that consumption varied within each group of mice assigned to a particular concentration.<br /> 3. The description of the PK data in Figure 3 did not specify if sex differences were examined. Prior studies have found that males and females can exhibit stark differences in brain and plasma levels of THC and metabolites, even when behavioral effects are similar. However, this does depend on species, route, timing of tissue collection. It would be helpful to describe the PK profile of males and females separately.<br /> 4. In Figure 5, it is unclear how the predicted i.p. THC dose could be 30 mg/kg when 30 mg/kg was not tested by the i.p. route according to the figure, and if it had been it would have likely been almost zero acoustic startle, not the increased startle that was observed in the 2 hr gel group. It seems more likely that it would be equivalent to 3 mg/kg i.p. Could there be an error in the modeling, or was it based on the model used for the triad effects? This should be clarified.

Reviewer #1 (Public Review):

Prior research demonstrated that vocal learning is sexually dimorphic in zebra finches; female song nuclei atrophy and fail to develop, but can be rescued with exogenous 17-𝛃-estradiol (E2) treatment. In previous research, the authors treated both male and female birds with exogenous E2. They laser-captured dissected tissue samples from the E2-treated individuals as well as untreated controls. They then extracted RNA and used RNA-seq to characterize the transcriptomes within and adjacent to four major song nuclei (HVC, LMAN, RA, Area X) in these birds. In this study, Davenport et al. remapped this massive amount of transcriptome data (n=3 birds per sex/treatment group) to fully resolve the genomic location of differentially expressed genes, which they assigned to several modules based on co-expression. Adequate read mapping to all chromosomes was previously impossible with zebra finch genome assemblies lacking W chromosome data. Using the high-quality zebra finch genome assembly with Z and W chromosomes (bTaeGut2.pat.W.v2), the authors were able to demonstrate the enrichment of certain modules on certain chromosomes; most interestingly, Z chromosome gene expression was increased in E2-treated females. This research greatly improves our understanding of the ontology and location of genes involved in song development in E2-treated females, providing insight into the development of vocal learning in the zebra finch.

The authors' main conclusions on the importance of certain gene modules in the vocal learning process are well warranted by their excellent data and thorough analyses, but should not be too broadly interpreted as necessarily applying to the gene expression involved in vocal learning in other species. While the data here further supports convergent evolution in vocal learning genes in humans and zebra finches, vocal learning is unusually sexually dimorphic in zebra finches compared to most other vocal learners.

The authors note the possibility of female haploinsufficiency of Z-linked genes such as the growth hormone receptor (GHR) and also imply there are potential effects of the fission of the ancestral chromosome into passerine chromosomes 1 and 1A impacting the typical development of male zebra finch song and the lack thereof in females. These thoughts are intriguing and should prompt further transcriptomic research in avian species with the same genomic features (ZW females, split 1 and 1A chromosomes) where females also learn song, i.e. female-singing passerine species. Currently, it is impossible to say if female-singing species are, as is likely with the E2-treated zebra finch females, using estrogen signaling pathways to regulate an increase in dosage of these genes. Alternately, these female-singing birds may be using different gene modules, which is also worthy of investigation. This research excels at elucidating the genomic underpinnings of vocal learning in a model organism; further research will demonstrate how broadly applicable these authors' findings are across other species.

Reviewer #1 (Public Review):

Summary:<br /> This paper presents an innovative decoding approach for brain-computer interfaces (BCIs), introducing a new method named MINT. The authors develop a trajectory-centric approach to decode behaviors across several different datasets, including eight empirical datasets from the Neural Latents Benchmark. Overall, the paper is well written and their method shows impressive performance compared to more traditional decoding approaches that use a simpler approach. While there are some concerns (see below), the paper's strengths, particularly its emphasis on a trajectory-centric approach and the simplicity of MINT, provide a compelling contribution to the field.

Strengths:<br /> The adoption of a trajectory-centric approach that utilizes statistical constraints presents a substantial shift in methodology, potentially revolutionizing the way BCIs interpret and predict neural behaviour. This is one of the strongest aspects of the paper.

The thorough evaluation of the method across various datasets serves as an assurance that the superior performance of MINT is not a result of overfitting. The comparative simplicity of the method in contrast to many neural network approaches is refreshing and should facilitate broader applicability.

Weaknesses:<br /> Scope: Despite the impressive performance of MINT across multiple datasets, it seems predominantly applicable to M1/S1 data. Only one of the eight empirical datasets comes from an area outside the motor/somatosensory cortex. It would be beneficial if the authors could expand further on how the method might perform with other brain regions that do not exhibit low tangling or do not have a clear trial structure (e.g. decoding of position or head direction from hippocampus)

When comparing methods, the neural trajectories of MINT are based on averaged trials, while the comparison methods are trained on single trials. An additional analysis might help in disentangling the effect of the trial averaging. For this, the authors could average the input across trials for all decoders, establishing a baseline for averaged trials. Note that inference should still be done on single trials. Performance can then be visualized across different values of N, which denotes the number of averaged trials used for training.

Reviewer #1 (Public Review):

This EEG study probes the prediction of a mechanistic account of P300 generation through the presence of underlying (alpha) oscillations with a non-zero mean. In this model, the P300 can be explained by a baseline shift mechanism. That is, the non-zero mean alpha oscillations induce asymmetries in the trial-averaged amplitudes of the EEG signal, and the associated baseline shifts can lead to apparent positive (or negative) deflections as alpha becomes desynchronized at around P300 latency. The present paper examines the predictions of this model in a substantial data set (using the typical P300-generating oddball paradigm and careful analyses). The results show that all predictions are fulfilled: the two electrophysiological events (P300, alpha desynchronization) share a common time-course, anatomical sources (from inverse solutions), and covariations with behaviour; plus relate (negatively) in amplitude, while the direction of this relationship is determined by the non-zero-mean deviation of alpha oscillations pre-stimulus (baseline shift index, BSI). This is indictive of a link of the P300 with underlying alpha oscillations through a baseline shift account, and hence that the P300 can be explained, at least in parts, by non-zero mean brain oscillations as they undergo post-stimulus changes.

Reviewer #1 (Public Review):

This paper aims to study the effects of choice history on action-selective beta band signals in human MEG data during a sensory evidence accumulation task. It does so by placing participants in three different stochastic environments, where the outcome of each trial is either random, likely to repeat, or likely to alternate across trials. The authors provide good behavioural evidence that subjects have learnt these statistics (even though they are not explicitly told about them) and that they influence their decision-making, especially on the most difficult trials (low motion coherence). They then show that the primary effect of choice history on lateralised beta-band activity, which is well-established to be linked to evidence accumulation processes in decision-making, is on the slope of evidence accumulation rather than on the baseline level of lateralised beta.

The strengths of the paper are that it is: (i) very well analysed, with compelling evidence in support of its primary conclusions; (ii) a well-designed study, allowing the authors to investigate the effects of choice history in different stochastic environments.

There are no major weaknesses to the study. On the other hand, investigating the effects of choice/outcome history on evidence integration is a fairly well-established problem in the field. As such, I think that this provides a valuable contribution to the field, rather than being a landmark study that will transform our understanding of the problem.

The authors have achieved their primary aims and I think that the results support their main conclusions. One outstanding question in the analysis is the extent to which the source-reconstructed patches in Figure 2 are truly independent of one another (as often there is 'leakage' from one source location into another, and many of the different ROIs have quite similar overall patterns of synchronisation/desynchronisation.). A possible way to investigate this further would be to explore the correlation structure of the LCMV beamformer weights for these different patches, to ask how similar/dissimilar the spatial filters are for the different reconstructed patches.

The revised paper now states explicitly how source-reconstructed patches are indeed affected by leakage, but also why the focus of the authors on differences (rather than similarities) between patches leaves their findings and conclusions essentially unaffected by this intrinsic limitation of cortical source reconstruction from surface MEG data.

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

Colin et al demonstrated that condensin is a key factor for the disjunction of sister-telomeres during mitosis and proposed that it is due to that condensin restrains the telomere association of cohesin. The authors first showed that condensin binds telomeres in mitosis evidenced by ChIP-qPCR and calibrated ChIP-seq. They further demonstrated that compromising condensin's activity leads to a failure in the disjunction of telomeres, with convincing cytological and HI-seq evidence. Two telomeric proteins Taz1 and Mit1 were identified to specifically regulate the telomere association of cohesin. Deletion of these genes decreased/increased condensin's telomere association and exacerbated/remedied the defected telomere disjunction in a condensin mutant, echoing the role of condensin in telomere disjunction. They proposed that the underlying mechanism is that condensin inhibits cohesin's accumulation at telomeres. However, the evidence for this claim might need to be further strengthened. Nevertheless, this study uncovered a novel role of condensin in the separation of telomeres of sister chromosomes and open a question of how condensin regulates the structure of chromosomal ends.

Reviewer #1 (Public Review):

Summary:<br /> In this paper the authors present genome-wide association analyses of 11 different cancers including time-to-event analyses. The authors use two recently published Bayesian methods, one of which is constructed to handle time-to-event data. The authors demonstrate that polygenic risk scores trained on these models give nominally better predictions than standard polygenic risk scores. Further they show that performing 11 GWASs in UKB while adjusting for the polygenic effects estimated by their improved predictor, they find seven novel loci are implicated by one or both of these methods of which the authors find that three replicate in Estonian Biobank.

Strengths:<br /> A clear strength is that the authors evaluate the performance of the model in a completely different dataset (Estonian Biobank) than the one it is trained in.

Weaknesses:<br /> The 11 phenotypes that the authors chose have the challenge that they are rare, particularly in healthy biobank participants, which means that (i) the benefit of modeling it as a time-to-event analysis is expected to be smaller and (ii) that models have to be stable under imbalanced case/control fractions. In GWAS analyses authors handle this second problem by using a recently published association test, which is robust to imbalanced data, which likely means that they avoid inflated test statistics, but also that they do not leverage the actual time-to-event information to its full potential.

The authors chose not to use the recently published methods BayesRR-RC and BayesW, but instead they run these models and then add an extra step where they run a logistic regression with an offset term set to the LOCO genomic values as estimated by GRMR-BayesW and GRMR-BayesRR-RC respectively. They write that this was because of the imbalanced case/control proportion, but not how the problem was detected. If the authors have insight about when the standard GRMR-BayesW and GRMR-BayesRR-RC become unreliable, I think it would be helpful to share in this paper. Further, if the associations implicated by standard GRMR-BayesW and GRMR-BayesRR-RC are not reliable, I think we need some justification that the variance components reported in Figure 1 are still reliable.

The authors chose to compare the two new GWAS methods, GMRM-BayesW-adjusted and GMRM-BayesRR-RC-adjusted, to REGENIE, so an obvious first question in my opinion is if GMRM-BayesW-adjusted and GMRM-BayesRR-RC-adjusted find more signal than REGENIE.<br /> a. We see that 7 loci where found by GMRM-BayesW but not by REGENIE, but how many were found by REGENIE but not by GMRM-BayesW?<br /> b. Figure S5 as I understand it is showing that the mean -log(p-value) is lower in GMRM-BayesW than REGENIE for variants that have a p-value in GMRM-BayesW that is lower than 5e-8. I don't think this is a valid way to check if GMRM-BayesW has more power. I have a feeling that there could be a winner's curse-like phenomenon here. I think a more principled comparison could be provided.

The title of the paper ("Novel discoveries and enhanced genomic prediction from modelling genetic risk of cancer age-at-onset") seems to imply that the age of onset informed model (GMRM-BayesW) does better. But I think the foundation for that statement could be strengthened.<br /> Figure S6 shows that 261 previously reported loci were replicated by GMRM-BayesW-adjusted whereas 256 were replicated by GMRM-BayesRR-RC. How were previously reported loci defined? did they include UKB data? and how many where there in total?<br /> In the PRS analyses presented in Figure 3a GMRM-BayesW does better than GMRM-BayesRR-RC in 8/11 phenotypes, which does not itself appear significant to me. And with overlapping confidence intervals the significance of the improvement is hard to see.

In Table 1 it says that rs35763415, rs117972357 and rs7902587 replicated in the Estonian Biobank but Figure 3b it says that rs35763415, rs117972357 and rs1015362 replicated in the Estonian Biobank. What is the difference between these two analyses? In the methods it says that you checked your findings for replication in FinnGen, but I don't see any results from FinnGen anywhere?

Reviewer #1 (Public Review):

Summary<br /> The authors investigated the antigenic diversity of recent (2009- 2017) A/H3N2 influenza neuraminidases (NAs), the second major antigenic protein after haemagglutinin. They used 27 viruses and 43 ferret sera and performed NA inhibition. This work was supported by a subset of mouse sera. Clustering analysis determined 4 antigenic clusters, mostly in concordance with the genetic groupings. Association analysis was used to estimate important amino acid positions, which were shown to be more likely close to the catalytic site. Antigenic distances were calculated and a random forest model was used to determine potential important sites.

This has the potential to be a very interesting piece of work. At present, there are inconsistencies in the methods, results and presentation that limit its impact. In particular, there are weaknesses in some of the computational work.

Strengths<br /> 1. The data cover recent NA evolution and a substantial number (43) of ferret (and mouse) sera were generated and titrated against 27 viruses. This is laborious experimental work and is the largest publicly available neuraminidase inhibition dataset that I am aware of. As such, it will prove a useful resource for the influenza community.

2. A variety of computational methods were used to analyse the data, which give a rounded picture of the antigenic and genetic relationships and link between sequence, structure and phenotype.

Weaknesses<br /> 1. Inconsistency in experimental methods<br /> Two ferret sera were boosted with H1N2, while recombinant NA protein for the others. This, and the underlying reason, are clearly explained in the manuscript. The authors note that boosting with live virus did not increase titres. Nevertheless, these results are included in the analysis when it would be better to exclude them (Figure 2 shows much lower titres to their own group than other sera).

2. Inconsistency in experimental results<br /> Clustering of the NA inhibition results identifies three viruses which do not cluster with their phylogenetic group. Again this is clearly pointed out in the paper. Further investigation of this inconsistency is required to determine whether this has a genetic basis or is an experimental issue. It is difficult to trust the remaining data while this issue is unresolved.

3. Inconsistency in group labelling<br /> A/Hatay/4990/2016 & A/New Caledonia/23/2016 are in phylogenetic group 1 in Figure 2 and phylogenetic group 1 in Figure 5 - figure supplement 1 panel a.<br /> A/Kansas/14/2017 is selected as a representative of antigenic group 2, when in Figure 2 it is labelled as AC1 (although Figure 2 - supplement 4 which the text is referring to shows data for A/Singapore/Infimh-16-0019/2016 as the representative of AC2). A/Kansas/14/2017 is coloured and labelled as AC2 in Figure 2 - supplement 5.<br /> The colouring is changed for Figure 3a at the bottom. A/Heilongjiang-Xiangyang/1134/2011 is coloured the same as AC4 viruses when it is AC1 in Figure 2.<br /> This lack of consistency makes the figures misleading.

4. Data not presented, without explanation<br /> The paper states that 44 sera and 27 H6N2 viruses were used (line 158). However, the results for the Kansas/14/2017 sera do not appear to be presented in any of the figures (e.g. Figure 2 phylogenetic tree, Figure 5 - figure supplement 1). It is not obvious why these data were not presented. The exclusion of this serum could affect the results as often the homologous titre is the highest and several heatmaps show the fold down from the highest titre.

5. The cMDS plot does not have sufficient quality assurance<br /> A cMDS plot is shown in Figure 5 - figure supplement 1, generated using classical MDS. The following support for the appropriateness of this visualisation is not given.<br /> a. Goodness of fit of the cMDS projection, including per point and per titre.<br /> b. Testing of the appropriate number of dimensions (the two sera from phylogenetic group 3 are clustered with phylogenetic group 2; additional dimensions might separate these groups).<br /> c. A measure of uncertainty in positioning, e.g. bootstrapping.<br /> d. A sensitivity analysis of the assumption about titres below the level of detection (i.e. that <20 = 10).<br /> Without this information, it is difficult to judge if the projection is reliable.

6. Choice of antigenic distance measure<br /> The measure of antigenic distance used here is the average difference between titres for two sera. This is dependent on which viruses have been included in the analysis and will be biased by the unbalanced number of viruses in the different clusters (12, 8, 2, 5).

7. Association analysis does not account for correlations<br /> For each H6N2 virus and position, significance was calculated by comparing the titres between sera that did or did not have a change at that position. This does not take into account the correlations between positions. For haemagglutinin, it can be impossible to determine the true antigenic effects of such correlated substitutions with mutagenesis studies.

8. Random forest method<br /> 25 features are used to classify 43 sera, which seems high (p/3 is typical for classification). By only considering mismatches, rather than the specific amino acid changes, some signals may be lost (for example, at a given position, one amino acid change might be neutral while another has a large antigenic effect). Features may be highly, or perfectly correlated, which will give them a lower reported importance and skew the results.

Reviewer #1 (Public Review):

Summary:<br /> In the present study, the authors examined the possibility of using phosphatidyl-inositol kinase 3-kinase alpha (PI3Ka) inhibitors for heterotopic ossification (HO) in fibrodysplasia ossificans progressiva (FOP). Administration of BYL719, a chemical inhibitor of PI3Ka, prevented HO in a mouse model of FOP that expressed a mutated ACVR1 receptor. Genetic ablation of PI3Ka (p110a) also suppressed HO in mice. BYL719 blocked osteochondroprogenitor specification and reduced inflammatory responses, such as pro-inflammatory cytokine expression and migration/proliferation of immune cells. The authors claimed that inhibition of PI3Ka is a safe and effective therapeutic strategy for HO.

Strengths:<br /> This manuscript reports an interesting finding that BYL719 inhibits HO in a mouse model of FOP.

Weaknesses:<br /> The molecular mechanisms of BYL719 were still unclear because BYL719 affected multiple events and many types of cells. Additional experimental data would be needed to show more clearly how PI3Ka regulates HO.

Reviewer #1 (Public Review):

This study compares visuospatial working (VWM) memory performance between patients with MS and healthy controls, assessed using analog report tasks that provide continuous measures of recall error. The aim is to advance on previous studies of VWM in MS that have used binary (correct/incorrect) measures of recall, such as from change detection tasks, that are not sensitive to the resolution with which features can be recalled, and to use mixture modelling to disentangle different contributions to overall performance. The results identify a specific decrease in the precision of VWM recall in MS, although the possibility that visual and/or motor impairments contribute to performance decrements on the memory task cannot be ruled out.

Reviewer #1 (Public Review):

The manuscript aims to provide mechanistic insight into the activation of PI3Kbeta by its known regulators tyrosine phosphorylated peptides, GTP-loaded Rac1 and G-protein beta-gamma subunits. To achieve this the authors have used supported lipid bilayers, engineered recombinant peptides and proteins (often tagged with fluorophores) and TIRF microscopy to enable bulk (averages of many molecules) and single molecule quantitation. The great strength of this approach is the precision and clarity of mechanistic insight. Although the study does not use "in transfecto" or in vivo models the experiments are performed using "physiologically-based" conditions and provide a powerful insight into core regulatory principles that will be relevant in vivo.

The results are beautiful, high quality, well controlled and internally consistent (and with other published work that overlaps on some points) and as a result are compelling. The primary conclusion is that the primary regulator of PI3Kbeta are tyrosine phosphorylated peptides (and by inference tyrosine phsophorylated receptors/adaptors) and that the other activators can synergise with that input but have relatively weak impacts on their own.

Although the methodology is not easily imported, for reasons of both cost and the experience needed to execute them well, the results have broad importance for the field and reverse an impression that had built in large parts of the broader signalling and PI3K communities that all of the inputs to PI3Kbeta were relatively equivalent, however, these conclusions were based on "in cell" or in vivo studies that were very difficult to interpret clearly.

Reviewer #1 (Public Review):

Warming and precipitation regime change significantly influences both above-ground and below-ground processes across Earth's ecosystems. Soil microbial communities, which underpin the biogeochemical processes that often shape ecosystem function, are no exception to this, and although research shows they can adapt to this warming, population dynamics and ecophysiological responses to these disturbances are not currently known. The Qinghai-Tibet Plateau, the Third Pole of the Earth, is considered among the most sensitive ecosystems to climate change. The manuscript described an integrated, trait-based understanding of these dynamics with the qSIP data. The experimental design and methods appear to be of sufficient quality. The data and analyses are of great value to the larger microbial ecological community and may help advance our understanding of how microbial systems will respond to global change. There are very few studies in which the growth rates of bacterial populations from multifactorial manipulation experiments on the Qinghai-Tibet Plateau have been investigated via qSIP, and the large quantity of data that comprises the study described in this manuscript, will substantially advance our knowledge of bacterial responses to warming and precipitation manipulations.

Joint Public Review:

Using Ts65Dn - the most commonly used mouse model of Down syndrome (DS) - the goal of this study is two-pronged: 1) to conduct a thorough assessment of DS-related genotypic, physiological, behavioral, and phenotypic measures in a longitudinal manner; and 2) to measure the effects of chronic GTE-EGCG on these measures in the Ts65Dn mouse model. Corroborating results from several previous studies on Ts65Dn mice, findings of this study show confirm the Ts65Dn mouse model exhibits the suite of traits associated with DS. The findings also suggest that the mouse model might have experienced drift, given the milder phenotypes than those reported by earlier studies. Results of the GTE-EGCG treatment do not support its therapeutic use and instead show that the treatment exacerbated certain DS-related phenotypes.

Strengths:<br /> The authors performed a rigorous assessment of treatment and examined treatment and genotypic alterations at multiple time points during growth and aging. Detailed analysis shows differences in genotype during aging as well as genotype with treatment. This study is solid in the overarching methodological approach (with the exception of RNAseq, described below). The biggest strength of the study is its approach and dataset, which corroborate results from a multitude of past studies on Ts65Dn mice, albeit on adult specimens. It would be beneficial for the dataset to be made available to other researchers using a public data repository.

Weaknesses:<br /> There are several primary weaknesses, described below:

Sex was not considered in the analyses<br /> The number of experimental animals of each sex are not clearly represented in the paper, but are buried in supplemental tables, and the Ns for the RNAseq are unclear. No analyses were done to examine sex differences in male/female DS or WT animals with or without treatment. Body measurements will greatly vary by sex, but this was not taken into consideration during assessments. As such, there is a high amount of variability within each cohort measured for body assessments (tibia, body weight, skeletal development etc.). Supplemental table 14 had the list of each animal, but not collated by sex, genotype or treatment, making it difficult to assess the strength of each measurement.

Key results are not clearly depicted in the main figures<br /> Rigorous assessment of each figure and the clarity of the figure to convey the results of the analysis needs to be performed. Many of the figures do not clearly represent the findings, with authors heavily relying on supplemental figures to present details to explain results. Figure legends do not adequately describe figures; rather, they are limited to describing how the analysis is performed. For example, LDA plots in Figure 4 do not clearly convey the results of metabolite analysis.<br /> Overall, the amount of data presented here is overwhelming, making it difficult to interpret the findings. Some assessments that do not add to the overall paper need to be removed. Clarifying the text, figures and trimming the supplement to represent the data in a manner that is easily understood will improve the readability of the paper. For example, perhaps measures which are not strongly impacted by genotype could be moved to the supplement, because they are not directly relevant to the question of whether GTE-EGCG reverses the impact of trisomy on the measures.

Lack of clarity in the behavioral analyses<br /> Behavioral assessments are not clearly written in the methods. For example, for the novel object recognition task, it isn't clear how preference was calculated. Is this simply the percent of time spent with the novel object, or is this a relative measure (novel:familiar ratio)? This matters because if it is simply the percent of time, the relevant measure is to compare each group to 50% (the absence of a preference). The key measures for each test need to be readily distinguished from the control measures.<br /> There are also many dependent behavioral measures. For example, speed and distance are directly related to each other, but these are typically reported as control measures to help interpret the key measure, which is the anxiety-like behavior. Similarly, some behavioral tests were used to represent multiple behavioral dimensions, such as anxiety and arousal. In general, the measurements of arousal seem atypical (speed and distance are typically reported as control measures, not measures of arousal). Similarly, measures of latency during training would not typically be used as a measure of long-term memory but instead reported as a control measure to show learning occurred. LDA analysis requires independence of the measures, as well as normality. It does not appear that all of the measures fed into this analysis would have met these assumptions, but the methods also do not clearly describe which measures were actually used in the LDA.

Unclear value of RNAseq<br /> RNAseq was performed in cerebellum, a relatively spared region in DS pathology at an early time point in disease. Further, the expression of 125 genes triplicated in DS was shown in a PCA plot to highly overlap with WT, indicating that there are minimal differences in gene expression in these genes. If these genes are not critical for cerebellar function, perhaps this could account for the lack of differences between WT and Ts65Dn mice. If the authors are interested in performing RNAseq, it would have made more sense to perform this in hippocampus (to compare with metabolites) and to perform more stringent bioinformatic analysis than assessment by PCA of a limited subset of genes. Supplementary Table S14, which shows the differentially expressed genes, appears to be missing from the manuscript and cannot be evaluated. Additionally, the methods of the RNAseq are not sufficiently described and lack critical details. For example, what was the normalization performed, and which groups were compared to identify differentially expressed genes? It would also be worthwhile to describe how animals were identified for RNAseq-were those animals representative of their groups across other measures?

Reviewer #1 (Public Review):

Wheeler et al. have discovered a new RNA circuit that regulates T-cell function. They found that the long non-coding RNA Malat1 sponges miR-15/16, which controls many genes related to T cell activation, survival, and memory. This suggests that Malat1 indirectly regulates T-cell function. They used CRISPR to mutate the miR-15/16 binding site in Malat1 and observed that this disrupted the RNA circuit and impaired cytotoxic T-cell responses. While this study presents a novel molecular mechanism of T-cell regulation by Malat1-miR-15/16, the effects of Malat1 are weaker compared to miR-15/16. This could be due to several reasons, including higher levels of miR-15/16 compared to Malat1 or Malat1 expression being mostly restricted to the nucleus. Although the role of miR15/16 in T-cell activation has been previously published, if the authors can demonstrate that miR15/16 and/or Malat1 affect the clearance of Listeria or LCMV, this will significantly add to the current findings and provide physiological context to the study.

Joint Public Review:

The authors of this manuscript studied cell-cell interaction between fibroblast and cancer cells as an intermediary model of tumor cell migration/invasion. The work focused on the mesenchymal cadherin-11 (CDH11) which is expressed in the later stages of the epithelial mesenchymal transition (EMT) in tumor cellular models, and whose expression is correlated with tumor progression in vivo. The authors employed 3-D matrix and live cell imaging to visualize the nutrient-dependent co-migration of fibroblast and cancer cells. By siRNA-based suppression of CDH11 expression in tumor cell line and/or fibroblast cells, the authors observed decreased co-movement and attenuated growth of mixed xenograft. Accordingly, the authors conclude that post-EMT cancer cells are capable of migrating/invading through CDH11-mediated cell-cell contact.

While the data point to the involvement of CDH11 in fibroblast mediated co-invasion, as it stands it is difficult to fully contextualize these observations within the broader context of the molecular mechanisms underlying metastasis, and in particular do not firmly establish a primary role for CDH11 at this time. The reviewers were specifically concerned about indirect effects of CDH11 manipulation on the physiology and cell biology of the tumor cells, and the possibility that several of the results could be consequences of these changes rather than due specifically to CDH11 mediated interactions.

The reviewers acknowledge the difficulty in fully controlling for these phenomena, and believe this work will be of interest to the large number of researchers investigating the molecular basis for metastasis and specifically of trans cell-type interactions. However until experiments establishing the specific formation and CDH11-mediated interactions in co-invasion are carried out, the author's conclusions about the prominent role of CDH11 should be treated as intriguing, but speculative.

Reviewer #1 (Public Review):

The overall tone of the rebuttal and lack of responses on several questions was surprising. Clearly, the authors took umbrage at the phrase 'no smoking gun' and provided a lengthy repetition of the fair argument about 'ticking boxes' on the classic list of criteria. They also make repeated historical references that descriptions of neurotransmitters include many papers, typically over decades, e.g. in the case of ACh and its discovery by Sir Henry Dale. While I empathize with the authors' apparent frustration (I quote: '...accept the reality that Rome was not built in a single day and that no transmitter was proven by a one single paper') I am a bit surprised at the complete brushing away of the argument, and in fact the discussion. In the original paper, the notion of a receptor was mentioned only in a single sentence and all three reviewers brought up this rather obvious question. The historical comparisons are difficult: Of course many papers contribute to the identification of a neurotransmitter, but there is a much higher burden of proof in 2023 compared to the work by Otto Loewi and Sir Henry Dale: most, if not all, currently accepted neurotransmitter have a clear biological function at the level of the brain and animal behavior or function - and were in fact first proposed to exist based on a functional biological experiment (e.g. Loewi's heart rate change). This, and the isolation of the chemical that does the job, were clear, unquestionable 'smoking guns' a hundred years ago. Fast forward 2023: Creatine has been carefully studied by the authors to tick many of the boxes for neurotransmitters, but there is no clear role for its function in an animal. The authors show convincing effects upon K+ stimulation and electrophysiological recordings that show altered neuronal activity using the slc6a8 and agat mutants as well as Cr application - but, as has been pointed out by other reviewers, these effects are not a clear-cut demonstration of a chemical transmitter function, however many boxes are ticked. The identification of a role of a neurotransmitter for brain function and animal behavior has reasonably more advanced possibilities in 2023 than a hundred years ago - and e.g. a discussion of approaches for possible receptor candidates should be possible.

Again, I reviewed this positively and agree that a lot of cumulative data are great to be put out there and allow the discovery to be more broadly discussed and tested. But I have to note, that the authors simply respond with the 'Rome was not built in a single day' statement to my suggestions on at least 'have some lead' how to approach the question of a receptor e.g. through agonists or antagonists (while clearly stating 'I do not think the publication of this manuscript should not be made dependent' on this). Similarly, in response to reviewer 2's concerns about a missing receptor, the authors' only (may I say snarky) response is ' We have deleted this sentence, though what could mediate postsynaptic responses other than receptors?' The bullet point by reviewer 3 ' • No candidate receptor for creatine has been identified postsynaptically.' is the one point by that reviewer that is simply ignored by the authors completely. Finally, I note that my reivew question on the K stimulation issues (e.g. 35 neurons that simply did not respond at all) was: ' Response: To avoid the disadvantage of K stimulation, we also performed optogenetic experiments recently and obtained encouraging preliminary results.' No details, not data - no response really.

In sum, I find this all a bit strange and the rebuttal surprising - all three reviewers were supportive and have carefully listed points of discussion that I found all valid and thoughtful. In response, the authors selectively responded scientifically to some experimental questions, but otherwise simply rather non-scientifically dismissed questions with 'Rome was not built in a day'-type answers, or less. I my view, the authors have disregarded the review process and the effort of three supportive reviewers, which should be part of the permanent record of this paper.

Reviewer #1 (Public Review):

Summary:

HP1 plays a pivotal role in orchestrating chromatin packaging through the creation of biomolecular condensates. The existence of distinct homologs offers an intriguing avenue for investigating the interplay between genetic sequence and condensate formation. In this study, the authors conducted extensive coarse-grained simulations to delve into the phase separation behavior of HP1 paralogs. Additionally, the researchers delved into the captivating possibility of various HP1 paralogs co-localizing within assemblies composed of multiple components. Importantly, the study also delved into the critical role of DNA in finely tuning this complex process.

Strengths:

I applaud the authors for their methodical approach in conducting simulations aimed at dissecting the contributions of hinges, CTE, NTE, and folded regions. The comprehensive insights unveiled in Figure 3 compellingly substantiate the significance of these protein components in facilitating the process of phase separation.

This systematic exploration has yielded several innovative revelations. Notably, the authors uncovered a nuanced interplay between the folded and disordered domains. Although disordered regions have traditionally been linked to driving phase separation through their capacity for forming multivalent interactions, the authors have demonstrated that the contribution of the CD cannot be overlooked, as it significantly impacts the saturation concentration.

The outcomes of this study serve to elucidate the intricate mechanisms and regulatory aspects governing HP1 LLPS.

Weaknesses:

The authors do not provide an assessment of the quantitative precision of their model. To illustrate, HP1a is anticipated to undergo phase separation primarily under low salt concentrations. Does the model effectively capture this sensitivity to salt conditions? Regrettably, the specific salt conditions employed in the simulations are not explicitly stated. While I anticipate that numerous findings in the manuscript remain valid, it could be beneficial to acknowledge potential limitations tied to the simulations. For instance, might the absence of quantitative precision impact certain predictions, such as the CD's influence on phase separation?

Reviewer #1 (Public Review):

The authors have generated a set of yeast S. cerevisiae strains containing different numbers of chromosomes. Elimination of telomerase activates homologous recombination (HR) to maintain telomeres in cells containing the original 16 chromosomes. However, elimination of telomerase leads to circularization of cells containing a single or two chromosomes. The authors examined whether the subtelomeric sequences X and Y' promote HR-mediated telomere maintenance using the strain SY12 carrying three chromosomes. They found that the subtelomeric sequences X and Y' are dispensable for cell proliferation and HR-mediated telomere maintenance in telomerase-minus SY12 cells. They conclude that subtelomeric X and Y' sequences do not play essential roles in both telomerase-proficient and telomerase-null cells and propose that these sequences represent remnants of genome evolution.<br /> Interestingly, telomerase-minus SY12 generate survivors that are different from Type I or Type II survivors.

Strengths: The authors examined whether the subtelomeric sequences X and Y' promote HR-mediated telomere maintenance using the strain SY12 carrying three chromosomes.

Weaknesses:<br /> It is not determined how atypical survivors or Type X survivors are generated in telomerase-deficient SY12 cells.<br /> Survivor generation of each type (Type I, Type II, Type X or atypical and circularization) is not quantitated.

Joint Public Review:

Smirnova et al. present a cryo-EM structure of human SIRT6 bound to a nucleosome as well as the results from molecular dynamics simulations. The results show that the combined conformational flexibilities of SIRT6 and the N-terminal tail of histone H3 limit the residues with access to the active site, partially explaining the substrate specificity of this sirtuin-class histone deacetylase. Two other groups have recently published cryo-EM structures of SIRT6:nucleosome complexes; this manuscript confirms and complements these previous findings, with the addition of some novel insights into the role of structural flexibility in substrate selection.

This manuscript is the third in a recent series of reports of cryo-EM structures of Sirt6:nucleosome complexes. The main conclusions of the three studies are similar, but this manuscript from Smirnova et al. includes additional molecular dynamics analysis of the histone tails. These studies suggest that part of the specificity for sites on the H3 tail is the result of only this tail having significant access to the active site. The results are partially validated by showing that H3-K27Ac is sometimes found near the active site in the simulations, and is a weak substrate for the deacetylase in vitro. All of the structures show Sirt6 contacting the acidic patch of H2A-H2B, partial displacement of the H2A C-terminal tail, and displacement of the DNA at the entry-exit site to "unclamp" the H3 N-terminal tail. This manuscript provides additional support for the conclusions drawn in the first two published structures, adds molecular dynamics simulations that provide further insight and includes a biochemical assay that helps to resolve an apparent conflict regarding the deacetylation of H3-K27Ac from the other two papers.

Reviewer #1 (Public Review):

In this study, Satake and colleagues endeavored to explore the rates and patterns of somatic mutations in wild plants, with a focus on their relationship to longevity. The researchers examined slow- and fast-growing tropical tree species, demonstrating that slow-growing species exhibited five times more mutations than their fast-growing counterparts. The number of somatic mutations was found to increase linearly with branch length. Interestingly, the somatic mutation rate per meter was higher in slow-growing species, but the rate per year remained consistent across both species. A closer inspection revealed a prevalence of clock-like spontaneous mutations, specifically cytosine-to-thymine substitutions at CpG sites. The author suggested that somatic mutations were identified as neutral within an individual, but subject to purifying selection when transmitted to subsequent generations. The authors developed a model to assess the influence of cell division on mutational processes, suggesting that cell-division independent mutagenesis is the primary mechanism.

The authors have gathered valuable data on somatic mutations, particularly regarding differences in growth rates among trees. Their meticulous computational analysis led to fascinating conclusions, primarily that most somatic mutations accumulate in a cell-division independent manner. The discovery of a molecular clock in somatic mutations significantly advances our comprehension of mutational processes that may generate genetic diversity in tropical ecosystems. The interpretation of the data appears to be based on the assumption that somatic mutations can be effectively transmitted to the next generation unless negative selection intervenes. However, accumulating evidence suggests that plants may also possess "effective germlines," which could render the somatic mutations detected in this study non-transmittable to progeny. Incorporating additional analyses/discussion in the context of plant developmental biology, particularly recent studies on cell lineage, could further enhance this study.

Specifically, several recent studies address the topics of effective germline in plants. For instance, Robert Lanfear published an article in PLoS Biology exploring the fundamental question, "Do plants have a segregated germline?" A study in PNAS posited that "germline replications and somatic mutation accumulation are independent of vegetative life span in Arabidopsis." A phylogenetic-based analysis titled "Rates of Molecular Evolution Are Linked to Life History in Flowering Plants" discovered that "rates of molecular evolution are consistently low in trees and shrubs, with relatively long generation times, as compared with related herbaceous plants, which generally have shorter generation times." Another compelling study, "The architecture of intra-organism mutation rate variation in plants," published in PLoS Biology, detected somatic mutations in peach trees and strawberries. Although some of these studies are cited in the current work, a deeper examination of the findings in relation to the existing literature would strengthen the interpretation of the data.

Reviewer #1 (Public Review):

The authors have employed a digital twin approach to show that depending on the underlying disease mechanism, a digital replica constructed from human data can both recapitulate clinical findings, but also provide important insights into the fundamental disease state by revealing underlying contributing mechanisms. Moreover, the authors are able to show that a disease state caused by two different underlying genetic anomalies exhibit different electrical and morphological profiles.

This is important information as it allows for potential stratification of treatment approaches in future cases based on underlying phenotype by linking it to specific genotype properties. One of the most innovative aspects of the study is the mismatch switching between personalized structure, remodeling and genotype specific electrophysiological properties. The approach is elegant and allows for further exposure of the key mechanisms that contribute to the development of ventricular tachycardia circuits. One addition that could add more insight is to predict the effect of structural remodeling alone well, considering only normal electrophysiological models. Another interesting approach would be a sensitivity analysis, to determine how sensitive the VT circuits are to the specific geometry of the patient and remodeling that occurs during the disease, such an approach could also be used to determine how sensitive the outputs are to electrophysiological model inputs.

Reviewer #1 (Public Review):

The research titled "Spatial and temporal distribution of ribosomes in single cells reveals aging differences between old and new daughters of Escherichia coli" by Lin Chao, Chun Kuen Chen, Chao Shi, and Camilla U. Rang addresses the asymmetric distribution of ribosomes in single E. coli cells during aging by time-lapse microscopy, as well as its correlation to protein misfolding. The presented research is an important contribution to the field of protein biosynthesis pathways and their link to aging, especially in regard to the thorough analysis of variation in cell elongation rate in old and new daughter cells derived from old and new mother cells. However, the imaging results, analysis, and methodologies require substantial elaboration, as in its current form several key characteristics remain unanswered. Furthermore, the results should be compared and discussed in regard to several other reports, which analyzed ribosome asymmetric distribution and inheritance in E.coli, see detailed comments below.

Major comments:<br /> *It is not clear from the results or the material and methods sections how the authors define and detect old vs. new mother cells up to 128 cells division, which is the limit the manuscript describes in line 574: "To avoid effects of crowding within the micro-colonies, movies were ended when micro-colonies exceeded 128 cells". The results described only refer to 3 cell divisions (Fig.1 for example). As this is the key issue the manuscript addresses this requires elaboration.

* The authors should present several representative images of the results described, including: "New daughters at birth from old mothers have more ribosomes" - this should include clear quantification, of normalized fluorescence intensity vs. normalized cell length, as well as for "Ribosomal asymmetry between daughters are spatially in place in mothers before division"(line 218) for example. This should include annotation of the exact time points in minutes. The quantification can be done and presented as in their previous work, which provides the basis for this study: (Figure 2b, for example) "Allocation of gene products to daughter cells is determined by the age of the mother in single Escherichia coli cells" Chao Shi, Lin Chao, Audrey Menegaz Proenca, Andrew Qiu, Jasper Chao and Camilla U. Rang, May 2020, https://doi.org/10.1098/rspb.2020.0569.

* Quantification of variations over generations time during the time lapse is required: the change in cell-length (y-axis, the length of full-grown cell normalized to 1) vs. ribosomes number (y-axis) relative to the generation time analysis should be presented, based on the time-lapse images. The mean from ~10 independent cells should be presented, as in many similar research, for example: "Organization of Ribosomes and Nucleoids in Escherichia coli Cells during Growth and in Quiescence" Qian Chai, Bhupender Singh, Kristin Peisker, Nicole Metzendorf, Xueliang Ge, Santanu Dasgupta, Suparna Sanyal, 2014, JBC (Figure 3b).

* The distribution of ribosomes should be compared to the nucleoid distribution, as this is a major factor in RNA and translation distribution in bacterial cells (for example Gray, W. T., Govers, S. K., Xiang, Y., Parry, B. R., Campos, M., Kim, S., & Jacobs-Wagner, C. (2019). Nucleoid size scaling and intracellular organization of translation across bacteria. Cell, 177(6), 1632-1648.e20. https://doi.org/10.1016/j.cell.2019.05.017 , as reviewed in RNA localization in prokaryotes: Where, when,how, and why, Mikel Irastortza-Olaziregi, Orna Amster-Choder, 2020). The authors should add and discuss this, or elaborate on the reasons to omit this analysis.

* The results should be compared and discussed in regard to several other reports, which analyzed ribosome asymmetric distribution and inheritance in E.coli by tagging different ribosomal proteins, as well as different methodologies, including:

Organization of Ribosomes and Nucleoids in Escherichia coli Cells during Growth and in Quiescence" Qian Chai, Bhupender Singh, Kristin Peisker, Nicole Metzendorf, Xueliang Ge, Santanu Dasgupta, Suparna Sanyal, 2014, JBC

Gray, W. T., Govers, S. K., Xiang, Y., Parry, B. R., Campos, M., Kim, S., & Jacobs-Wagner, C. (2019). Nucleoid size scaling and intracellular organization of translation across bacteria. Cell, 177(6), 1632-1648.e20. https://doi.org/10.1016/j.cell.2019.05.017

Spatiotemporal Organization of the E. coli Transcriptome: Translation Independence and Engagement in Regulation Graphical Abstract Highlights d RNAs in E. coli exhibit asymmetric distribution on a transcriptome-wide scale, Shanmugapriya Kannaiah, Jonathan Livny, Orna Amster-Choder, 2019

Several of the findings reported, including asymmetric ribosome distribution and inheritance levels seem different than the ones reported here. This should be discussed in regard to the different methodologies.

Reviewer #1 (Public Review):

Summary:<br /> Here the authors have tethered a Pgp substrate to strategically place cysteine residues in the protein. Notably, the cysteine-linked substrate (ANC-DNPT)- stimulates ATP hydrolyse and so is able to undergo IF to OF transitions. The authors then determined cryo-EM structures of these complexes and MD simulations of bound states. By capturing unforeseen OF conformations with substate they propose that TM1 undergoes local conformational changes that are sufficient to translocate substrates, rather than large bundle movements.

Strengths:<br /> This paper provides the first substrate (ANC-DNPT)- bound conformations of PgP and a new mechanistic model of how substrates are translocated.

Weaknesses:<br /> Although the cross-links stimulate ATP hydrolysis, further controls are needed to convince me that the TM1 conformations observed in the structures are physiologically relevant, since they have been trapped by "large" substrates covalently-tethered by cross-links.

Reviewer #1 (Public Review):

Summary. In this investigation Kapustin et al. demonstrate that vascular smooth muscle cells (VSMCs) exposed to the extracellular matrix fibronectin stimulates the release of small extracellular vesicles (sEVs). The authors provide experimental evidence that stimulation of the actin cytoskeleton boosts sEV secretion and posit that sEVs harbor both fibronectin and collagen IV protein themselves which also, in turn, alter cell migration parameters. It is well established that fibronectin is associated with increased cell migration and adherence; therefore, this association with VSMCs is not novel. The authors purport that sEV are largely born of filopodia origin; however, this data is not well executed and seems generally at odds with the presented data. Similarly, the effect of sEVs on parameters of cell migration has almost no magnitude of effect, making mechanism exploration somewhat nebulous. Lastly, the proposed mechanism of VSMCs responding to, and depositing, ECM proteins via sEVs was not rigorously executed; again, making the conclusions challenging for the reader to interpret.

Strengths. The authors provide a comprehensive battery of cytoskeletal experiments to test how fibronectin and sEVs impact both sEV release and vascular smooth muscle cell migratory activation.

Weaknesses. Unfortunately, this article suffers from many weaknesses. First, the rigor of the experimental approach is low, which calls into question the merit of the conclusions. In this vein, there is a lack of proper controls or inclusion of experiments addressing alternative explanations for the phenotype or lack thereof.

Reviewer #1 (Public Review):

In their article, "Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings," Tendolkar and colleagues explore Ubx regulation in butterflies. The authors investigated how Ubx expression is restricted to the hindwing in butterflies through a series of genomic analyses and genetic perturbations. The authors provide evidence that a Topoologiacally Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin around Ubx, largely through an apparent boundary element. CRISPR mutations of this boundary element led to ectopic Ubx expression in forewings, resulting in homeotic transformation in the wings. The authors also explore the results of the mutation in two non-coding RNA regions as well as a possible enhancer module. Each of these induces homeotic phenotypes. Finally, the authors describe a number of homeotic phenotypes in butterflies, which they relate to their work.

Together, this was an interesting paper with compelling initial data. That said, I have several items that I feel would warrant further discussion, presentation, or data.

First, I would not state, "Little is known about how Hox genes are regulated outside of flies." They should add "in insects" since so much in known in vertebrates

For Figure 1, it would aid the readers if the authors could show the number of RNAseq reads across the locus. This would allow the readership to evaluate the frequency of the lncRNAs, splice variants, etc.

How common are boundary elements within introns? Typically, boundary elements are outside gene bodies, so this could be explored further. This seems like an interesting bit of biology which, following from the above point, it would be interesting to, at a minimum, discuss, but also relate to how transcription occurs through a possible boundary element (are there splice variants, for example?).

The CRISPR experiments led to compelling phenotypes. However, as a Drosophila biologist, I found it hard to interpret the data from mosaic experiments. For example, in control experiments, how often do butterflies die? Are there offsite effects? It's striking that single-guide RNAs led to such strong effects. Is this common outside of this system? Is it possible to explore the function effects at the boundary element - are these generating large deletions (for example, like Mazo-Vargas et al., 2022)?

For the mosaic experiments, how frequent are these effects in nature or captive stocks? Would it be possible to resequence these types of effects? At the moment, this data, while compelling, was hard to put into the context of the experiments above without understanding how common the effects are. Ideally, there would be resequencing of these tissues, which could be targeted, but it was not clear to me the general rates of these variants.

In sum, I enjoyed the extensive mosaic perturbations. However, I feel that more molecular descriptions would elevate the work and make a larger impact on the field.