Reviewer #1 (Public review):

In the manuscript by Su et al., the authors present a massively parallel reporter assay (MPRA) measuring the stability of in vitro transcribed mRNAs carrying wild-type or mutant 5' or 3' UTRs transfected into two different human cell lines. The goal presented at the beginning of the manuscript was to screen for effects of disease-associated point mutations on the stability of the reporter RNAs carrying partial human 5' or 3' UTRs. However, the majority of the manuscript is dedicated to identifying sequence components underlying the differential stability of reporter constructs. This analysis showed that UA dinucleotides are the most predictive feature of RNA stability in both cell lines and both UTRs.

The effect of AU rich elements (AREs) on RNA stability is well established in multiple systems, and the present study confirms this general trend, but points out variability in the consequence of seemingly similar motifs on RNA stability. For example, the authors report that a long stretch of Us has extreme opposite effects on RNA stability depending on whether it is preceded by an A (strongly destabilizing) or followed by an A (strongly stabilizing). While the authors interpretation of a context-dependence of the effect is certainly well-founded, it seems counterintuitive that the preceding or following A would be the (only) determining factor. This points to a generally reductionist approach taken by the authors in the analysis of the data and in their attempt to dissect the contribution of "AU rich sequences" to RNA stability, with a general tendency to reduce the size and complexity of the features (e.g. to dinucleotides). While this certainly increases the statistical power of the analysis due to the number of occurrences of these motifs, it limits the interpretability of the results. How do UA dinucleotides per se contribute to destabilizing the RNA, both in 5' and 3' UTRs, but (according to limited data presented) not in coding sequences? What is the mechanism? RBPs binding to UA dinucleotide containing sequences are suggested to "mask" the destabilizing effect, thereby leading to a more stable RNA. Gain of UA dinucleotides is reported to have a destabilizing effect, but again no hypothesis is provided as to the underlying molecular mechanism. In addition to reducing the motif length to dinucleotides, the notion of "context dependence" is used in a very narrow sense.

The present MPRA measures the effect of UTR sequences in one specific reporter context and using one experimental approach (following the decay of in vitro transcribed and transfected RNAs). While this method certainly has its merits compared to other approaches, it also comes with some caveats: RNA is delivered naked, without bound RBPs and no nuclear history, e.g. of splicing (no EJCs), editing and modifications. Therefore, it remains to be seen whether UA dinucleotide frequency is a substantial factor in determining the half-lives of endogenous mRNAs.

The authors conclude their study with a meta-analysis of genes with increased UA dinucleotides in 5' and 3'UTRs, showing that specific functional groups are overrepresented among these genes. In addition, they provide evidence for an effect of disease-associated UTR mutations on endogenous RNA stability. While these elements link back to the original motivation of the study (screening for effects of point mutations in 5' and 3' UTRs), they provide only a limited amount of additional insights.

In summary, this manuscript presents an interesting addition to the long-standing attempts at dissecting the sequence basis of RNA stability in human cells. The analysis is in general comprehensive and sound; however, it remains unclear to what extent the findings can be generalized beyond the method and the experimental system used here.

Comments on revisions:

Parts of my original comments have been adequately addressed by the reviewers.<br /> After reading the revised manuscript and the rebuttal, my main concern is related to the figure comparing the half-lives as measured in the two different cell lines that was included in the response to reviewer 2, but not in the revised manuscript. The complete lack of correlation between the half-lives of the 3'UTR library measured in the two cell lines is concerning. While variability and cell type-specific effects can be expected, some principles should be the same (such as the effect of UA dinucleotides that the authors report), leading to at least some correlation.<br /> In addition, it is unclear to me why the half-lives measured for the two libraries in HEK cells are shifted (median ln(t 1/2)=6-7 for the 5'UTR library and ln(t 1/2)=4-4.5 for the 3'UTR library), but not in SH.

I feel that this figure contains important information that should be included in the final manuscript.

Reviewer #1 (Public review):

This study tests whether Little Swifts exhibit optimal foraging, which the data seem to indicate is the case. This is unsurprising as most animals would be expected to optimize the energy income : expenditure ratio, however it hasn't been explicitly quantified before the way it was in this manuscript.

The major strength of this work is the sheer volume of tracking data and the accuracy of those data. The ATLAS tracking system really enhanced this study and allowed for pinpoint monitoring of the tracked birds. These data could be used to ask and answer many questions beyond just the one tested here.

The major weakness of this work lies in the sampling of insect prey abundance at a single point on the landscape, 6.5 km from the colony. This sampling then requires the authors to work under the assumption that prey abundance is simultaneously even across the study region. It may be fair to say that prey populations might be correlated over space but are not equal. It is uncertain whether other aspects of the prey data are problematic. For example, the radar only samples insects at 50m or higher from the ground - how often do Little Swifts forage under 50m high?

The finding that Little Swifts forage optimally is indeed supported by the data, notwithstanding some of the shortcomings in the prey abundance data. The authors achieved their aims and the results support their conclusions.

At its centre, this work adds to our understanding of Little Swift foraging and extends to a greater understanding of aerial insectivores in general. While unsurprising that Little Swifts act as optimal foragers, it is good to have quantified this and show that the population declines observed in so many aerial insectivores are not necessarily a function of inflexible foraging habits. Further, the methods used in this research have great potential for other work. For example, the ATLAS system poses some real advantages and an exciting challenge to existing systems, like MOTUS. The radar that was used to quantify prey abundance also presents exciting possibilities if multiple units could be deployed to get a more spatially-explicit view.

To improve the context of this work, it is worth noting that this research goes into much further depth than any previous studies on a similar topic in several flycatcher and swallow species. A further justification is posited that this research is needed due to dramatic insect population declines, however, the magnitude and extent of such declines are fiercely debated in the literature.

Reviewer #1 (Public review):

This study asks whether the phenomenon of crossmodal temporal recalibration, i.e. the adjustment of time perception by consistent temporal mismatches across the senses, can be explained by the concept of multisensory causal inference. In particular they ask whether the explanation offered by causal inference better explains temporal recalibration better than a model assuming that crossmodal stimuli are always integrated, regardless of how discrepant they are.

The study is motivated by previous work in the spatial domain, where it has been shown consistently across studies that the use of crossmodal spatial information is explained by the concept of multisensory causal inference. It is also motivated by the observation that the behavioral data showcasing temporal recalibration feature nonlinearities that, by their nature, cannot be explained by a fixed integration model (sometimes also called mandatory fusion).

To probe this the authors implemented a sophisticated experiment that probed temporal recalibration in several sessions. They then fit the data using the two classes of candidate models and rely model criteria to provide evidence for their conclusion. The study is sophisticated, conceptually and technically state-of-the-art and theoretically grounded. The data clearly support the authors conclusions.

I find the conceptual advance somewhat limited. First, by design the fixed integration model cannot explain data with a nonlinear dependency on multisensory discrepancy, as already explained in many studies on spatial multisensory perception. Hence, it is not surprising that the causal inference model better fits the data. Second, and again similar to studies on spatial paradigms, the causal inference model fails to predict the behavioral data for large discrepancies. The model predictions in Figure 5 show the (expected) vanishing recalibration for large delta, while the behavioral data don't' decay to zero. Either the range of tested SOAs is too small to show that both the model and data converge to the same vanishing effect at large SOAs, or the model's formula is not the best for explaining the data. Again, the studies using spatial paradigms have the same problem, but in my view this poses the most interesting question here.

In my view there is nothing generally wrong with the study, it does extend the 'known' to another type of paradigm. However, it covers little new ground on the conceptual side.<br /> On that note, the small sample size of n=10 is likely not an issue, but still it is on the very low end for this type of study.

Comments on revision:

The revision has addressed most of these points and makes for a much stronger contribution. The issue of sample size remains.

Reviewer #1 (Public review):

Summary:

In this manuscript by Bimbard et al., a new method to perform stable recordings over long periods of time with neuropixels as well as the technical details on how the electrodes can be explanted for a follow up reuse is provided. I think the description of all parts of the method are very clear, and the validation analyses (n of units per day over time, RMS over recording days...) are very convincing. I however missed a stronger emphasis on why this could provide a big impact on the ephys community, by enabling new analyses, new behavior correlation studies or neurophysiological mechanisms across temporal scales that were previously inaccessible with high temporal resolution (i.e. not with imaging).

Strengths:

Open source method. Validation across laboratories. Across species (mice and rats) demonstration of its use and in different behavioral conditions (head-fixed and freely moving). The implant offers a major advance compared to previous methods and that will help the community generate richer datasets.

Weaknesses:

None noted.

Reviewer #1 (Public review):

Summary:

Parsing speech into meaningful linguistic units is a fundamental yet challenging task that infants face while acquiring the native language. Computing transitional probabilities (TPs) between syllables is a segmentation cue well-attested since birth. In this research, the authors examine whether newborns compute TPs over any available speech feature (linguistic and non-linguistic), or whether by contrast newborns favor computation of TPs over linguistic content over non-linguistic speech features such as speaker voice. Using EEG and the artificial language learning paradigm, they record the neural responses of two groups of newborns presented with speech streams in which either phonetic content or speaker voice are structured to provide TPs informative of word boundaries, while the other dimension provides uninformative information. They compare newborns' neural responses to these structured streams to their processing of a stream in which both dimensions vary randomly. After the random and structured familiarization streams, the newborns are presented with (pseudo)words as defined by their informative TPs, as well as partwords (that is, sequences that straddle a word boundary), extracted from the same streams. Analysis of the neural responses show that while newborns neural activity entrained to the syllabic rate (2 Hz) when listening to the random and structured streams, it additionally entrained at the word rate (4 Hz) only when listening to the structured streams, finding no differential response between the streams structured around voice or phonetic information. Newborns showed also different neural activity in response to the words and part words. In sum, the study reveals that newborns compute TPs over linguistic and non-linguistic features of speech, these are calculated independently, and linguistic features do not lead to a processing advantage.

Strengths:

This interesting research furthers our knowledge of the scope of the statistical learning mechanism, which is confirmed to be a general-purpose powerful tool that allows humans to extract patterns of co-occurring events while revealing no apparent preferential processing for linguistic features. To answer its question, the study combines a highly replicated and well-established paradigm, i.e. the use of an artificial language in which pseudowords are concatenated to yield informative TPs to word boundaries, with a state-of-the-art EEG analysis, i.e. neural entrainment. The sample size of the groups is sufficient to ensure power, and the design and analysis are solid and have been successfully employed before.

Weaknesses:

There are no significant weaknesses to signal in the manuscript. However, in order to fully conclude that there is no obvious advantage for the linguistic dimension in neonates, future studies should pit both dimensions against each other, to determine whether statistical learning weighs linguistic and non-linguistic features equally, or whether phonetic content is preferentially processed.

To sum up, the authors achieved their central aim of determining whether TPs are computed over both linguistic and non-linguistic features, and their conclusions are supported by the results. This research is important for researchers working on language and cognitive development, and language processing, as well as for those working on cross-species comparative approaches.

Comments on revisions:

The authors have addressed my suggestions. I have no further comments.

Reviewer #1 (Public review):

Summary:

Numerous mechanism and structural studies reported the cooperative role of Oct4 and Sox2 during the establishment of pluripotency during reprogramming. Due to the difficulty in sample collection and RNA-seq with low-number cells, the precise mechanisms remain in early embryos. This manuscript reported the role of OCT4 and SOX2 in mouse early embryos using knockout models with low-input ATAC-seq and RNA-seq. Compared to the control, chromatin accessibility and transcriptome were affected when Oct4 and Sox2 were deleted in early ICM. Specifically, decreased ATAC-seq peaks showed enrichment of Motifs of TF such as OCT, SOX, and OCT-SOX, indicating their importance during early development. Moreover, by deep analysis of ATAC-seq and RNA-seq data, they found Oct4 and Sox2 target enhancer to activate their downstream genes. In addition, they also uncovered the role of OS during development from the morula to ICM, which provided the scientific community with a more comprehensive understanding.

Strengths:

On the whole, the manuscript is innovative, and the conclusions of this paper are mostly well supported by data.

Weaknesses:

Major Points:<br /> (1) In Figure 1, a more detailed description of the knockout strategy should be provided to clarify itself. The knockout strategy in Fig1 is somewhat obscure, such as how is OCT4 inactivated in Oct4mKO2 heterozygotes. As shown in Figure 1, the exon of OCT4 is not deleted, and its promoter is not destroyed. Therefore, how does OCT4 inactivate to form heterozygotes?<br /> (2) Is ZP 3-Cre expressed in the zygotes? Is there any residual protein?<br /> (3) What motifs are enriched in the rising ATAC-seq peaks after knocking out of OCT4 and SOX2?<br /> (4) The ordinate of Fig4c is lost.<br /> (5) Signals of H3K4me1, H3K27ac, and so on are usually used to define enhancers, and the loci of enhancers vary greatly in different cells. In the manuscript, the authors defined ATAC-seq peaks far from the TSS as enhancers. The definition in this manuscript is not strictly an enhancer.<br /> (6) If Oct4 and Sox2 truly activate sap 30 and Uhrf 1, what effect does interfering with both genes have on gene expression and chromatin accessibility？

Comments on revisions:

The authors have addressed my concerns so I am fine with revision in principle.

Reviewer #1 (Public review):

Summary:

This noteworthy paper examines the role of planar cell polarity and Wnt signalling in the body axis formation of the hydrozoan Clytia. In contrast to the freshwater polyp Hydra or the sea anemone Nematostella, Clytia represents a cnidarian model system with a complete life cycle (planula-polyp-medusa). In this species, classical experiments have demonstrated that a global polarity is established from the oral end of the embryos (Freeman, 1981). Prior research has demonstrated that Wnt3 plays a role in the formation of the oral organiser in Clytia and other cnidarians, acting in an autocatalytic feedback loop with β-catenin. However, the question of whether and to what extent an oral-aboral gradient of Wnt activity is established remained unanswered. This gradient is thought to control both tissue differentiation and tissue polarity. The planar cell polarity (PCP) pathway has been linked to this polarity, although it is generally considered to be β-catenin independent.

The authors have conducted a series of sophisticated experiments utilising morpholinos, mRNA microinjection, and immunofluorescent visualisation of PCP. The objective of these experiments was to address the function of Wnt3, β-catenin, and PCP core proteins in the coordination of the global polarity of Clytia embryos. The authors conclude that PCP plays a role in regulating polarity along the oral-aboral axis of embryos and larvae. This offers a conceivable explanation for how polarity information is established and distributed globally during Clytia embryogenesis, with implications for our understanding of axis formation in cnidarians and the evolution of Wnt signalling in general. While the experiments are well-designed and executed, there are some criticisms, questions, or suggestions that should be addressed.

Comments:

Beautiful and solid experiments to clarify the role of canonical Wnt signalling and PCP core factors in coordinating planar cell polarity. However, there are also several points that should be addressed.

(1) Wnt3 cue and global PCP. PCP has been described in detail in a previous paper on Clytia (Momose et al, 2012): its orientation along the oral-aboral body axis (ciliary basal body positioning studies), and its function in directional polarity during gastrulation (Stbm-, Fz1-, and Dsh-MO experiments). I wonder if this part could be shortened. What is new, however, are the knockdown and Wnt3-mRNA rescue experiments, which provide a deeper insight into the link between Wnt3 function in the blastopore organiser as a source or cue for axis formation. These experiments demonstrate that the Wnt3 knockdown induces defects equivalent to PCP factor knockdown, but can be rescued by Wnt3-mRNA injection, even at a distance of 200 µm away from the Wnt-positive area. The experimental set-up of these new molecular experiments follows in important aspects those of Freeman's experiments of 1981 (who in turn was motivated to re-examine Teissier's work of 1931/1933 ...). Freeman did not use the term "global polarity" but the concept of an axis-inducing source and a long-range tissue polarity can be traced back to both researchers.

(2) PCP propagation and β-catenin. The central but unanswered question in this study focuses on the interaction between Wnt3 and PCP and the propagation of PCP. Wnt3 has been described in cnidarians but also in vertebrates and insects as a canonical Wnt interacting with β-catenin in an autocatalytic loop. The surprising result of this study is that the action of Wnt3 on PCP orientation is not inhibited in the presence of a dominant-negative form of CheTCF (dnTCF) ruling out a potential function of β-catenin in PCP. This was supported by studies with constitutively active β-catenin (CA-β-cat) mRNA which was unable to restore PCP coordination nor elongation of Wnt3-depleted embryos but did restore β-catenin-dependent gastrulation. Based on these data, the authors conclude that Wnt3 has two independent roles: Wnt/β-catenin activation and initial PCP orientation (two-step model for PCP formation). However, the molecular basis for the interaction of Wnt3 with the PCP machinery and how the specificity of Wnt3 for both pathways is regulated at the level of Wnt-receiving cells (Fz-Dsh) remain unresolved. Also, with respect to PCP propagation, there is no answer with respect to the underlying mechanisms. The authors found that PCP components are expressed in the mid-blastula stage, but without any further indication of how the signal might be propagated, e.g., by a wavefront of local cell alignment. Here, it is necessary to address the underlying possible cellular interactions more explicitly.

(3) The proposed two-step model for PCP formation has important evolutionary implications in that it excludes the current alternate model according to which a long-range Wnt3-gradient orients PCP ("Wnt/β-catenin-first"). Nevertheless, the initial PCP orientation by Wnt3 - as proposed in the two-step model - is not explained at all on the molecular level. Another possible, but less well-discussed and studied option for linking Wnt3 with PCP action could be the role of other Wnt pathways. The authors present compelling evidence that Wnt3 is the most highly expressed Wnt in Clytia at all stages of development. The authors convincingly show that Wnt3 is the most highly expressed Wnt in Clytia at all stages of development (Figure S1). However, Wnt7 is also more highly expressed, which makes it a candidate for signal transduction from canonical Wnts to PCP Wnts. An involvement of Wnt7 in PCP regulation has been described in vertebrates (http://dx.doi.org/10.1016/j.celrep.2013.12.026). This would challenge the entire discussion and speculation on the evolutionary implications according to which PCP Wnt signaling comes first (PCP-first scenario") and canonical Wnt signaling later in metazoan evolution.

(4) The discussion, including Figure 6, is strongly biased towards the traditional evolutionary scenario postulating a choanzoan-sponge ancestry of metazoans. Chromosome-linkage data of pre-metazoans and metazoans (Schulz et al., 2023; https://doi.org/10 (1038/s41586-023-05936-6) now indicate a radically different scenario according to which ctenophores represent the ancestral form and are sister to sponges, cnidarians and bilaterians (the Ctenophora-sister hypothesis). This has also implications for the evolution of Wnt signalling, as discussed in the recent Nature Genetics Review by Holzem et al. (2024) (https://doi.org/10.1038/s41576-024-00699-w). Furthermore, it calls into question the hypothesis of a filter-feeding multicellular gastrula-like ancestor as proposed by Haeckel (Maegele et al., 2023). These papers have not yet been referenced, but they would provide a more robust discussion.

Reviewer #1 (Public Review):

In this work, Kanie and colleagues explored the composition, structure, and assembly hierarchy of distal appendage proteins. The microscopy was well executed and appropriately quantified. Importantly, the quality of individual antibodies was documented with a discussion of how this might complicate results. The hierarchy of assembly was established by careful quantification of assembly in an extensive set of knockout cell lines. This work will be of interest to cell biologists exploring organelle assembly as well as human geneticists trying to understand the clinical implications of mutations.

Reviewer #1 (Public review):

Summary:

The manuscript asks the question of whether astrocytes contribute to behavioral deficits triggered by early life stress. This question is tested by experiments that monitor the effects of early life stress on anxiety-like behaviors, long-term potentiation in the lateral amygdala, and immunohistochemistry of astrocyte-specific (GFAP, Cx43, GLT-1) and general activity (c-Fos ) markers. Secondarily, astrocyte activity in the lateral amygdala is impaired by viruses that suppress gap-junction coupling or reduce astrocyte Ca2+ followed by behavioral, synaptic plasticity, and c-Fos staining. Early life stress is found to reduce expression of GFAP, Cx43 and induce translocation of the glucocorticoid receptor to astrocytic nuclei. Both early life stress and astrocyte manipulations are found to result in generalization of fear to neutral auditory cues. All of the experiments are done well with appropriate statistics and control groups. The manuscript is very well-written and the data are presented clearly. The authors' conclusion that lateral amygdala astrocytes regulate amygdala-dependent behaviors is strongly supported by the data as is the conclusion that cellular and behavioral outcomes provoked by early life stress are similar to the outcomes provoked by astrocyte dysfunction. However, the extent to which early life stress requires astrocytes to generate these outcomes remains open to debate.

Strengths:

A strong combination of behavioral, electrophysiology, and immunostaining approaches is utilized and possible sex-differences in behavioral data are considered. The experiments clearly demonstrate that disruption of astrocyte networks or reduction of astrocyte Ca2+ provoke generalization of fear and impair long-term potentiation in lateral amygdala. The provocative finding that astrocyte dysfunction accounts for a subset of behavioral effects of early life stress (e.g. not elevated plus or distance traveled observations) is also perceived as a strength.

Weaknesses:

The main weakness is absence of direct evidence that behavioral and neuronal plasticity after early life stress can be attributed to astrocytes. It remains unknown what would happen if astrocyte activity were disrupted concurrently with early life stress or if changes in astrocyte Ca2+ could attenuate early life stress outcomes. As is, the only presented evidence that early life stress involves astrocytes is nuclear translocation of GR and downregulation of GFAP and Cx43 in Figure 3 which may or may not cause the reported astrocyte activity changes.

Reviewer #1 (Public review):

Summary:

The authors attempt to validate Fisher Kernels on the top of HMM as a way to better describe human brain dynamics at resting-state. The objective criterion was the better prediction of the proposed pipeline of the individual traits.

Comments on revisions:

The authors addressed adequately all my comments.

Reviewer #1 (Public review):

Summary:

This study adapts a previously published model of the cat spinal locomotor network to make predictions of how phase durations of swing and stance at different treadmill speeds in tied-belt and split-belt conditions would be altered following a lateral hemisection. The simulations make several predictions that are replicated in experimental settings. This updated manuscript addressed well many of the reviewer comments made to the first version.

Strengths:

-Despite only altering the connections in the model, the model is able to replicate very well several experimental findings. This provides strong validation for the model and highlights its utility as a tool to investigate the operations of mammalian spinal locomotor networks.

-The study provides insights about interactions between the left and right side of the spinal locomotor networks, and how these interactions depend on the mode of operation, as determined by speed and state of the nervous system.

-The writing is logical, clear and easy to follow.

Comments on revisions:

My concerns were well addressed by the authors. I have no additional concerns

Reviewer #1 (Public review):

Summary:

This is a high-quality, well-thought through analysis of STEC transmission in Alberta, Canada.

Strengths:

* The combined human and animal sampling is a great foundation for this kind of study.<br /> * Phylogenetic analyses seem to have been carried out in a high quality fashion.

Comments on the revised version:

I'd like to thank the authors for the diligence with which they addressed my comments. I agree with their points and am happy for the manuscript to proceed.

Reviewer #1 (Public review):

Summary:

Can a plastic RNN serve as a basis function for learning to estimate value. In previous work this was shown to be the case, with a similar architecture to that proposed here. The learning rule in previous work was back-prop with an objective function that was the TD error function (delta) squared. Such a learning rule is non-local as the changes in weights within the RNN, and from inputs to the RNN depends on the weights from the RNN to the output, which estimates value. This is non-local, and in addition, these weights themselves change over learning. The main idea in this paper is to examine if replacing the values of these non-local changing weights, used for credit assignment, with random fixed weights can still produce similar results to those obtained with complete bp. This random feedback approach is motivated by a similar approach used for deep feed-forward neural networks.

This work shows that this random feedback in credit assignment performs well but is not as well as the precise gradient-based approach. When more constraints due to biological plausibility are imposed performance degrades. These results are not surprising given previous results on random feedback. This work is incomplete because the delay times used were only a few time steps, and it is not clear how well random feedback would operate with longer delays. Additionally, the examples simulated with a single cue and a single reward are overly simplistic and the field should move beyond these exceptionally simple examples.

Strengths:

• The authors show that random feedback can approximate well a model trained with detailed credit assignment.<br /> • The authors simulate several experiments including some with probabilistic reward schedules and show results similar to those obtained with detailed credit assignments as well as in experiments.<br /> • The paper examines the impact of more biologically realistic learning rules and the results are still quite similar to the detailed back-prop model.

Weaknesses:

• The authors also show that an untrained RNN does not perform as well as the trained RNN. However, they never explain what they mean by an untrained RNN. It should be clearly explained. These results are actually surprising. An untrained RNN with enough units and sufficiently large variance of recurrent weights can have a high-dimensionality and generate a complete or nearly complete basis, though not orthonormal (e.g: Rajan&Abbott 2006). It should be possible to use such a basis to learn this simple classical conditioning paradigm. It would be useful to measure the dimensionality of network dynamics, in both trained and untrained RNN's.

• The impact of the article is limited by using a network with discrete time-steps, and only a small number of time steps from stimulus to reward. What is the length of each time step? If it's on the order of the membrane time constant, then a few time steps are only tens of ms. In the classical conditioning experiments typical delays are of the order to hundreds of milliseconds to seconds. Authors should test if random feedback weights work as well for larger time spans. This can be done by simply using a much larger number of time steps.

• In the section with more biologically constrained learning rules, while the output weights are restricted to only be positive (as well as the random feedback weights), the recurrent weights and weights from input to RNN are still bi-polar and can change signs during learning. Why is the constraint imposed only on the output weights? It seems reasonable that the whole setup will fail if the recurrent weights were only positive as in such a case most neurons will have very similar dynamics, and the network dimensionality would be very low. However, it is possible that only negative weights might work. It is unclear to me how to justify that bipolar weights that change sign are appropriate for the recurrent connections and inappropriate for the output connections. On the other hand, an RNN with excitatory and inhibitory neurons in which weight signs do not change could possibly work.

• Like most papers in the field this work assumes a world composed of a single cue. In the real world there many more cues than rewards, some cues are not associated with any rewards, and some are associated with other rewards or even punishments. In the simplest case, it would be useful to show that this network could actually work if there are additional distractor cues that appear at random either before the CS, or between the CS and US. There are good reasons to believe such distractor cues will be fatal for an untrained RNN, but might work with a trained RNN, either using BPPT or random feedback. Although this assumption is a common flaw in most work in the field, we should no longer ignore these slightly more realistic scenarios.

DOI: 10.1016/j.celrep.2024.115195

Resource: None

Curator: @sjvitug

SciCrunch record: RRID:ZFIN_ZDB-ALT-200131-1

What is this?

Reviewer #1 (Public review):

This is an interesting manuscript where the authors systematically measure rG4 levels in brain samples at different ages of patients affected by AD. To the best of my knowledge this is the first time that BG4 staining is used in this context and the authors provide compelling evidence to show an association with BG4 staining and age or AD progression, which interestingly indicates that such RNA structure might play a role in regulating protein homeostasis as previously speculated. The methods used and the results reported seems robust and reproducible. There were two main things that needed addressing:

(1) Usually in BG4 staining experiments to ensure that the signal detected is genuinely due to rG4 an RNase treatment experiment is performed. This does not have to be extended to all the samples presented but having a couple of controls where the authors observe loss of staining upon RNase treatment will be key to ensure with confidence that rG4s are detected under the experimental conditions. This is particularly relevant for this brain tissue samples where BG4 staining has never been performed before.

(2) The authors have an association between rG4-formation and age/disease progression. They also observe distribution dependency of this, which is great. However, this is still an association which does not allow the model to be supported. This is not something that can be fixed with an easy experiment and it is what it is, but my point is that the narrative of the manuscript should be more fair and reflect the fact that, although interesting, what the authors are observing is a simple correlation. They should still go ahead and propose a model for it, but they should be more balanced in the conclusion and do not imply that this evidence is sufficient to demonstrate the proposed model. It is absolutely fine to refer to the literature and comment on the fact that similar observations have been reported and this is in line with those, but still this is not an ultimate demonstration.

Comments on current version:

The authors have now addressed my concerns.

Reviewer #2 (Public review):

Summary:

This paper by Olah et al., uncovers a previously unknown role of HCN channels in shaping synaptic inputs to L2/3 cortical neurons. The authors demonstrate using slice electrophysiology and computational modeling that unlike layer 5 pyramidal neurons, L2/3 neurons have an enrichment of HCN channels in the proximal dendrites. This location provides a locus of neuromodulation for inputs onto the proximal dendrites from L4 without an influence on distal inputs from L1. the authors use pharmacology to demonstrate the effect of HCN channels on NMDA-mediated synaptic inputs from L4. The authors further demonstrate the developmental time course of HCN function in L2/3 pyramidal neurons. Taken together, this a well constructed investigation of HCN channel function and the consequences of these channels on synaptic integration in L2/3 pyramidal neurons.

Strengths:

The authors use careful, well-constrained experiments using multiple pharmacological agents to asses HCN channel contributions to synaptic integrations. The authors also use voltage-clamp to directly measure the current through HCN channels across developmental ages. The authors also provide supplemental data showing that their observation is consistent across multiple areas of the cerebral cortex.

Weaknesses:

The gradient of HCN channel function is based almost exclusively on changes in EPSP width measured at the soma. While providing strong evidence for the presence of HCN current in L2/3 neurons, there are space clamp issues related to the use of somatic whole-cell voltage clamp that should be considered in the discussion. One omission by the authors is related to cell morphology. They make a point of normalizing the current injections to cell capacitance to account for variability in neuronal morphology. It is not clear however, how, if at all, this variability would affect EPSP propagation and modulation by proximal HCN channels. This should at least be discussed. Also, if there is high variability in cell morphology, was this considered in the modeling experiments?

Reviewer #1 (Public review):

Summary:

The use of antalarmin, a selective CRF1 receptor antagonist, prevents the deficits in sociability in (acutely) morphine-treated males, but not in females. In addition, cell attached experiments show a rescue to control levels of the morphine-induced increased firing in PVN neurons from morphine-treated males. Similar results are obtained in CRF receptor 1-/- male mice, confirming the involvement of CRF receptor 1-mediated signaling in both sociability deficits and neuronal firing changes in morphine-treated male mice.

Strengths:

In the revised version of the paper the authors respond to some reviewers's points with a new statistical analysis of behavioral data and a new discussion of previous literature.

Weaknesses:

Following reviewers' comments, the authors provided mechanistic insights of their findings with new experiments.

Reviewer #1 (Public Review):

Summary:

This manuscript from Clayton and co-authors aims to clarify the molecular mechanism of BRAF dimer selectivity. Indeed, first generation BRAF inhibitors, targeting monomeric BRAFV600E, are ineffective in treating resistant dimeric BRAF isoforms. Here, the authors employed molecular dynamics simulations to study the conformational dynamics of monomeric and dimeric BRAF, in the presence and absence of inhibitors. Multi-microseconds MD simulations showed an inward shift of the αC helix in the BRAFV600E mutant dimer. This helped identify a hydrogen bond between the inhibitors and the BRAF residue Glu501 as critical for dimer compatibility. The stability of the aforementioned interaction seems to be important to distinguish between dimer-selective and equipotent inhibitors.

Strengths:

The study is overall valuable and robust. The authors used the recently developed particle mesh Ewald constant pH molecular dynamics, a state-of-the-art method, to investigate the correct histidines protonation considering the dynamics of the protein. Then, multi-microsecond simulations showed differences in the flexibility of the αC helix and DFG motif. The dimerization restricts the αC position in the inward conformation, in agreement with the result that dimer-compatible inhibitors are able to stabilize the αC-in state. Noteworthy, the MD simulations were used to study the interactions between the inhibitors and the protein, suggesting a critical role for a hydrogen bond with Glu501. Finally, simulations of a mixed state of BRAF (one protomer bound to the inhibitor and the other apo) indicate that the ability to stabilize the inward αC state of the apo protomer could be at the basis of the positive cooperativity of PHI1.

Reviewer #1 (Public review):

Summary:

The authors test the "OHC-fluid-pump" hypothesis by assaying the rates of kainic acid dispersal both in quiet and in cochleae stimulated by sounds of different levels and spectral content. The main result is that sound (and thus, presumably, OHC contractions and expansions) result in faster transport along the duct. OHC involvement is corroborated using salicylate, which yielded results similar to silence. Especially interesting is the fact that some stimuli (e.g., tones) seem to provide better/faster pumping than others (e.g., noise), ostensibly due to the phase profile of the resulting cochlear traveling-wave response.

Strengths:

The experiments appear well controlled and the results are novel and interesting. Some elegant cochlear modeling that includes coupling between the organ of Corti and the surrounding fluid as well as advective flow supports the proposed mechanism.

The current limitations and future directions of the study, including possible experimental tests, extensions of the modeling work, and practical applications to drug delivery, are thoughtfully discussed.

Reviewer #1 (Public review):

Summary:

The Major Histocompatibility Complex (MHC) region is a collection of numerous genes involved in both innate and adaptive immunity. MHC genes are famed for their role in rapid evolution and extensive polymorphism in a variety of vertebrates. This paper presents a summary of gene-level gain and loss of orthologs and paralogs within MHC across the diversity of primates, using publicly available data.

Strengths:

This paper provides a strong case that MHC genes are rapidly gained (by paralog duplication) and lost over millions of years of macroevolution. The authors are able to identify MHC loci by homology across species, and from this infer gene duplications and losses using phylogenetic analyses. There is a remarkable amount of genic turnover, summarized in Figure 6 and Figure 7, either of which might be a future textbook figure of immune gene family evolution. The authors draw on state-of-the-art phylogenetic methods, and their inferences are robust insofar as the data might be complete enough to draw such conclusions.

Weaknesses:

One concern about the present work is that it relies on public databases to draw inferences about gene loss, which is potentially risky if the publicly available sequence data are incomplete. To say, for example, that a particular MHC gene copy is absent in a taxon (e.g., Class I locus F absent in Guenons according to Figure 1), we need to trust that its absence from the available databases is an accurate reflection of its absence in the genome of the actual organisms. This may be a safe assumption, but it rests on the completeness of genome assembly (and gene annotations?) or people uploading relevant data. This reviewer would have been far more comfortable had the authors engaged in some active spot-checking, doing the lab work to try to confirm absences at least for some loci and some species. Without this, a reader is left to wonder whether gene loss is simply reflecting imperfect databases, which then undercuts confidence in estimates of rates of gene loss.

Some context is useful for comparing rates of gene turnover in MHC, to other loci. Changing gene copy numbers, duplications, and loss of duplicates, are common it seems across many loci and many organisms; is MHC exceptional in this regard, or merely behaving like any moderately large gene family? I would very much have liked to see comparable analyses done for other gene families (immune, like TLRs, or non-immune), and quantitative comparisons of evolutionary rates between MHC versus other genes. Does MHC gene composition evolve any faster than a random gene family? At present readers may be tempted to infer this, but evidence is not provided.

While on the topic of making comparisons, the authors make a few statements about relative rates. For instance, lines 447-8 compare gene topology of classical versus non-classical genes; and line 450 states that classical genes experience more turnover. But there are no quantitative values given to these rates to provide numerical comparisons, nor confidence intervals provided (these are needed, given that they are estimates), nor formal statistical comparisons to confirm our confidence that rates differ between types of genes.

More broadly, the paper uses sophisticated phylogenetic methods, but without taking advantage of macroevolutionary comparative methods that allow model-based estimation of macroevolutionary rates. I found the lack of quantitative measurements of rates of gene gain/loss to be a weakness of the present version of the paper, and something that should be readily remedied. When claiming that MHC Class I genes "turn over rapidly" (line 476) - what does rapidly mean? How rapidly? How does that compare to rates of genetic turnover at other families? Quantitative statements should be supported by quantitative estimates (and their confidence intervals).

The authors refer to 'shared function of the MHC across species' (e.g. line 22); while this is likely true, they are not here presenting any functional data to confirm this, nor can they rule out neofunctionalization or subfunctionalization of gene duplicates. There is evidence in other vertebrates (e.g., cod) of MHC evolving appreciably altered functions, so one may not safely assume the function of a locus is static over long macroevolutionary periods, although that would be a plausible assumption at first glance.

Reviewer #1 (Public review):

The authors present their new bioinformatic tool called TEKRABber, and use it to correlate expression between KRAB ZNFs and TEs across different brain tissues, and across species. While the aims of the authors are clear and there would be significant interest from other researchers in the field for a program that can do such correlative gene expression analysis across individual genomes and species, the presented approach and work display significant shortcomings. In the current state of the analysis pipeline, the biases and shortcomings mentioned below, for which I have seen no proof that they are accounted for by the authors, are severely impacting the presented results and conclusions. It is therefore essential that the points below are addressed, involving significant changes in the TEKRABber program as well as the analysis pipeline, to prevent the identification of false positive and negative signals, that would severely affect the conclusions one can raise about the analysis.

My main concerns are provided below:

One important shortcoming of the biocomputational approach is that most TEs are not actually expressed, and others (Alus) are not a proxy of the activity of the TE class at all. I will explain: While specific TE classes can act as (species-specific) promoters for genes (such as LTRs) or are expressed as TE derived transcripts (LINEs, SVAs), the majority of other older TE classes do not have such behavior and are either neutral to the genome or may have some enhancer activity (as mapped in the program they refer to 'TEffectR'. A big focus is on Alus, but Alus contribute to a transcriptome in a different way too: They often become part of transcripts due to alternative splicing. As such, the presence of Alu derived transcripts is not a proxy for the expression/activity of the Alu class, but rather a result of some Alus being part of gene transcripts (see also next point). The bottom line is that the TEKRABber software/approach is heavily prone to picking up both false positives (TEs being part of transcribed loci) and false negatives (TEs not producing any transcripts at all), which has a big implication for how reads from TEs as done in this study should be interpreted: The TE expression used to correlate the KRAB ZNF expression is simply not representing the species-specific influences of TEs where the authors are after.

With the strategy as described, a lot of TE expression is misinterpreted: TEs can be part of gene-derived transcripts due to alternative splicing (often happens for Alus) or as a result of the TE being present in an inefficiently spliced out intron (happens a lot) which leads to TE-derived reads as a result of that TE being part of that intron, rather than that TE being actively expressed. As a result, the data as analysed is not reliably indicating the expression of TEs (as the authors intend to) and should be filtered for any reads that are coming from the above scenarios: These reads have nothing to do with KRAB ZNF control, and are not representing actively expressed TEs and therefore should be removed. Given that from my lab's experience in the brain (and other) tissues, the proportion of RNA sequencing reads that are actually derived from active TEs is a stark minority compared to reads derived from TEs that happen to be in any of the many transcribed loci, applying this filtering is expected to have a huge impact on the results and conclusions of this study.

Another potential problem that I don't see addressed is that due to the high level of similarity of the many hundreds of KRAB ZNF genes in primates and the reads derived from them, and the inaccurate annotations of many KZNFs in non-human genomes, the expression data derived from RNA-seq datasets cannot be simply used to plot KZNF expression values, without significant work and manual curation to safeguard proper cross species ortholog-annotation: The work of Thomas and Schneider (2011) has studied this in great detail but genome-assemblies of non-human primates tend to be highly inaccurate in appointing the right ortholog of human ZNF genes. The problem becomes even bigger when RNA-sequencing reads are analyzed: RNA-sequencing reads from a human ZNF that emerged in great apes by duplication from an older parental gene (we have a decent number of those in the human genome) may be mapped to that older parental gene in Macaque genome: So, the expression of human-specific ZNF-B, that derived from the parental ZNF-A, is likely to be compared in their DESeq to the expression of ZNF-A in Macaque RNA-seq data. In other words, without a significant amount of manual curation, the DE-seq analysis is prone to lead to false comparisons which make the strategy and KRABber software approach described highly biased and unreliable.

There is no doubt that there are differences in expression and activity of KRAB-ZNFs and TEs respectively that may have had important evolutionary consequences. However, because all of the network analyses in this paper rely on the analyses of RNA-seq data and the processing through the TE-KRABber software with the shortcomings and potential biases that I mentioned above, I need to emphasize that the results and conclusions are likely to be significantly different if the appropriate measures are taken to get more accurate and curated TE and KRAB ZNF expression data.

Finally, there are some minor but important notes I want to share:

The association with certain variations in ZNF genes with neurological disorders such as AD, as reported in the introduction is not entirely convincing without further functional support. Such associations could merely happen by chance, given the high number of ZNF genes in the human genome and the high chance that variations in these loci happen to associate with certain disease-associated traits. So using these associations as an argument that changes in TEs and KRAB ZNF networks are important for diseases like AD should be used with much more caution.

There are a number of papers where KRAB ZNF and TE expression are analysed in parallel in human brain tissues. So the novelty of that aspect of the presented study may be limited.

Reviewer #1 (Public review):

In this manuscript, Hoon Cho et al. presents a novel investigation into the role of PexRAP, an intermediary in ether lipid biosynthesis, in B cell function, particularly during the Germinal Center (GC) reaction. The authors profile lipid composition in activated B cells both in vitro and in vivo, revealing the significance of PexRAP. Using a combination of animal models and imaging mass spectrometry, they demonstrate that PexRAP is specifically required in B cells. They further establish that its activity is critical upon antigen encounter, shaping B cell survival during the GC reaction.

Mechanistically, they show that ether lipid synthesis is necessary to modulate reactive oxygen species (ROS) levels and prevent membrane peroxidation.

Highlights of the Manuscript:

The authors perform exhaustive imaging mass spectrometry (IMS) analyses of B cells, including GC B cells, to explore ether lipid metabolism during the humoral response. This approach is particularly noteworthy given the challenge of limited cell availability in GC reactions, which often hampers metabolomic studies. IMS proves to be a valuable tool in overcoming this limitation, allowing detailed exploration of GC metabolism.

The data presented is highly relevant, especially in light of recent studies suggesting a pivotal role for lipid metabolism in GC B cells. While these studies primarily focus on mitochondrial function, this manuscript uniquely investigates peroxisomes, which are linked to mitochondria and contribute to fatty acid oxidation (FAO). By extending the study of lipid metabolism beyond mitochondria to include peroxisomes, the authors add a critical dimension to our understanding of B cell biology.

Additionally, the metabolic plasticity of B cells poses challenges for studying metabolism, as genetic deletions from the beginning of B cell development often result in compensatory adaptations. To address this, the authors employ an acute loss-of-function approach using two conditional, cell-type-specific gene inactivation mouse models: one targeting B cells after the establishment of a pre-immune B cell population (Dhrs7b^f/f, huCD20-CreERT2) and the other during the GC reaction (Dhrs7b^f/f; S1pr2-CreERT2). This strategy is elegant and well-suited to studying the role of metabolism in B cell activation.

Overall, this manuscript is a significant contribution to the field, providing robust evidence for the fundamental role of lipid metabolism during the GC reaction and unveiling a novel function for peroxisomes in B cells. However, several major points need to be addressed:

Major Comments:

Figures 1 and 2

The authors conclude, based on the results from these two figures, that PexRAP promotes the homeostatic maintenance and proliferation of B cells. In this section, the authors first use a tamoxifen-inducible full Dhrs7b knockout (KO) and afterwards Dhrs7bΔ/Δ-B model to specifically characterize the role of this molecule in B cells. They characterize the B and T cell compartments using flow cytometry (FACS) and examine the establishment of the GC reaction using FACS and immunofluorescence. They conclude that B cell numbers are reduced, and the GC reaction is defective upon stimulation, showing a reduction in the total percentage of GC cells, particularly in the light zone (LZ).

The analysis of the steady-state B cell compartment should also be improved. This includes a more detailed characterization of MZ and B1 populations, given the role of lipid metabolism and lipid peroxidation in these subtypes.

Suggestions for Improvement:

- B Cell compartment characterization: A deeper characterization of the B cell compartment in non-immunized mice is needed, including analysis of Marginal Zone (MZ) maturation and a more detailed examination of the B1 compartment. This is especially important given the role of specific lipid metabolism in these cell types. The phenotyping of the B cell compartment should also include an analysis of immunoglobulin levels on the membrane, considering the impact of lipids on membrane composition.

- GC Response Analysis Upon Immunization: The GC response characterization should include additional data on the T cell compartment, specifically the presence and function of Tfh cells. In Fig. 1H, the distribution of the LZ appears strikingly different. However, the authors have not addressed this in the text. A more thorough characterization of centroblasts and centrocytes using CXCR4 and CD86 markers is needed.<br /> The gating strategy used to characterize GC cells (GL7+CD95+ in IgD− cells) is suboptimal. A more robust analysis of GC cells should be performed in total B220+CD138− cells.

- The authors claim that Dhrs7b supports the homeostatic maintenance of quiescent B cells in vivo and promotes effective proliferation. This conclusion is primarily based on experiments where CTV-labeled PexRAP-deficient B cells were adoptively transferred into μMT mice (Fig. 2D-F). However, we recommend reviewing the flow plots of CTV in Fig. 2E, as they appear out of scale. More importantly, the low recovery of PexRAP-deficient B cells post-adoptive transfer weakens the robustness of the results and is insufficient to conclusively support the role of PexRAP in B cell proliferation in vivo.

- In vitro stimulation experiments: These experiments need improvement. The authors have used anti-CD40 and BAFF for B cell stimulation; however, it would be beneficial to also include anti-IgM in the stimulation cocktail. In Fig. 2G, CTV plots do not show clear defects in proliferation, yet the authors quantify the percentage of cells with more than three divisions. These plots should clearly display the gating strategy. Additionally, details about histogram normalization and potential defects in cell numbers are missing. A more in-depth analysis of apoptosis is also required to determine whether the observed defects are due to impaired proliferation or reduced survival.

Reviewer #1 (Public review):

Summary:

The authors present an interesting study using RL and Bayesian modelling to examine differences in learning rate adaptation in conditions of high and low volatility and noise respectively. Through "lesioning" an optimal Bayesian model, they reveal that apparently a suboptimal adaptation of learning rates results from incorrectly detecting volatility in the environment when it is not in fact present.

Strengths:

The experimental task used is cleverly designed and does a good job of manipulating both volatility and noise. The modelling approach takes an interesting and creative approach to understanding the source of apparently suboptimal adaptation of learning rates to noise, through carefully "lesioning" and optimal Bayesian model to determine which components are responsible for this behaviour.

Weaknesses:

The study has a few substantial weaknesses; the data and modelling both appear robust and informative, and it tackles an interesting question. The model space could potentially have been expanded, particularly with regard to the inclusion of alternative strategies such as those that estimate latent states and adapt learning accordingly.

Reviewer #1 (Public review):

Summary:

In this study, the authors advance our understanding of neurodevelopmental changes in the brain's structural and functional connectivity, as well as their coupling. The paper presents evidence of alterations in and stability of the principal organizational gradients of structure and function across development (age) and contrasts them between neurotypical and neurodivergent individuals. The authors further extend their findings by exploring links with graph theory measures of brain connectivity and indices of nodal structure-function coupling. Finally, the developmental shifts in structural and functional brain organization are examined for potential associations with cognitive and psychopathological markers. The results suggest that structure-function coupling, both brain-wide and within specific functional networks, is associated with certain cognitive dimensions but not with measures of psychopathology.

Strengths:

This manuscript makes a significant contribution to the field by synthesizing previous research while offering novel insights into the developmental trajectories of brain organization. A key strength of this study lies in its integration of both structural and functional connectivity data, providing a comprehensive view of brain changes throughout development. The authors present findings that challenge earlier reports of shifts in principal gradients during late childhood and early adolescence (e.g., Dong et al., 2021; Xia et al., 2022), underscoring an important inconsistency that could have broader implications for our understanding of developmental brain reorganization. The introduction and discussion sections are well-crafted, offering a thorough review of relevant prior studies and effectively situating the current findings within the broader context of the literature. Additionally, the study design and methodology are detailed and adhere to recommended best practices, demonstrating a commendable level of rigor in the formulation of the study and its various assessments.

Weaknesses:

Despite these strengths, I think there are aspects of the manuscript that would benefit from further refinement. Below is detailed feedback and suggestions provided point-by-point.

Lack of Sensitivity Analyses for some Key Methodological Decisions:<br /> Certain methodological choices in this manuscript diverge from approaches used in previous works. In these cases, I recommend the following: (i) The authors could provide a clear and detailed justification for these deviations from established methods, and (ii) supplementary sensitivity analyses could be included to ensure the robustness of the findings, demonstrating that the results are not driven primarily by these methodological changes. Below, I outline the main areas where such evaluations are needed:<br /> - Use of Communicability Matrices for Structural Connectivity Gradients: The authors chose to construct structural connectivity gradients using communicability matrices, arguing that diffusion map embedding "requires a smooth, fully connected matrix." However, by definition, the creation of the affinity matrix already involves smoothing and ensures full connectedness. I recommend that the authors include an analysis of what happens when the communicability matrix step is omitted. This sensitivity test is crucial, as it would help determine whether the main findings hold under a simpler construction of the affinity matrix. If the results significantly change, it could indicate that the observations are sensitive to this design choice, thereby raising concerns about the robustness of the conclusions. Additionally, if the concern is related to the large range of weights in the raw structural connectivity (SC) matrix, a more conventional approach is to apply a log-transformation to the SC weights (e.g., log(1+𝑆𝐶𝑖𝑗)), which may yield a more reliable affinity matrix without the need for communicability measures.<br /> - Individual-Level Gradients vs. Group-Level Gradients: Unlike previous studies that examined alterations in principal gradients (e.g., Xia et al., 2022; Dong et al., 2021), this manuscript focuses on gradients derived directly from individual-level data. In contrast, earlier works have typically computed gradients based on grouped data, such as using a moving window of individuals based on age (Xia et al.) or evaluating two distinct age groups (Dong et al.). I believe it is essential to assess the sensitivity of the findings to this methodological choice. Such an evaluation could clarify whether the observed discrepancies with previous reports are due to true biological differences or simply a result of different analytical strategies.<br /> - Procrustes Transformation: It is unclear why the authors opted to include a Procrustes transformation in this analysis, especially given that previous related studies (e.g., Dong et al.) did not apply this step. I believe it is crucial to evaluate whether this methodological choice influences the results, particularly in the context of developmental changes in organizational gradients. Specifically, the Procrustes transformation may maximize alignment to the group-level gradients, potentially masking individual-level differences. This could result in a reordering of the gradients (e.g., swapping the first and second gradients), which might obscure true developmental alterations. It would be informative to include an analysis showing the impact of performing vs. omitting the Procrustes transformation, as this could help clarify whether the observed effects are robust or an artifact of the alignment procedure. (Please also refer to my comment on adding a subplot to Figure 1)<br /> - SC-FC Coupling Metric: The approach used to quantify nodal SC-FC coupling in this study appears to deviate from previously established methods in the field. The manuscript describes coupling as the "Spearman-rank correlation between Euclidean distances between each node and all others within structural and functional manifolds," but this description is unclear and lacks sufficient detail. Furthermore, this differs from what is typically referred to as SC-FC coupling in the literature. For instance, the cited study by Park et al. (2022) utilizes a multiple linear regression framework, where communicability, Euclidean distance, and shortest path length are independent variables predicting functional connectivity (FC), with the adjusted R-squared score serving as the coupling index for each node. On the other hand, the Baum et al. (2020) study, also cited, uses Spearman correlation, but between raw structural connectivity (SC) and FC values. If the authors opt to introduce a novel coupling metric, it is essential to demonstrate its similarity to these previous indices. I recommend providing an analysis (supplementary) showing the correlation between their chosen metric and those used in previous studies (e.g., the adjusted R-squared scores from Park et al. or the SC-FC correlation from Baum et al.). Furthermore, if the metrics are not similar and results are sensitive to this alternative metric, it raises concerns about the robustness of the findings. A sensitivity analysis would therefore be helpful (in case the novel coupling metric is not similar to previous ones) to determine whether the reported effects hold true across different coupling indices.

Methodological ambiguity/lack of clarity in the description of certain evaluation steps:<br /> Some aspects of the manuscript's methodological descriptions are ambiguous, making it challenging for future readers to fully reproduce the analyses based on the information provided. I believe the following sections would benefit from additional detail and clarification:<br /> - Computation of Manifold Eccentricity: The description of how eccentricity was computed (both in the results and methods sections) is unclear and may be problematic. The main ambiguity lies in how the group manifold origin was defined or computed. Specifically:<br /> (1) In the results section, it appears that separate manifold origins were calculated for the NKI and CALM groups, suggesting a dataset-specific approach.<br /> (2) Conversely, the methods section implies that a single manifold origin was obtained by somehow combining the group origins across the three datasets, which seems contradictory.<br /> Moreover, including neurodivergent individuals in defining the central group manifold origin is conceptually problematic. Given that neurodivergent participants might exhibit atypical brain organization (as suggested by Fig. 1), this inclusion could skew the definition of what should represent a typical or normative brain manifold. A more appropriate approach might involve constructing the group manifold origin using only the neurotypical participants from both the NKI and CALM datasets. Given the reported similarity between group-level manifolds of neurotypical individuals in CALM and NKI, it would be reasonable to expect that this combined origin should be close to the origin computed within neurotypical samples of either NKI or CALM. As a sanity check, I recommend reporting the distance of the combined neurotypical manifold origin to the centers of the neurotypical manifolds in each dataset. Moreover, if the manifold origin was constructed while utilizing all samples (including neurodivergent samples) I think this needs to be reconsidered.<br /> - Computation of SC-FC coupling: As noted in a previous comment, the explanation of this procedure is vague. The description lacks detail on the specific steps taken and differs from previous standard approaches in the field. I suggest clarifying the methodology and comparing with previous SC-FC coupling metrics.<br /> - Performing Procrustes transformation: The brief explanation in the first paragraph of page 30 does not provide enough information about the procedure or its justification. Since the Procrustes transformation alters the shape of individual gradients, it could artificially inflate consistency across development. I recommend including a rationale for using the Procrustes transformation and conducting a sensitivity analysis to assess its impact on the findings. Additionally, clarifying how exactly the transformation was applied to align gradients across hemispheres, individuals, and or datasets would help resolve ambiguity.

Insufficient Supporting Evaluations for Certain Claims:<br /> There are instances where additional analyses are necessary to substantiate the claims made in the manuscript. Without these evaluations, some conclusions may be premature or potentially misleading. I believe the following points need further analysis or, alternatively, adjustments to the claims:<br /> - Evaluating the Consistency of Gradients Across Development: The results shown in Fig. 1.e are used as evidence suggesting that gradients are consistent across ages. However, I believe additional analyses are required to identify potential sources of the observed inconsistency compared to previous works. The claim that the principal gradient explains a similar degree of variance across ages does not necessarily imply that the spatial structure of the gradient remains stable. The observed variance explanation is hence not enough to ascertain inconsistency with findings from Dong et al., as the spatial configuration of gradients may still change over time. Moreover, the introduction of the Procrustes transformation (not used by Dong et al.) further ambiguates the cause of this inconsistency. I suggest the following additional analyses to strengthen this claim: (1) Alignment to Group-Level Gradients: Assess how much of the variance in individual FC matrices is explained by each of the group-level gradients (G1, G2, and G3, for both FC and SC). This analysis could be visualized similarly to Fig. 1.e, with age on the x-axis and variance explained on the y-axis. If the explained variance varies as a function of age, it may indicate that the gradients are not as consistent as currently suggested. (2) For each individual's gradients (G1, G2, and G3, separately for FC and SC, without Procrustes transformation), evaluate their spatial similarity to the corresponding group-level gradients using a similarity metric (e.g., correlation coefficient). High spatial similarity, without a Procrustes transformation, would support the claim of stable gradient structures across development. On the other hand, if the similarities alter during development (e.g. such that at a certain age, individual G1 is less similar to group G1) this would contradict the stability of gradients during development. These additional analyses could potentially be included as additional panels in Fig. 1. In case significant deviations are observed, it might help refine the interpretation of the results and provide a more nuanced understanding of developmental changes in gradient organization.<br /> - Prediction vs. Association Analysis: The term "prediction" is used throughout the manuscript to describe what appear to be in-sample association tests. This terminology may be misleading, as prediction generally implies an out-of-sample evaluation where models trained on a subset of data are tested on a separate, unseen dataset. If the goal of the analyses is to assess associations rather than make true predictions, I recommend refraining from using the term "prediction" and instead clarifying the nature of the analysis. Alternatively, if prediction is indeed the intended aim (which would be more compelling), I suggest conducting the evaluations using a k-fold cross-validation framework. This would involve training the Generalized Additive Mixed Models (GAMMs) on a portion of the data and testing their predictive accuracy on a held-out sample (i.e., different individuals). Additionally, the current design appears to focus on predicting SC-FC coupling using cognitive or pathological dimensions. This is contrary to the more conventional approach of predicting behavioral or pathological outcomes from brain markers like coupling. Could the authors clarify why this reverse direction of analysis was chosen? Understanding this choice is crucial, as it impacts the interpretation and potential implications of the findings.

Methodological considerations<br /> - In typical applications of diffusion map embedding, sparsification (e.g., retaining only the top 10% of the strongest connections) is often employed at the vertex-level resolution to ensure computational feasibility. However, since the present study performs the embedding at the level of 200 brain regions (a considerably coarser resolution), this step may not be necessary or justifiable. Specifically, for FC, it might be more appropriate to retain all positive connections rather than applying sparsification, which could inadvertently eliminate valuable information about lower-strength connections. Whereas for SC, as the values are strictly non-negative, retaining all connections should be feasible and would provide a more complete representation of the structural connectivity patterns. Given this, it would be helpful if the authors could clarify why they chose to include sparsification despite the coarser regional resolution, and whether they considered this alternative approach (using all available positive connections for FC and all non-zero values for SC). It would be interesting if the authors could provide their thoughts on whether the decision to run evaluations at the resolution of brain regions could itself impact the functional and structural manifolds, their alteration with age, and or their stability (in contrast to Dong et al. which tested alterations in high-resolution gradients).

The Issue of Abstraction and Benefits of the Gradient-Based View:<br /> - The manuscript interprets the eccentricity findings as reflecting changes along the segregation-integration spectrum. Given this, it is unclear why a more straightforward analysis using established graph-theory measures of segregation-integration was not pursued instead. Mapping gradients and computing eccentricity adds layers of abstraction and complexity. If similar interpretations can be derived directly from simpler graph metrics, what additional insights does the gradient-based framework offer? While the manuscript argues that this approach provides "a more unifying account of cortical reorganization," it is not evident why this abstraction is necessary or advantageous over traditional graph metrics. Clarifying these benefits would strengthen the rationale for using this method.

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Reviewer #1 (Public review):

Summary:

The study dissects distinct pools of diacylglycerol (DAG), continuing a line of research on the central concept that there is a major lipid metabolism DAG pool in cells, but also a smaller signaling DAG pool. It tests the hypothesis that the second pool is regulated by Dip2, which influences Pkc1 signaling. The group shows that stressed yeast increase specific DAG species C36:0 and 36:1, and propose this promotes Pkc1 activation via Pck1 binding 36:0. The study also examines how perturbing the lipid metabolism DAG pool via various deletions such as lro1, dga1, and pah1 deletion impacts DAG and stress signaling. Overall this is an interesting study that adds new data to how different DAG pools influence cellular signaling.

Strengths:

The study nicely combined lipidomic profiling with stress signaling biochemistry and yeast growth assays.

Weaknesses:

One suggestion to improve the study is to examine the spatial organization of Dip2 within cells, and how this impacts its ability to modulate DAG pools. Dip2 has previously been proposed to function at mitochondria-vacuole contacts (Mondal 2022). Examining how Dip2 localization is impacted when different DAG pools are manipulated such as by deletion Pah1 (also suggested to work at yeast contact sites such as the nucleus-vacuole junction), or with Lro1 or Dga1 deletion would broaden the scope of the study.

Reviewer #1 (Public review):

Summary:

This manuscript by Alonso-Caraballo et al, is a novel piece of work that examines the impact of oxycodone self-administration on neural plasticity within the paraventricular thalamic (PVT) to nucleus accumbens shell (Shell) pathway - two regions shown to play a key role in cue-induced drug seeking on their own, and whether this plasticity varies based on abstinence period and biological sex.

Strengths:

The authors show using a clinically relevant long-access model of opioid self-administration promotes dependence and acute withdrawal in both male and female rats. During subsequent cue-induced relapse tests at 1 or 14 days following the conclusion of self-administration, data show that while both males and females demonstrate drug-seeking behavior at both time points, females show a further elevation in responding on day 14 versus day 1 which is not observed in the males. When accounting for past work showing elevations in drug-seeking in males after 30 days, these data indicate that craving-induced relapse for opioids may develop faster and may be more pronounced in females compared to males.

These behavioral findings were paralleled by the use of ex vivo acute slice electrophysiology and circuit-specific ex vivo optogenetics to examine the impact of oxycodone self-administration on synaptic strength within the paraventricular thalamus (PVT) to nucleus accumbens shell (NAcSh) pathway(s). Data support a time-dependent but sex-independent strengthening of glutamatergic signaling at PVT-to-NAcSh medium spiny neurons (MSNs) that is only present following a relapse test at 14 days post abstinence in males versus females, providing the first evidence that opioid self-administration and/or cue-induced drug-seeking augments this pathway. Using an extensive set of physiological measures, the authors show that this increased synaptic strength reflects an upregulation of presynaptic release probability. Further, this upregulation of excitatory signaling aligned temporally with an increase in MSN excitability, as assessed by increases in action potential firing frequency. Finally, the authors provide the first evidence that similar to other inputs to the NAcSh, PVT projections innervate both MSN as well as local interneurons, promoting a GABA-A-specific feedforward inhibitory circuit. Interestingly, unlike direct excitatory inputs to MSNs, no changes were observed ostensibly within this feedforward circuit, highlighting a selective enhancement of excitatory drive and output of MSNs with protracted abstinence.

Overall, these data highlight a potential role for heightened synaptic strength within the PVT-NAcSh pathway in cue-induced relapse behavior during protracted abstinence and identify a potential therapeutic target during abstinence to reduce relapse risk in abstaining individuals.

Weaknesses:

Overall, the experimental approach and data provided appear rigorous and support their overall conclusions and achieve their goal of understanding how opioid self-administration impacts synaptic strength within the PVT-NAcSh pathway. Although not undermining these data, there are a few potential weaknesses that reduce the impact of the work. For example, the inability to directly assess whether cue-induced drug-seeking is in fact augmented compared to daily intake during self-administration in the maintenance face only permits the authors to denote that reexposure to cues and the context is sufficient to promote active lever pressing without demonstrating whether seeking behavior is in fact elevated further during a cue test. This is notably understandable as drug available sessions were 6-hours versus a 1-hour relapse test. Importantly, it is clearly demonstrated that drug seeking is higher on average in female mice after 14 days versus 1 day.

With regard to the interpretation of electrophysiology findings, the lack of inclusion of an abstinence-only group does not permit interpretations to parse out whether observed increases in synaptic strength (or the lack of) reflect abstinence or an interaction between abstinence period and re-exposure to the operant chamber, as slices were taken 30-45 min post relapse test. While much literature has shown that drug-induced adaptations in the NAc require a post-drug period for plasticity to measurably emerge, studies have also shown that re-exposure to heroin-associated cues following abstinence seemingly "reverses" increases in cell excitability in prelimbic-NAc pyramidal neurons (Kokane et al., 2023) and that depotentiation of morphine-induced increases in synaptic strength in the NAc shell can be depotentiated by drug re-exposure - an effect also observed with cocaine re-exposure (Madayag et al., 2019). Notably, the lack of effect at 14 but not 1 day supports the likelihood that the relapse test does not in fact influence the plasticity within the PVT-NAcSh circuit.

While the lack of effect on AMPAR:NMDAR ratio and rectification indices do support the notion that enhanced EPSC amplitudes in input-output curves do not reflect a change in AMPAR subunit expression (i.e., increased GluA2-lacking receptors that exhibit inward rectification at depolarized potential) nor a change in postsynaptic sensitivity to glutamate, without direct assessment of AMPAR-specific and NMDAR-specific input-output curves, it doesn't definitively exclude the possibility that both AMPA and NMDA receptor currents are being upregulated, thus negating an observable change in postsynaptic strength.

Overall, these findings provide novel insight into how the PVT-NAcSh pathway is altered by opioid self-administration and whether this is unique based on abstinence period and sex. Importantly, these were the primary objectives stated by the author. Data highlight a potential role for the observed adaptations in relapse behavior and identify a potential therapeutic target during abstinence to reduce relapse risk in abstaining individuals. However, it should be noted that no causal link is demonstrated without experiments to reduce/prevent relapse.

Reviewer #1 (Public review):

In this manuscript, the role of orexin receptors in dopamine transmission is studied. It extends previous findings suggesting an interplay between these two systems in regulating behaviour by first characterizing the expression of orexin receptors in the midbrain and then disrupting orexin transmission in dopaminergic neurons by deleting its predominant receptor, OX1R (Ox1R fl/fl, Dat-Cre tg/wt mice). Electrophysiological and calcium imaging data suggest that orexin A acutely and directly stimulates SN and VTA dopaminergic neurons but does not seem to induce c-Fos expression. Behavioral effects of depleting OX1R from dopaminergic neurons include enhanced novelty-induced locomotion and exploration, relative to littermate controls (Ox1R fl/fl, Dat-Cre wt/wt). However, no difference between groups is observed in tests that measure reward processing, anxiety, and energy homeostasis. To test whether the depletion of OX1R alters overall orexin-triggered activation across the brain, PET imaging is used in OX1R∆DAT knockout and control mice. This analysis reveals that several regions show higher neuronal activation after orexin injection in OX1R∆DAT mice, but the authors focus their follow-up study on the dorsal bed nucleus of the stria terminalis (BNST) and lateral paragigantocellular nucleus (LPGi). Dopaminergic inputs and expression of dopamine receptors type-1 and -2 (DRD1 & DRD2) are assessed and compared to control demonstrating a moderate decrease in DRD1 and DRD2 expression in the BNST of OX1R∆DAT mice and unaltered expression of DRD2, with absence of DRD1 expression in LPGi of both groups. Overall, this study is valuable for the information it provides on orexin receptor expression and function in behaviour, as well as for the new tools it generated for the specific study of this receptor in dopaminergic circuits.

Strengths:

The use of a transgenic line that lacks OX1R in dopamine-transporter expressing neurons is a strong approach to dissect the direct role of orexin in modulating dopamine signaling in the brain. The battery of behavioral assays used to study this line provides valuable information for researchers interested in the interplay between dopamine and orexin systems and their role in animal physiology.

Weaknesses:

This study falls short in providing evidence for an anatomical substrate and mechanism underlying the altered behavior observed in mice lacking orexin receptor subtype 1 in dopaminergic neurons. How orexin transmission in dopaminergic neurons regulates the expression of postsynaptic dopamine receptors (as observed in the BNST of OX1R∆DAT mice) is an intriguing question not addressed in this study. An important aspect not investigated in this study is whether the disruption of orexin activity affects dopamine release in target areas.

Reviewer #1 (Public review):

Summary:

This study sought to reveal the potential roles of m6A RNA methylation in gene dosage regulatory mechanisms, particularly in the context of aneuploid genomes in Drosophila. Specifically, this work looked at the relationships between expression of m6A regulatory factors, RNA methylation status, classical and inverse dosage effects, and dosage compensation. Using RNA sequencing and m6A mapping experiments, an in depth analysis was performed to reveal changes in m6A status and expression changes across multiple aneuploid Drosophila models. The authors propose that m6A methylation regulates MOF and, in turn, deposition of H4K16Ac, critical regulators of gene dosage in the context of genomic imbalance.

Strengths:

This study seeks to address an interesting question with respect to gene dosage regulation and the possible roles of m6A in that process. Previous work has linked m6A to X-inactivation in humans through the Xist lncRNA, and to the regulation of the Sxl in flies. This study seeks to broaden that understanding beyond these specific contexts to more broadly understand how m6A impacts imbalanced genomes in other contexts.

Weaknesses:

The methods being used particularly for analysis of m6A at both the bulk and transcript-specific level are not sufficiently specific or quantitative to be able to confidently draw the conclusions the authors seek to make. MeRIP m6A mapping experiments can be very valuable, but differential methylation is difficult to assess when changes are small (as they often are, in this study but also m6A studies more broadly). For instance based on the data presented and the methods described, it is not clear that the statement that "expression levels at m6A sites in aneuploidies are significantly higher than that in wildtype" is supported. In my initial review I pointed out that MeRIP experiments are not quantitative and can be difficult to interpret when small changes are present. The data as presented still show only RPKM in IP samples, and the text alludes to changes in IP enrichment that are significant but the data do not appear to have been included in the figure. Concerns about the bulk-level m6A measurements also remain, as the new data showing m6A levels in mRNA show changes that are even smaller than those initially demonstrated in total RNA. Yet the data are still presented as significant, biologically relevant changes. The conclusions about mRNA m6A levels are not strengthened by measurements.

Reviewer #1 (Public review):

Summary:

How reconsolidation works - particularly in humans - remains largely unknown. With an elegant, 3-day design, combining fMRI and psychopharmacology, the authors provide evidence for a certain role for noradrenaline in the reconsolidation of memory for neutral stimuli. All memory tasks were performed in the context of fMRI scanning, with additional resting state acquisitions performed before and after recall testing on Day 2. On Day 1, 3 groups of healthy participants encoded word-picture associates (with pictures being either scenes or objects) and then performed an immediate cued recall task to presentation of the word (answering is the word old or new, and was it paired with a scene or an object). On Day 2, the cued recall task was repeated using half of the stimulus set words encoded on Day 1 (only old words were presented, with subjects required to indicate prior scene vs object pairing). This test was immediately preceded by the oral administration of placebo, cortisol, or yohimibine (to raise noradrenaline levels) depending on group assignment. On Day 3, all words presented on Day 1 were presented. As expected, on Day 3, memory was significantly enhanced for associations that were cued and successfully retrieved on Day 2 compared to uncued associations. However, for associative d', there was no Cued × Group interaction nor a main effect of Group, i.e., on the standard measure of memory performance, post-retrieval drug presence on Day 2 did not affect memory reconsolidation. As further evidence for a null result, fMRI univariate analyses showed no Cued × Group interactions in whole-brain or ROI activity.

Strengths:

There are some aspects of this study that I find impressive. The study is well-designed and the fMRI analysis methodology innovative and sound. The authors have made meticulous and thorough physiological measurements, and assays of mood, throughout the experiment. By doing so, they have overcome, to a considerable extent, the difficulties inherent in timing of human oral drug delivery in reconsolidation tasks, where it is difficult to have drug present in the immediate recall period without affecting recall itself. This is beautifully shown in Fig. 3. I also think that having some neurobiological assay of memory reactivation when studying reconsolidation in humans is critical, and the authors provide this. While multi-voxel patterns of hemodynamic responses are, in my view, very difficult to equate with an "engram", these patterns do have something to do with memory.

Weaknesses:

I have major issues regarding the behavioral results and the framing of the manuscript:

(1) To arrive at group differences in memory performance, the authors performed median splitting of Day 3 trials by short and long reaction times during memory cueing on Day 2, as they took this as a putative measure of high/low levels of memory reactivation. Associative category hits on Day 3 showed a Group by Day 2 Reaction time (short, long) interaction, with post-hocs showing (according to the text) worse memory for short Day 2 RTs in the yohimbine group. These post-hocs should be corrected for multiple comparisons, as the result is not what would be predicted (see point 2). My primary issue here is that we are not given RT data for each group, nor is the median splitting procedure described in the methods. Was this across all groups, or within groups? Are short RTs in the yohimbine group any different from short RTs in the other two groups? Unfortunately, we are not given Day 2 picture category memory levels or reaction times for each group. This is relevant because (as given in Supplemental Table S1) memory performance (d´) for the Yohimbine group on Day 1 immediate testing is (roughly speaking) 20% lower than the other 2 groups (independently of whether the pairs will be presented again the following day). I appreciate that this is not significant in a group x performance ANOVA but how does this relate to later memory performance? What were the group-specific RTs on Day 1? So, before the reader goes into the fMRI results, there are questions regarding the supposed drug-induced changes in behavior. Indeed, in the discussion, there is repeated mention of subsequent memory impairment produced by yohimbine but the nature of the impairment is not clear.

This weakness was satisfactorily addressed in one revision round. As RT data are often not normally distributed, were they transformed prior to entry into linear models?

(2) The authors should be clearer as to what their original hypotheses were, and why they did the experiment. Despite being a complex literature, I would have thought the hypotheses would be reconsolidation impairment by cortisol and enhancement by yohimbine. Here it is relevant to point out that - only when the reader gets to the Methods section - there is mention of a paper published by this group in 2024. In this publication, the authors used the same study design but administered a stress manipulation after Day 2 cued recall, instead of a pharmacological one. They did not find a difference in associative hit rate between stress and control groups, but - similar to the current manuscript - reported that post-retrieval stress disrupts subsequent remembering (Day 3 performance) depending on neural memory reinstatement during reactivation (specifically driven by the hippocampus and its correlation with neocortical areas).

Instead of using these results, and other human studies, to motivate the current work, reference is made to a recent animal study: Line 169 "Building on recent findings in rodents (Khalaf et al. 2018), we hypothesized that the effects of post-retrieval noradrenergic and glucocorticoid activation would critically depend on the reinstatement of the neural event representation during retrieval". It is difficult to follow that a rodent study using contextual fear conditioning and examining single neuron activity to remote fear recall and extinction would be relevant enough to motivate a hypothesis for a human psychopharmacological study on emotionally neutral paired associates.

Minor comments<br /> - Related to Major issue 2. In the introduction, it would be helpful to be specific about the type of memory being probed in the different studies referenced (episodic vs conditioning). For the former, please make it clear whether stimuli to be remembered were emotional or neutral, and for which stimulus class drug effects were observed. This is particularly important given that in the first paragraph you describe memory reactivation in the context of traumatic memories via mention of PTSD. It would also be helpful to know to which species you refer. For example, in line 115, "timing of drug administration..." a rodent and a human study are cited.

This weakness was addressed in one revision round, resulting in an excellent introduction, highlighting the importance of studying post-retrieval effects for memory researchers and healthcare workers.

Reviewer #1 (Public review):

Summary:

Jin, Briggs and colleagues use light sheet imaging to reconstruct the islet three-dimensional Ca2+ network. The authors find that early/late responding (leader) cells are dynamic over time, and located at the islet periphery. By contrast, highly connected or hub cells are stable, and located toward the islet center. Suggesting that the two subpopulations are differentially regulated by fuel input, glucokinase activation only influences leader cell phenotype, whereas hubs remain stable.

Strengths:

The studies are novel in providing the first three-dimensional snapshot of the beta cell functional network, as well as determining the localization of some of the different subpopulations identified to date. The studies also provide some consensus as to the origin, stability and role of such subpopulations in islet function.

Weaknesses:

Experiments with metabolic enzyme activators do not take into account the influence of cell viability on the observed Ca2+ network data. Limitations of the imaging approach used need to be recognised and evaluated/discussed.

Comments on revisions:

The authors have addressed the majority of the points raised.

Reviewer #1 (Public review):

SARS-CoV-2 encodes a macrodomain (Mac1) within the nsp3 protein that removes ADP-ribose groups from proteins. However, its role during infection is not well understood. Evidence suggests that Mac1 antagonizes the host interferon response by counteracting the wave of ADP ribosylation that occurs during infection. Indeed, several PARPs are interferon-stimulated genes. While multiple targets have been proposed, the mechanistic links between ADP ribosylation and a robust antiviral response remain unclear.

Genetic inactivation of Mac1 abrogates viral replication in vivo, suggesting that small-molecule inhibitors of Mac1 could be developed into antivirals to treat COVID-19 and other emerging coronaviruses. The authors report a potent and selective small molecule inhibitor targeting Mac1 (AVI-4206) that demonstrates efficacy in human airway organoids and animal models of SARS-CoV-2 infection. While these results are compelling and provide proof of concept for the therapeutic targeting of Mac1, I am particularly intrigued by the potential of this compound as a probe to elucidate the mechanistic connections between infection-induced ADP ribosylation and the host antiviral response.

The precise function of Mac1 remains unclear. Given its presence in multiple viruses, it likely acts on a fundamental host immune pathway(s). AVI-4206, while promising as a lead compound for the development of antivirals targeting coronaviruses, could also be a valuable tool for uncovering the function of the Mac1 domain. This may lead to fundamental insights into the host immune response to viral infection.

Reviewer #1 (Public review):

Summary:

In this paper, the authors develop a biologically plausible recurrent neural network model to explain how the hippocampus generates and uses barcode-like activity to support episodic memory. They address key questions raised by recent experimental findings: how barcodes are generated, how they interact with memory content (such as place and seed-related activity), and how the hippocampus balances memory specificity with flexible recall. The authors demonstrate that chaotic dynamics in a recurrent neural network can produce barcodes that reduce memory interference, complement place tuning, and enable context-dependent memory retrieval, while aligning their model with observed hippocampal activity during caching and retrieval in chickadees.

Strengths:

(1) The manuscript is well-written and structured.<br /> (2) The paper provides a detailed and biologically plausible mechanism for generating and utilizing barcode activity through chaotic dynamics in a recurrent neural network. This mechanism effectively explains how barcodes reduce memory interference, complement place tuning, and enable flexible, context-dependent recall.<br /> (3) The authors successfully reproduce key experimental findings on hippocampal barcode activity from chickadee studies, including the distinct correlations observed during caching, retrieval, and visits.<br /> (4) Overall, the study addresses a somewhat puzzling question about how memory indices and content signals coexist and interact in the same hippocampal population. By proposing a unified model, it provides significant conceptual clarity.

Weaknesses:

The recurrent neural network model incorporates assumptions and mechanisms, such as the modulation of recurrent input strength, whose biological underpinnings remain unclear. The authors acknowledge some of these limitations thoughtfully, offering plausible mechanisms and discussing their implications in depth.

One thread of questions that authors may want to further explore is related to the chaotic nature of activity that generates barcodes when recurrence is strong. Chaos inherently implies sensitivity to initial conditions and noise, which raises questions about its reliability as a mechanism for producing robust and repeatable barcode signals. How sensitive are the results to noise in both the dynamics and the input signals? Does this sensitivity affect the stability of the generated barcodes and place fields, potentially disrupting their functional roles? Moreover, does the implemented plasticity mitigate some of this chaos, or might it amplify it under certain conditions? Clarifying these aspects could strengthen the argument for the robustness of the proposed mechanism.

It may also be worth exploring the robustness of the results to certain modeling assumptions. For instance, the choice to run the network for a fixed amount of time and then use the activity at the end for plasticity could be relaxed.

for - article - LinkedIn - Breaking up with Psychology - Jason Ross PhD - 2024 Jul 1

source - I was searching for existential phenomenological psychology this morning on both Google and LinkedIn and going Jason's article on LinkedIn

// - summary - This is an interesting article that overlaps a number of areas I have been attracted to. - As he wrote, I thought of how Zen teacher and scholar David Loy also focuses on - emptiness and - that sense of lack - at the core of each of us - He didn't cite Loy's work. I wonder if he knows of it? - It was nice to see how he connected - Husserl and the German school of phenomenology and existentialism with - the French school of Sarte, Lacan, Derrida, etc - It gives a big picture of house all these thinkers were connected and timebound - It's was also interesting to read about Leonard Cohen as well. - Question - I wonder if the author knows about the decades long sexual abuse story of Cohen's teacher, Joshua Sasaki?<br /> - Along with all the abuse going in the Catholic church, it's sure to sow a lot of distrust and doubt about professed gurus and religion in general

Reviewer #1 (Public review):

(1a) Summary:

The author studied metabolic networks for central metabolism, focusing on how system trajectories returned to their steady state. To quantify the response, systematic perturbation was performed in simulation and the maximal destabilization away from steady state (compared with initial perturbation distance) was characterized. The author analyzed the perturbation response and found that sparse network and networks with more cofactors are more "stable", in the sense that the perturbed trajectories have smaller deviation along the path back to the steady state.

(1b) Strengths and major contributions:

The author compared three metabolic models and performed systematic perturbation analysis in simulation. This is the first work characterized how perturbed trajectories deviate from equilibrium in large biochemical systems and illustrated interesting findings about the difference between sparse biological systems and randomly simulated reaction networks.

(1c) Discussion and impact for the field:

Metabolic perturbation is an important topic in cell biology and has important clinical implication in pharmacodynamics. The computational analysis in this study provides an initiative for future quantitative analysis on metabolism and homeostasis.

Comments on revised version:

The revised version of this manuscript made some clarifications, while I think the analysis of response coefficients is still numerical and model-specific, being unclear under dynamical systems of views.

Joint Public Review:

Automatically identifying single cell types in heterogeneous mixed cell populations hold great promise to characterize mixed cell populations and to discover new rules of spatial organization and cell-cell communication. Although the current manuscript focuses on the application of quality control of iPSC cultures, the same approach can be extended to a wealth of other applications including in depth study of the spatial context. The simple and high-content assay democratizes use and enables adoption by other labs.

The authors also propose a new nucleocentric phenotyping pipeline, where a convolutional neural network is trained on the nucleus and some margins around it. This nucleocentric approach improves classification performance at high densities because nuclear segmentation is less prone to errors in dense cultures.

The manuscript is supported by comprehensive experimental and computational validations that raises the bar beyond the current state of the art in the field of high-content phenotyping and makes this manuscript especially compelling. These include (i) Explicitly assessing replication biases (batch effects); (ii) Direct comparison of feature-based (a la cell profiling) versus deep-learning-based classification (which is not trivial/obvious for the application of cell profiling); (iii) Systematic assessment of the contribution of each fluorescent channel; (iv) Evaluation of cell-density dependency; (v) explicit examination of mistakes in classification; (vi) Evaluating the performance of different spatial contexts around the cell / nucleus; (vii) generalization of models trained on cultures containing a single cell type (mono-cultures) to mixed co-cultures; (viii) application to multiple classification tasks.

Reviewer #1 (Public review):

Summary:

The authors of this study set out to find RNA binding proteins in the CNS in cell-type specific sequencing data and discover that the cardiomyopathy-associated protein RBM20 is selectively expressed in olfactory bulb glutamatergic neurons and PV+ GABAergic neurons. They make an HA-tagged RBM20 allele to perform CLIP-seq to identify RBM20 binding sites and find direct targets of RBM20 in olfactory bulb glutmatergic neurons. In these neurons, RBM20 binds intronic regions. RBM20 has previously been implicated in splicing, but when they selectively knockout RBM20 in glutamatergic neurons they do not see changes in splicing, but they do see changes in RNA abundance, especially of long genes with many introns, which are enriched for synapse-associated functions. These data show that RBM20 has important functions in gene regulation in neurons, which was previously unknown, and they suggest it acts through a mechanism distinct from what has been studied before in cardiomyocytes.

Strengths:

The study finds expression of the cardiomyopathy-associated RNA binding protein RBM20 in specific neurons in the brain, opening new windows into its potential functions there.

The study uses CLIP-seq to identify RBM20 binding RNAs in olfactory bulb neurons.

Conditional knockout of RBM20 in glutamatergic or PV neurons allows the authors to detect mRNA expression that is regulated by RBM20.

The data include substantial controls and quality control information to support the rigor of the findings.

Weaknesses:

The authors do not fully identify the mechanism by which RBM20 acts to regulate RNA expression in neurons, though they do provide data suggesting that neuronal RBM20 does not regulate alternate splicing in neurons, which is an interesting contrast to its proposed mechanism of function in cardiomyocytes. Discovery of the RNA regulatory functions of RBM20 in neurons is left as a question for future studies.

The study does not identify functional consequences of the RNA changes in the conditional knockout cells, so this is also a question for the future.

Joint Public Reviews:

Summary:

The authors examine the eigenvalue spectrum of the covariance matrix of neural recordings in the whole-brain larval zebrafish during hunting and spontaneous behavior. They find that the spectrum is approximately power law, and, more importantly, exhibits scale-invariance under random subsampling of neurons. This property is not exhibited by conventional models of covariance spectra, motivating the introduction of the Euclidean random matrix model. The authors show that this tractable model captures the scale invariance they observe. They also examine the effects of subsampling based on anatomical location or functional relationships. Finally, they briefly discuss the benefit of neural codes which can be subsampled without significant loss of information.

Strengths:

With large-scale neural recordings becoming increasingly common, neuroscientists are faced with the question: how should we analyze them? To address that question, this paper proposes the Euclidean random matrix model, which embeds neurons randomly in an abstract feature space. This model is analytically tractable and matches two nontrivial features of the covariance matrix: approximate power law scaling, and invariance under subsampling. It thus introduces an important conceptual and technical advance for understanding large-scale simultaneously recorded neural activity.

Weaknesses:

The downside of using summary statistics is that they can be hard to interpret. Often the finding of scale invariance, and approximate power law behavior, points to something interesting. But here caution is in order: for instance, most critical phenomena in neural activity have been explained by relatively simple models that have very little to do with computation (Aitchison et al., PLoS CB 12:e1005110, 2016; Morrell et al., eLife 12, RP89337, 2014). Whether the same holds for the properties found here remains an open question.

Reviewer #1 (Public review):

This work regards the role of Aurora Kinase A (AurA) in trained immunity. The authors claim that AurA is essential to the induction of trained immunity. The paper starts with a series of experiments showing the effects of suppressing AurA on beta-glucan-trained immunity. This is followed by an account of how AurA inhibition changes the epigenetic and metabolic reprogramming that are characteristic of trained immunity. The authors then zoom in on specific metabolic and epigenetic processes (regulation of S-adenocylmethionine metabolism & histone methylation). Finally, an inhibitor of AurA is used to reduce beta-glucan's anti-tumour effects in a subcutaneous MC-38 model.

Strengths:

With the exception of my confusion around the methods used for relative gene expression measurements, the experimental methods are generally well-described. I appreciate the authors' broad approach to studying different key aspects of trained immunity (from comprehensive transcriptome/chromatin accessibility measurements to detailed mechanistic experiments). Approaching the hypothesis from many different angles inspires confidence in the results (although not completely - see weaknesses section). Furthermore, the large drug-screening panel is a valuable tool as these drugs are readily available for translational drug-repurposing research.

Weaknesses

(1) The manuscript contains factual inaccuracies such as:<br /> (a) Intro: the claim that trained cells display a shift from OXPHOS to glycolysis based on the paper by Cheng et al. in 2014; this was later shown to be dependent on the dose of stimulation and actually both glycolysis and OXPHOS are generally upregulated in trained cells (pmid 32320649)<br /> (b) Discussion: Trained immunity was first described as such in 2011, not decades ago.

(2) The authors approach their hypothesis from different angles, which inspires a degree of confidence in the results. However, the statistical methods and reporting are underwhelming.<br /> (a) Graphs depict mean +/- SEM, whereas mean +/- SD is almost always more informative.<br /> (b) The use of 1-tailed tests is dubious in this scenario. Furthermore, in many experiments/figures the case could be made that the comparisons should be considered paired (the responses of cells from the same animal are inherently not independent due to their shared genetic background and, up until cell isolation, the same host factors like serum composition/microbiome/systemic inflammation etc).<br /> (c) It could be explained a little more clearly how multiple testing correction was done and why specific tests were chosen in each instance.<br /> (d) Most experiments are done with n = 3, some experiments are done with n = 5. This is not a lot. While I don't think power analyses should be required for simple in vitro experiments, I would be wary of drawing conclusions based on n = 3. It is also not indicated if the data points were acquired in independent experiments. ATAC-seq/RNA-seq was, judging by the figures, done on only 2 mice per group. No power calculations were done for the in vivo tumor model.<br /> (e) Furthermore, the data spread in many experiments (particularly BMDM experiments) is extremely small. I wonder if these are true biological replicates, meaning each point represents BMDMs from a different animal? (disclaimer: I work with human materials where the spread is of course always much larger than in animal experiments, so I might be misjudging this.).

(3) Maybe the authors are reserving this for a separate paper, but it would be fantastic if the authors would report the outcomes of the entire drug screening instead of only a selected few. The field would benefit from this as it would save needless repeat experiments. The list of drugs contains several known inhibitors of training (e.g. mTOR inhibitors) so there must have been more 'hits' than the reported 8 Aurora inhibitors.

(4) Relating to the drug screen and subsequent experiments: it is unclear to me in supplementary figure 1B which concentrations belong to secondary screens #1/#2 - the methods mention 5 µM for the primary screen and "0.2 and 1 µM" for secondary screens, is it in this order or in order of descending concentration?<br /> (a) It is unclear if the drug screen was performed with technical replicates or not - the supplementary figure 1B suggests no replicates and quite a large spread (in some cases lower concentration works better?)

(5) The methods for (presumably) qPCR for measuring gene expression in Figure 1C are missing. Which reference gene was used and is this a suitably stable gene?

(6) From the complete unedited blot image of Figure 1D it appears that the p-Aurora and total Aurora are not from the same gel (discordant number of lanes and positioning). This could be alright if there are no/only slight technical errors, but I find it misleading as it is presented as if the actin (loading control to account for aforementioned technical errors!) counts for the entire figure.

(7) Figure 2: This figure highlights results that are by far not the strongest ones - I think the 'top hits' deserve some more glory. A small explanation on why the highlighted results were selected would have been fitting.

(8) Figure 3 incl supplement: the carbon tracing experiments show more glucose-carbon going into TCA cycle (suggesting upregulated oxidative metabolism), but no mito stress test was performed on the seahorse.

(9) Inconsistent use of an 'alisertib-alone' control in addition to 'medium', 'b-glucan', 'b-glucan + alisertib'. This control would be of great added value in many cases, in my opinion.

(10) Figure 4A: looking at the unedited blot images, the blot for H3K36me3 appears in its original orientation, whereas other images appear horizontally mirrored. Please note, I don't think there is any malicious intent but this is quite sloppy and the authors should explain why/how this happened (are they different gels and the loading sequence was reversed?)

(11) For many figures, for example prominently figure 5, the text describes 'beta-glucan training' whereas the figures actually depict acute stimulation with beta-glucan. While this is partially a semantic issue (technically, the stimulation is 'the training-phase' of the experiment), this could confuse the reader.

(12) Figure 6: Cytokines, especially IL-6 and IL-1β, can be excreted by tumour cells and have pro-tumoral functions. This is not likely in the context of the other results in this case, but since there is flow cytometry data from the tumour material it would have been nice to see also intracellular cytokine staining to pinpoint the source of these cytokines.

Reviewer #1 (Public Review):

HLA genes have long been known to harbor trans-species polymorphism (TSP). This manuscript aimed to use state-of-the-art analyses and updated genotyping data to rigorously test for the presence of TSP in HLA genes, quantify the timescales associated with HLA TSP, and relate HLA disease associations to evolutionary rates. To do this, the authors chose HLA alleles across great apes, old world monkeys, and new world monkeys on which to perform phylogenetic analyses, alongside non-parametric tests that compare patterns of synonymous diversity. Finally, HLA genetic associations with the disease were correlated with evolutionary rate.

Strengths:

The manuscript is well written and neatly organized, the figures are clear, and there are many supplementary analyses that will make this paper a great resource for MHC phylogenetics at allelic resolution.

Deployment of modern methodology such as BEAST2 can also test if the hypothesis of TSP is supported while accounting for uncertainties in tree topology and evolutionary rates, necessary additions to analyses of the MHC.

Weaknesses:

Because TSP has already been convincingly demonstrated to occur in the MHC, the primary benefit of the current study is to ensure these previous observations are still supported by the wealth of genetic data that is now available and modern phylogenetic approaches. However, the benefit of using the robust BEAST2 method comes with the weakness of not using all available data. Focusing on single gene trees with only a small subset of alleles may bias results, and inclusion/exclusion criteria should be better defined.

One major point that is somewhat overlooked is the presence of multiple copy numbers for the MHC genes through classic birth and death evolution. For example, MHC-B in new world monkeys is duplicated many times (up to 10; PMID: 23715823). This duplication is naturally accompanied by gene loss and pseudogene formation. All of these things muddy the waters considerably yet are not addressed here. A good example is MHC-A, where it has been very difficult to apportion orthologs, even amongst closely related species, due to alternative or incomplete duplication/loss across the species, or region configuration polymorphism (e.g. PMID: 26371256). An example is chimpanzee Patr-AL which shares similarities with human HLA-A*02 lineage, but is a separate locus, could this show up as TSP under the current analysis?

Similarly, an alternative hypothesis for TSP is convergent gene conversion mutations: intergenic gene conversion has been repeatedly observed in HLA genes and the possibility of it occurring with the same two genes becomes more realistic over 45 million years. If the same two MHC genes recombined in humans and in an NWM, each on their own lineages, this would appear as TSP and would cause an overlap of pairwise synonymous divergence between human-human and human-NWM allele comparisons. This might be especially possible in MHC-DR and MHC-DQ genes presented in Figure 2 since both humans and NWM have multiple MHC-DRB and DQB genes (unless e.g. were genes besides HLA/MHC-DRB1 such as DRB3,4,5 included in the DRB phylogenies?). While BEAST2 may be a good way of robustly modeling and identifying TSP, and I understand these analyses cannot support many more sequences, the authors should consider adding an analysis that rules out gene conversion as an explanation for their results (especially the often repeated claim of 45 million year TSP). For example, can the authors use BLAST to ensure that the alleles that underlie 45 million years of TSP do not share close similarities to other HLA genes present in their respective human and NWM genomes? This seems like it could be fairly quickly performed for all genes, and even if it argued against TSP, it would be an interesting finding.

Finally, the authors have limited themselves to a small subset of HLA/MHC alleles and do not provide sufficient information in the methods to understand how these were chosen nor sufficient discussion surrounding how inclusion/exclusion criteria could bias results. For example, the authors say the alleles were chosen at 2-digit (i.e. 1 field) resolution, but in the phylogenies of Fig. 2, I see variable numbers of alleles chosen for each 2-digit allele family - what metric was used to decide on these alleles?

"We also collected associations between amino acids and TCR phenotypes". It is not clear either what was analyzed, or the results for this part of the analysis. This is a topic of much debate and none of the previous work has been discussed (PMID: 18304006, PMID: 29636542 as primers for this contentious subject)

MHC class I also interact with NK cell receptors, including polymorphic KIR. Through their interactions during infection control and reproduction, the two complexes co-evolve across primates, contributing to the maintenance of MHC diversity. Interaction with KIR likely has a greater impact on HLA polymorphism than interactions with TCR, yet this is not factored into any of the models, or indeed mentioned in the text.

One additional reason inclusion of the KIR binding is important relates to the point above about gene conversion, where it is established that gene conversion reproducibly swaps KIR-binding motifs among MHC class I alleles and genes. HLA-A*23, *24, and *25, *32, for example, are characterized by the acquisition of the 'Bw4' motif from HLA-B (PMID: 26284483), likely followed by positive natural selection. For exon 2 (which encodes the motif), these alleles turn up in a clade distinct from other human HLA-A (Fig 2-S1). What is the impact of the Bw4 motif on this phylogeny? Could this shuffling of motifs be interpreted as indirect TSP?

The analysis that shows the most rapidly evolving sites occur in the peptide binding domain brings little new to the field. This has been established by the Hughes and Nei (cited) and Parham, Lawlor, etc of 1988 (e.g. PMID: 3375250), and replicated multiple times across human populations and many other species.<br /> Likewise, the disease association part. It is nice to have a summary of the known associations, but there are others out there and this one is far from thorough. Here, 50% of the information about infectious diseases appears to be taken from one reference, leaving out some major bodies of work; for example identifying specific peptide binding residues or peptides that associate with HIV (PMID: 22896606) or malaria control (PMID: 1280333). It is also missing some major concepts -such as the DRB1 'shared epitope' of peptide binding residues that predispose to Rheumatoid Arthritis and protects from Parkinson's disease (35 years of work from PMID: 2446635 through PMID: 30910980). The nasopharyngeal carcinoma and EBV story (e.g. PMID: 23209447). Another huge gap here is the pregnancy syndromes -associations of specific HLA C and NK cell receptor allotypes with preeclampsia for example. There are thousands of HLA associations not considered in this section, and to do them justice would likely require an enormous amount of work.<br /> Thus - neither the idea that HLA/MHC polymorphism is focused on peptide binding nor that this binding drives resistance to infection and associations with the disease are new concepts. The previous work in these areas is inadequately acknowledged.

The paper is written in a very approachable language, which is nice to read and friendly to non-experts, but perhaps a little too much so in places. I find that the paper follows a very non-traditional format with respect to for example the results section, which seems a mixture of Introduction/methods/figure legends/discussion with no real solid result description.

Reviewer #1 (Public review):

Summary:

The manuscript discusses the role of phosphorylated ubiquitin (pUb) by PINK1 kinase in neurodegenerative diseases. It reveals that elevated levels of pUb are observed in aged human brains and those affected by Parkinson's disease (PD), as well as in Alzheimer's disease (AD), aging, and ischemic injury. The study shows that increased pUb impairs proteasomal degradation, leading to protein aggregation and neurodegeneration. The authors also demonstrate that PINK1 knockout can mitigate protein aggregation in aging and ischemic mouse brains, as well as in cells treated with a proteasome inhibitor. While this study provided some interesting data, several important points should be addressed before being further considered.

Strengths:

(1) Reveals a novel pathological mechanism of neurodegeneration mediated by pUb, providing a new perspective on understanding neurodegenerative diseases.<br /> (2) The study covers not only a single disease model but also various neurodegenerative diseases such as Alzheimer's disease, aging, and ischemic injury, enhancing the breadth and applicability of the research findings.

Weaknesses:

(1) PINK1 has been reported as a kinase capable of phosphorylating Ubiquitin, hence the expected outcome of increased p-Ub levels upon PINK1 overexpression. Figures 5E-F do not demonstrate a significant increase in Ub levels upon overexpression of PINK1 alone, whereas the evident increase in Ub expression upon overexpression of S65A is apparent. Therefore, the notion that increased Ub phosphorylation leads to protein aggregation in mouse hippocampal neurons is not yet convincingly supported.<br /> (2) The specificity of PINK1 and p-Ub antibodies requires further validation, as a series of literature indicate that the expression of the PINK1 protein is relatively low and difficult to detect under physiological conditions.<br /> (3) In Figure 6, relying solely on Western blot staining and golgi staining under high magnification is insufficient to prove the impact of PINK1 overexpression on neuronal integrity and cognitive function. The authors should supplement their findings with immunostaining results for MAP2 or NeuN to demonstrate whether neuronal cells are affected.<br /> (4) The authors should provide more detailed figure captions to facilitate the understanding of the results depicted in the figures.<br /> (5) While the study proposes that pUb promotes neurodegeneration by affecting proteasomal function, the specific molecular mechanisms and signaling pathways remain to be elucidated.

Reviewer #1 (Public review):

Previous experimental studies demonstrated that membrane association drives avidity for several potent broadly HIV-neutralizing antibodies and its loss dramatically reduces neutralization. In this study, the authors present a tour de force analysis of molecular dynamics (MD) simulations that demonstrate how several HIV-neutralizing membrane-proximal external region (MPER)-targeting antibodies associate with a model lipid bilayer.

First, the authors compared how three MPER antibodies, 4E10, PGZL1, and 10E8, associated with model membranes, constructed with two lipid compositions similar to native viral membranes. They found that the related antibodies 4E10 and PGZL1 strongly associate with a phospholipid near heavy chain loop 1, consistent with prior crystallographic studies. They also discovered that a previously unappreciated framework region between loops 2-3 in the 4E10/PGZL1 heavy chain contributes to membrane association. Simulations of 10E8, an antibody from a different lineage, revealed several differences from published X-ray structures. Namely, a phosphatidylcholine binding site was offset and includes significant interaction with a nearby framework region. The revised manuscript demonstrates that these lipid interactions are robust to alterations in membrane composition and rigidity. However, it does not address the reverse-that phospholipids known experimentally not to associate with these antibodies (if any such lipids exist) also fail to interact in MD simulations.

Next, the authors simulate another MPER-targeting antibody, LN01, with a model HIV membrane either containing or missing an MPER antigen fragment within. Of note, LN01 inserts more deeply into the membrane when the MPER antigen is present, supporting an energy balance between the lowest energy conformations of LN01, MPER, and the complex. These simulations recapitulate lipid binding interactions solved in published crystallographic studies but also lead to the discovery of a novel lipid binding site the authors term the "Loading Site", which could guide future experiments with this antibody.

The authors next established course-grained (CG) MD simulations of the various antibodies with model membranes to study membrane embedding. These simulations facilitated greater sampling of different initial antibody geometries relative to membrane. These CG simulations , which cannot resolve atomistic interactions, are nonetheless compelling because negative controls (ab 13h11, BSA) that should not associate with membrane indeed sample significantly less membrane.

Distinct geometries derived from CG simulations were then used to initialize all-atom MD simulations to study insertion in finer detail (e.g., phospholipid association), which largely recapitulate their earlier results, albeit with more unbiased sampling. The multiscale model of an initial CG study with broad geometric sampling, followed by all-atom MD, provides a generalized framework for such simulations.

Finally, the authors construct velocity pulling simulations to estimate the energetics of antibody membrane embedding. Using the multiscale modelling workflow to achieve greater geometric sampling, they demonstrate that their model reliably predicts lower association energetics for known mutations in 4E10 that disrupt lipid binding. However, the model does have limitations: namely, its ability to predict more subtle changes along a lineage-intermediate mutations that reduce lipid binding are indistinguishable from mutations that completely ablate lipid association. Thus, while large/binary differences in lipid affinity might be predictable, the use of this method as a generative model are likely more limited.

The MD simulations conducted throughout are rigorous and the analysis are extensive, creative, and biologically inspired. Overall, these analyses provide an important mechanistic characterization of how broadly neutralizing antibodies associate with lipids proximal to membrane-associated epitopes to drive neutralization.

Reviewer #1 (Public review):

In this work, the authors examine the mechanism of action of MOTS-c and its impact on monocyte-derived macrophages. In the first part of the study, they show that MOTS-c acts as a host defense peptide with direct antibacterial activity. In the second part of the study, the authors aim to demonstrate that MOTS-c influences monocyte differentiation into macrophages via transcriptional regulation.

Major strengths. Methods used to study the bactericidal activity of MOTS-c are appropriate and the results convincing.

Major weaknesses. Methods used to study the impact on monocyte differentiation are inappropriate and the conclusions not fully supported by the data shown. A major issue is the use of the THP-1 cell line, a transformed monocytic line which does not mimic physiological monocyte biology. In particular, THP-1 differentiation is induced by PMA, which is a completely artificial system and conclusions from this approach cannot be generalized to monocyte differentiation. The authors would need to perform this series of experiments using freshly isolated monocytes, either from mouse or human. The read-out used for macrophage differentiation (adherence to plastic) is also not very robust, and the authors would need to analyze other parameters such as cell surface markers. It is also not clear whether MOTS-c could act in a cell-intrinsic fashion, as the authors have exposed cells to exogenous MOTS-c in all their experiments. The authors have also analyzed the transcriptomic changes induced by MOTS-c exposure in macrophages derived from young or old mice. While the results are potentially interesting, the differences observed seem independent from MOTS-c and mainly related to age, therefore the conclusions from this figure are not clear. The physiological relevance of this study is also unclear.

Reviewer #1 (Public review):

Summary:

This paper examines patterns of diversity and divergence in two closely related sub-species of Zea mays. While the data are interesting and the authors have tried to exclude multiple confounding factors, many patterns cannot clearly be ascribed to one cause or another.

Strengths:

The paper presents interesting data from sets of sympatric populations of the two sub-species, maize and teosinte. This sampling offers unique insights into the diversity and divergence between the two, as well as the geographic structure of each. Many analyses and simulations to check analyses have been carried out.

Weaknesses:

The strength of conclusions that can be drawn from the analyses was low, partly because there are many strange patterns. The authors have done a good job of adding caveats, but clearly, these species do not meet many assumptions of our methods

Reviewer #1 (Public review):

Summary:

In this manuscript, Shao et al. investigate the contribution of different cortical areas to working memory maintenance and control processes, an important topic involving different ideas about how the human brain represents and uses information when no longer available to sensory systems. In two fMRI experiments, they demonstrate that human frontal cortex (area sPCS) represents stimulus (orientation) information both during typical maintenance, but even more so when a categorical response demand is present. That is, when participants have to apply an added level of decision control to the WM stimulus, sPCS areas encode stimulus information more than conditions without this added demand. These effects are then expanded upon using multi-area neural network models, recapitulating the empirical gradient of memory vs control effects from visual to parietal and frontal cortices. Multiple experiments and analysis frameworks provide support for the authors' conclusions, and control experiments and analysis are provided to help interpret and isolate the frontal cortex effect of interest. While some alternative explanations/theories may explain the roles of frontal cortex in this study and experiments, important additional analyses have been added that help ensure a strong level of support for these results and interpretations.

Strengths:

- The authors use an interesting and clever task design across two fMRI experiments that is able to parse out contributions of WM maintenance alone along with categorical, rule-based decisions. Importantly, the second experiment only uses one fixed rule, providing both an internal replication of Experiment 1's effects and extending them to a different situation when rule switching effects are not involved across mini-blocks.

- The reported analyses using both inverted encoding models (IEM) and decoders (SVM) demonstrate the stimulus reconstruction effects across different methods, which may be sensitive to different aspects of the relationship between patterns of brain activity and the experimental stimuli.

- Linking the multivariate activity patterns to memory behavior is critical in thinking about the potential differential roles of cortical areas in sub-serving successful working memory. Figure 3's nicely shows a similar interaction to that of Figure 2 in the role of sPCS in the categorization vs. maintenance tasks. This is an important contribution to the field when we consider how a distributed set of interacting cortical areas supports successful working memory behavior.

- The cross-decoding analysis in Figure 4 is a clever and interesting way to parse out how stimulus and rule/category information may be intertwined, which would have been one of the foremost potential questions or analyses requested by careful readers.

- Additional ROI analyses in more anterior regions of the PFC help to contextualize the main effects of interest in the sPCS (and no effect in the inferior frontal areas, which are also retinotopic, adds specificity). And, more explanation for how motor areas or preparation are likely not involved strengthens the takeaways of the study (M1 control analysis).

Weaknesses:

- An explicit, quantitative link between the RNN and fMRI data is perhaps a last point that would integrate the RNN conclusion and analyses in line with the human imaging data.

- As Rev 2 mentions, multiple types of information codes may be present, and the response letter Figure 5 using representational similarity (RSA) gets at this question. It would strengthen the work to, at minimum, include this analysis as an extended or supplemental figure.

To sum up the results, a possible, brief schematic of each cortical area analyzed and its contribution to information coding in WM and successful subsequent behavior may help readers take away important conclusions of the cortical circuitry involved.

Reviewer #1 (Public review):

Summary:

This paper proposes a new model of perceptual habituation and tests it over two experiments with both infants and adults. The model combines a neural network for visual processing with a Bayesian rational model for attention (i.e., looking time) allocation. This Bayesian framework allows the authors to measure elegantly diverse factors that might drive attention, such as expected information gain, current information gain, and surprise. The model is then fitted to infant and adult participants' data over two experiments, which systematically vary the amount of habituation trials (Experiment 1) and the type of dishabituation stimulus (familiarity, pose, number, identity, and animacy). Results show that a model based on (expected) information gain performs better than a model based on surprise. Additionally, while novelty preference is observed when exposure to familiar stimuli is elevated, no familiarity preference is observed when exposure to familiar stimuli is low or intermediate, which is in contrast with past work.

Strengths:

There are three key strengths of this work:

(1) It integrates a neural network model with a Bayesian rational learner, thus bridging the gap between two fields that have often been disconnected. This is rarely seen in the cognitive science field, but the advantages are very clear from this paper: It is possible to have computational models that not only process visual information, but also actively explore the environment based on overarching attentional processes.

(2) By varying parametrically the amount of stimulus exposure and by testing the effects of multiple novel stimulus types, this work allowed the authors to put classical theories of habituation to the test on much finer scales than previous research has done.

(3) The Bayesian model allows the authors to test what specific aspects are different in infants and adults, showing that infants display greater values for the noise parameter.

Weaknesses:

Although a familiarity preference is not found, it is possible that this is related to the nature of the stimuli and the amount of learning that they offer. While infants here are exposed to the same perceptual stimulus repeatedly, infants can also be familiarised to more complex stimuli or scenarios. Classical statistical learning studies for example expose infants to specific pseudo-words during habituation/familiarisation, and then test their preference for familiar vs novel streams of pseudo-words. The amount of learning progress in these probabilistic learning studies is greater than in perceptual studies, and familiarity preferences may thus be more likely to emerge there. For these reasons, I think it is important to frame this as a model of perceptual habituation. This would also fit well with the neural net that was used, which is processing visual stimuli rather than probabilistic structures. If statements in the discussion are limited to perceptual paradigms, they would make the arguments more compelling.

Reviewer #1 (Public review):

In the manuscript "Identification of neurodevelopmental organization of the cell populations of Juvenile Huntington's disease using dorso-ventral HD organoids and HD mouse embryos," the authors establish a fused dorso-ventral system that mimics cortex-striatum interactions within a single organoid and use this system to investigate neurodevelopmental impairments caused by HD. Specifically, they describe certain phenotypes in 60-day HD organoids and the brains of humanized mouse embryos, utilizing both wet-lab and single-cell sequencing techniques. The authors also develop dorsal/ventral and ventral/dorsal mosaic control/HD organoids, showing a capacity to rescue some HD phenotypes.

The manuscript could be a valuable contribution to the field, however it has relevant drawbacks, the most significant being a lack of clarity regarding the replicates used for each genotype in the sequencing analyses. The lack of information on replicates raises the possibility that only a single replicate was analyzed for each organoid and brain sample. This approach may lead to concerns regarding the reproducibility of the findings, and it may be necessary for the authors to generate additional data to strengthen their conclusions. In addition, the analysis of the HD samples was conducted by pooling distinct cell populations from different brain regions (CTX, HIP, ChP for the dorsal brain, and STR, HYP, TH for the ventral brain). It is unclear why scRNA seq was used on pooled brain regions, which could obscure region-specific insights.

Another issue pertains to their proposed outcome: "Finally, we found that TTR protein, a choroid plexus marker, is elevated in the adult HD mouse serum, indicating that TTR may be a promising marker for detecting HD". This statement appears to lack statistical support, which makes this set of data potentially misleading and inconclusive.

The authors are encouraged to provide evidence of biological replicates, remove outcomes that lack statistical support, and address a series of points as detailed elsewhere.

Reviewer #1 (Public review):

Shin et al. conduct extensive electrophysiological and behavioral experiments to study the mechanisms of short-term synaptic plasticity at excitatory synapses in layer 2/3 of the rat medial prefrontal cortex. The authors interestingly find that short-term facilitation is driven by progressive overfilling of the readily releasable pool, and that this process is mediated by phospholipase C/diacylglycerol signaling and synaptotagmin-7 (Syt7). Specifically, knockdown of Syt7 not only abolishes the refilling rate of vesicles with high fusion probability, but it also impairs the acquisition of trace fear memory.

Overall, the authors offer novel insight to the field of synaptic plasticity through well-designed experiments that incorporate a range of techniques.

Reviewer #1 (Public review):

Summary:

In this study, the authors explore a novel mechanism linking aging to chromosome mis-segregation and aneuploidy in yeast cells. They reveal that, in old yeast mother cells, chromosome loss occurs through asymmetric partitioning of chromosomes to daughter cells, a process coupled with the inheritance of an old Spindle Pole Body. Remarkably, the authors identify that remodeling of the nuclear pore complex (NPC), specifically the displacement of its nuclear basket, triggers these asymmetric segregation events. This disruption also leads to the leakage of unspliced pre-mRNAs into the cytoplasm, highlighting a breakdown in RNA quality control. Through genetic manipulation, the study demonstrates that removing introns from key chromosome segregation genes is sufficient to prevent chromosome loss in aged cells. Moreover, promoting pre-mRNA leakage in young cells mimics the chromosome mis-segregation observed in old cells, providing further evidence for the critical role of nuclear envelope integrity and RNA processing in aging-related genome instability.

Strengths:

The findings presented are not only intriguing but also well-supported by robust experimental data, highlighting a previously unrecognized connection between nuclear envelope integrity, RNA processing, and genome stability in aging cells, deepening our understanding of the molecular basis of chromosome loss in aging.

Weaknesses:

Further analysis of yeast aging data from microfluidic experiments will provide important information about the dynamic features and prevalence of the key aging phenotypes, e.g. pre-mRNA leakage and chromosome loss, reported in this work. In addition, a discussion would be needed to clarify the relationship between "chromosome loss" in this study and "genomic missegregation" reported previously in yeast aging.

Reviewer #1 (Public review):

Summary:

The manuscript uses state-of-the-art analysis technology to document the spatio-temporal dynamics of brain activity during the processing of threats. The authors offer convincing evidence that complex spatio-temporal aspects of brain dynamics are essential to describe brain operations during threat processing.

Strengths:

Rigorous complex analyses well suited to the data.

Weaknesses:

Lack of a simple take-home message about discovery of a new brain operation.

Reviewer #1 (Public review):

Summary:

The topic of nanobody-based PET imaging is important and holds great potential for real-world applications since nanobodies have many advantages over full sized immunoglobulins and small molecules.

Strengths:

The submitted manuscript contains quite a bit of interesting data from a collaborative team of well-respected researchers. The authors are to be congratulated for presenting results that may not have turned out the way they had hoped, and doing so in a transparent fashion.

Weaknesses:

However, the manuscript could be considered to be a collection of exploratory findings rather than a complete and mature scientific exposition. Most of the sample sizes were 3 per group, which is fine for exploratory work, but insufficient to draw strong statistically robust conclusions for definitive results.

Reviewer #1 (Public review):

Summary:

The paper is well-organized, with clearly defined sections. The systematic review methodology is thorough, with clear eligibility criteria, search strategy, and data collection methods. The risk of bias assessment is also detailed and useful for evaluating the strength of evidence. The involvement of a patient panel is noticeable and positive, ensuring the research addresses real-world concerns and aligning scientific inquiry with patient perspectives. The statistical approach used for analyzing seems appropriate.

The authors are encouraged to take into account the following points:

As the authors have acknowledged, there is a high risk of bias across all included studies, particularly in randomization, selective outcome reporting, and incomplete data, which could be highlighted more explicitly in the paper's discussion section, particularly the potential implications for the generalizability of the results. The authors can also suggest mitigation strategies for future studies (e.g., better randomization, blinding, reporting standards, etc.). None of the studies include female animals, and the use of young adult animals (instead of aged models) limits the applicability of the findings to the human stroke population, where stroke incidence is higher in older adults and perhaps the gender issue must be included to reflect the translational aspects. The authors can add to the paper's discussion section that perhaps future preclinical studies should include both sexes and aged animals to align better with the clinical population and improve the translation of findings. Another point is the comorbidity. Comorbidities such as diabetes and hypertension are prevalent in stroke patients. How can these be considered in preclinical designs? The authors should emphasize the importance of future research incorporating such comorbid models to enhance clinical relevance.

None of the studies had independent replication of their findings, which is a key limitation, especially for a field with high translational expectations. This should be highlighted as a critical next step for validating the efficacy of CCR5 antagonists.

The studies accessed limited cognitive outcomes (only one reported a cognitive outcome). Given the importance of cognitive recovery post-stroke, this is a gap to highlight in the discussion. Future studies should include more diverse and comprehensive behavioral assessments, including cognitive and emotional domains, to fully evaluate the therapeutic potential.

The timing of CCR5 administration across studies varies widely (from pre-stroke to several days post-stroke) complicating the interpretation and comparison of results. The authors are encouraged to add that future preclinical studies could focus on narrowing the therapeutic window to more clinically relevant time points.<br /> The paper identifies some alignment with clinical trials, but there are several gaps, too, particularly in the types of behavioral tests used in preclinical studies versus those in clinical trials. If this systematic review and meta-analysis aim to formulate a set of recommendations for future studies, it is important that the authors also propose specific preclinical behavioral tasks that could better align with clinical measures used in trials, like functional assessments related to human stroke outcomes.

The discussion needs some revisions. It could benefit from an expanded explanation of CCR5's mechanistic role in neuroplasticity and stroke recovery. For instance, linking CCR5 antagonism more closely with molecular pathways related to synaptic repair and remyelination would enhance the quality of the discussion and understanding of the drugs' potential.

While the tool is used to assess the risk of bias, it might be helpful to integrate a broader framework for evaluating the quality of included studies. This could include sample size justifications, statistical power analysis, or the use of pre-registration in animal studies. These elements can also introduce bias or minimize those if in place.

Please also highlight confounding factors that might have influenced the results in the included studies, such as variation in stroke models, dosing regimens, or behavioral assessment methods.

There is some discussion of the meta-analysis' limitations due to the few studies, but this point could be more thoroughly addressed. Please consider including a more critical discussion of the limitations of pooling data from heterogeneous study designs, stroke models, and outcome measures. What can this lead to? Is it reliable to do so, or does it lack scientific rigor? The authors are encouraged to formulate a balanced discussion adding, positive and negative aspects.<br /> The conclusion should more explicitly acknowledge that while CCR5 antagonists show potential, the findings are still preliminary due to the limitations in the preclinical studies (high bias risk, lack of diverse animal models). Overall, the conclusion can end with a call for rigorous, well-controlled, and replicated studies with improved alignment to clinical populations and trials to show that the conclusion remains inconclusive, considering what has been analyzed here.

Reviewer #1 (Public review):

Summary:

This work made a lot of efforts to explore the multifaceted roles of the inferior colliculus (IC) in auditory processing, extending beyond traditional sensory encoding. The authors recorded neuronal activity from the IC at single unit level when monkeys were passively exposed or actively engaged in behavioral task. They concluded that 1）IC neurons showed sustained firing patterns related to sound duration, indicating their roles in temporal perception, 2) IC neuronal firing rates increased as sound sequences progress, reflecting modulation by behavioral context rather than reward anticipation, 3) IC neurons encode reward prediction error and their capability of adjusting responses based on reward predictability, 4) IC neural activity correlates with decision-making. In summary, this study tried to provide a new perspective on IC functions by exploring its roles in sensory prediction and reward processing, what are not traditionally associated with this structure.

Strengths:

The major strength of this work is that the authors performed electrophysiological recordings from the IC of behaving monkeys. Compared with the auditory cortex and thalamus, the IC in monkeys has not been adequately explored.

Comments on revised version:

The authors have adequately addressed all my concerns.

Reviewer #1 (Public review):

In this manuscript, the authors recorded cerebellar unipolar brush cells (UBCs) in acute brain slices. They confirmed that mossy fiber (MF) inputs generate a continuum of UBC responses. Using systematic and physiological trains of MF electrical stimulation, they demonstrated that MF inputs either increased or decreased UBC firing rates (UBC ON vs. OFF) or induced complex, long-lasting modulation of their discharges. The MF influence on UBC firing was directly associated with a specific combination of metabotropic glutamate receptors, mGluR2/3 (inhibitory) and mGluR1 (excitatory). Ultimately, the amount and ratio of these two receptors controlled the time course of the effect, yielding specific temporal transformations such as phase shifts. The experiments are well-executed and properly analyzed.

Strengths:

(1) A wide range of MF stimulation based on activity patterns observed in vivo was explored, including burst duration and frequency dependency, which could serve as a valuable foundation for explicit modeling of temporal transformations in the granule cell layer.<br /> (2) The pharmacological blockade of mGluR2/3, mGluR1, AMPA, and NMDA receptors helped identify the specific roles of these glutamate receptors.<br /> (3) The experiments convincingly demonstrate the key role of mGluR1 receptors in temporal information processing by UBCs.

Weaknesses:

(1) This study is a follow up of previous work (Guo et al., Nat. Commun., 2021).<br /> (2) The MF activity used to mimic natural stimulation was previously collected from primates, whereas the recordings were conducted in mice.

Comments on revisions:

The authors included a discussion about inhibition, but I still disagree with their claim that it was not possible to study the MF-UBC connection with inhibition unblocked. This group has already conducted experiments on Golgi cell inhibition in slices.

Reviewer #1 (Public review):

Summary:

In the submitted manuscript, Solomon et al carefully detail shifts in tissue-specific myeloid populations associated with trained immunity using intraperitoneal BCG injection as a model for induction. They define the kinetics of shifts in myeloid populations within the spleen and the transcriptional response associated with IP BCG exposure. In lineage tracing experiments, they demonstrate that tissue-resident macrophages, red-pulp macrophages (RPM) that are rapidly depleted after BCG exposure, are replenished from recruited monocytes and expansion of tissue-resident cells; they use transcriptional profiling to characterize those cells. In contrast to previous descriptions of BCG-driven immune training, they do not find BCG in the bone marrow in their model, suggesting that there is not direct training of myeloid precursor populations in the bone marrow. They then link the observed trained immunity phenotype (restriction of heterologous infection with ST) with early activation of STAT1 through IFN-γ.

Strengths:

The work includes careful detaining of shifts and origins of myeloid populations within tissue associated with trained immunity and is a meaningful advance for the field.

Caveats:<br /> Given that the authors demonstrate that BCG persists in the spleen, it is possible that some level of BCG persistence in the spleen is a necessary contributor (together with signaling through STAT1) to the observed tissue-specific T1 phenotype.

Whether ongoing signaling through the axes are required for ongoing protection is not specifically addressed in this work. There is recent work by other groups that partially addresses these caveats, and it would be helpful context to reference those papers.

Reviewer #1 (Public review):

This manuscript by Kleinman & Foster investigates the dependence of hippocampal replay on VTA activity. They recorded neural activity from the dorsal CA1 region of the hippocampus while chemogenetically silencing VTA dopamine neurons as rats completed laps on a linear track with reward delivery at each end. Reward amount changed across task epochs within a session on one end of the track. The authors report that VTA activity is necessary for an increase in sharp-wave rate to remain localized to the feeder that undergoes a change in reward magnitude, an effect that was especially pronounced in a novel environment. They follow up on this result with a second experiment in which reward magnitude varies unpredictably at one end of the linear track and report that changes in sharp-wave rate at the variable location reflect both the amount of reward rats just received there, in addition to a smaller modulation that is reminiscent of reward prediction error coding, in which the previous reward rats received at the variable location affects the magnitude of the subsequent change in sharp-wave rate that occurs on the present visit.

This work is technically innovative, combining neural recordings with chemogenetic inactivation. The question of how VTA activity affects replay in the hippocampus is interesting and important given that much of the work implicating hippocampal replay in memory consolidation and planning comes from reward-motivated behavioral tasks.

Comments on revisions:

Overall, I think the authors have done everything they could to address reviewer concerns, short of collecting more data. The more consistent statistical approach makes the paper easier to read and follow. It's helpful to have more details/rationale for the variability in CNO dose and timing. I think some of the results are still not fully convincing, especially the reward volatility experiment (which the authors also note requires additional validation). Given the small number of rats, the small effect sizes, and the complexity of the experimental manipulations, I still have concerns about whether these effects would hold with larger groups sizes.

Reviewer #1 (Public review):

The paper proposes an interesting perspective on the spatio-temporal relationship between FC in fMRI and electrophysiology. The study found that while similar networks configurations are found in both modalities, there is a tendency for the networks to spatially converge more commonly at synchronous than asynchronous timepoints. However, my confidence in the findings and their interpretation is undermined by an incomplete justification for the expected outcomes for each of the proposed scenarios.

Main Concern

Fig 1 makes sense to me conceptually, including the schematics of the trajectories, i.e.:

- Scenario1. Temporally convergent, same trajectories through connectome state space<br /> - Scenario2. Temporally divergent, different trajectories through connectome state space

However, based on my understanding (and apologies if I am mistaken), I am concerned that these scenarios do not necessarily translate into the schematic CRP plots shown in fig 2C, or the statements in the main text, i.e.:

- For scenario1, "epochs of cross-modal spatial similarity should occur more frequently at on-diagonal (synchronous) than off-diagonal (asynchronous) entries, resulting in an on-/off-diagonal ratio larger than unity"<br /> - For scenario2, "epochs of spatial similarity could occur equally likely at on-diagonal and off-diagonal entries (ratio≈1)"

Where do the authors get these statements and the schematics in fig2C from? They do not seem to be fully justified via previous literature, theory, or simulations?

In particular, I am not convinced based on the evidence currently in the paper, that the ratio of off- to on-diagonal entries (and under what assumptions) is a definitive way to discriminate between scenarios 1 and 2.

For example, what about the case where the same network configuration reoccurs in both modalities at multiple time points. It seems to me that you would get a CRP with entries occurring equally on the on-diagonal as on the off-diagonal, regardless of whether the dynamics are matched between the two modalities or not (i.e. regardless of scenario 1 or 2 being true).

This thought experiment example might have a flaw in it, and the authors might ultimately be correct, but nonetheless a systematic justification needs to be provided for using the ratio of off- to on-diagonal entries to discriminate between scenario 1 and 2 (and under what assumptions it is valid).

In the absence of theory, the authors could use surrogate data for scenario 1 and 2. For example:

a. For scenario 1, run the CRP using a single modality. E.g. feed in the EEG into the analysis as both modality 1 AND modality 2. This should provide at least one example of CRP under scenario 1 (although it does not ensure that all CRPs under this scenario will look like this, it is at least a useful sanity check).<br /> b. For scenario 2, run the CRP using a single modality plus a shuffled version. E.g. feed in the EEG into the analysis as both modality 1 AND a temporally shuffled version of the EEG as modality 2. The temporal shuffling of the EEG could be done by simple splitting the data into blocks of say ~10s and then shuffling them into a new order. This should provide a version of the CRP under scenario 2 (although it does not ensure that all CRPs under this scenario will look like this, it is at least a useful sanity check)

The authors have provided CRP plots for option a. It shows a CRP, as expected, consistent with scenario 1. This is a useful sanity check. However, as mentioned above, it does not ensure that all CRPs under this scenario will look like this.

However, the authors have not shown a CRP as per option b. As such, there is an incomplete justification for the expected outcomes of the scenarios.

Note that another option, which has not been carried out, is to use full simulations, with clearly specified assumptions, for scenario1 and 2. One way of doing this is to use a simplified (state-space) setup where you randomly simulate N spatially fixed networks that are independently switching on and off over time (i.e. "activation" is 0 or 1). Note that this would result in a N-dimensional connectome state space.

Using this, you can simulate and compute the CRPs for the two scenarios:

a. Scenario 1: where the simulated activation timecourses are set to be the same between both modalities<br /> b. Scenario 2: where the simulated activation timecourses are simulated separately for each of the modalities

Minor Concern

Leakage correction. The paper states: "To mitigate this issue, we provide results from source-localized data both with and without leakage correction (supplementary and main text, respectively)." It is great that the authors provide both. However, given that FC in EEG is almost totally dominated by spatial leakage (see Hipp paper), the main results/figures for the scalp EEG should be done using spatial leakage corrected EEG data.

Reviewer #1 (Public review):

Summary:

The anatomical connectivity of the claustrum and the role of its output projections has, thus far, not been studied in detail. The aim of this study was to map the outputs of the endopiriform (EN) region of the claustrum complex, and understand their functional role. Here the authors have combined sophisticated intersectional viral tracing techniques, and ex vivo electrophysiology to map the neural circuitry of EN outputs to vCA1, and shown that optogenetic inhibition of the EN→vCA1 projection impairs both social and object recognition memory. Interestingly the authors find that the EN neurons target inhibitory interneurons providing a mechanism for feedforward inhibition of vCA1.

Strengths:

The strength of this study was the application of a multilevel analysis approach combining a number of state-of-the-art techniques to dissect the contribution of the EN→vCA1 to memory function.

In addition the authors conducted behavioural analysis of locomotor activity, anxiety and fear memory, and complemented the analysis of discrimination with more detailed description of the patterns of exploratory behaviour.

Reviewer #1 (Public review):

Summary:

Liu and colleagues applied the hidden Markov model on fMRI to show three brain states underlying speech comprehension. Many interesting findings were presented: brain state dynamics were related to various speech and semantic properties, timely expression of brain states (rather than their occurrence probabilities) was correlated with better comprehension, and the estimated brain states were specific to speech comprehension but not at rest or when listening to non-comprehensible speech.

Strengths:

Recently, the HMM has been applied to many fMRI studies, including movie watching and rest. The authors cleverly used the HMM to test the external/linguistic/internal processing theory that was suggested in comprehension literature. I appreciated the way the authors theoretically grounded their hypotheses and reviewed relevant papers that used the HMM on other naturalistic datasets. The manuscript was well written, the analyses were sound, and the results had clear implications.

Reviewer #1 (Public review):

The authors in this paper investigate the nature of the activity in the rodent EPN during a simple freely moving cue-reward association task. Given that primate literature suggest movement coding whereas other primate and rodent studies suggest mainly reward outcome coding in the EPNs, it is important try to tease apart the two views. Through careful analysis of behavior kinematics, position, and the neural activity in the EPNs, the authors reveal an interesting and complex relationship between the EPN and mouse behavior.

Strengths:

(1) The authors use a novel freely moving task to study EPN activity, which displays rich movement trajectories and kinematics. Given that previous studies have mostly looked at reward coding during head fixed behavior, this study adds a valuable dataset to the literature.

(2) The neural analysis is rich and thorough. Both single neuron level and population level (i.e. PCA) analysis are employed to reveal what EPN encodes.

Discussion:<br /> EPN is one of the major output nuclei of the basal ganglia. What information is present within EPN is still unclear, and under investigation. The authors have used electrophysiology to determine the nature of information present within EPN that is likely to be valuable to the field. Future studies should try to address whether this information is specific to certain cell types within EPN or whether there is topography within EPN that reflects the kinematic information present within EPN. This will require more careful dissection of EPN activity based on anatomy. Future experiments should also consider tasks that isolate a single limb (i.e. joystick tasks) in order to better understand the kinematic encoding of forelimb movement. This, combined with recording in forelimb encoding region of EPN, should give us insights into the nature of kinematic control of EPN. Overall, this study will be useful to inspire future investigations in the function of EPN.

Reviewer #1 (Public review):

Kreeger and colleagues have explored the balance of excitation and inhibition in the cochlear nucleus octopus cells of mice using morphological, electrophysiological and computational methods. On the surface, the conclusion, that synaptic inhibition is present, does not seem like a leap. However, the octopus cells have been in the past portrayed as lacking synaptic inhibition. This view was supported by the paucity of glycinergic fibers in the octopus cell area and the lack of apparent IPSPs. Here, Kreeger et al., used beautiful immunohistochemical and mouse genetic methods to quantify the inhibitory and excitatory boutons over the complete surface of individual octopus cells and further analyzed the proportions of the different subtypes of spiral ganglion cell inputs. I think the analysis of synaptic distribution and the origin of the excitatory inputs stands as one of the most complete descriptions of any neuron, leaving little doubt about the presence of glycinergic boutons.

Kreeger et al then examined inhibition physiologically. Recordings from these neurons are notoriously difficult to make because of the enormous leak currents that shunt membrane stimuli and currents, and complicate voltage clamp. The authors have tried to overcome these limitations using drugs to block leak conductances, and computational approaches based on realistic parameters. They conclude that dendritic inhibition can modify the size and kinetics of excitatory signals, and may play out in computations made on temporally dispersed stimuli as might be experienced during a ramp in sound frequency or complex natural sounds like vocalizations.

Reviewer #1 (Public review):

Summary:

The authors use an innovative behavior assay (chamber preference test) and standard calcium imaging experiments on cultured dorsal root ganglion (DRG) neurons to evaluate the consequences of global knockout of TRPV1 and TRPM2, and overexpression of TRPV1, on warmth detection. They find a profound effect of TRPM2 elimination in the behavioral assay, whereas the elimination of TRPV1 has the largest effect on the neuronal responses. These findings are very important, as there is substantial ongoing discussion in the field regarding the contribution of TRP channels to different aspects of thermosensation.

Strengths:

The chamber preference test is an important innovation compared to the standard two-plate test, as it depends on thermal information sampled from the entire skin, as opposed to only the plantar side of the paws. With this assay, and the detailed analysis, the authors provide strong supporting evidence for a role of TRPM2 in warmth avoidance. The conceptual framework using the Drift Diffusion Model provides a first glimpse of how this decision of a mouse to change between temperatures can be interpreted and may form the basis for further analysis of thermosensory behavior.

Weaknesses:

The authors juxtapose these behavioral data with calcium imaging data using isolated DRG neurons. As the authors acknowledge, it remains unclear whether the clear behavioral effect seen in the TRPM2 knockout animals is directly related to TRPM2 functioning as a warmth sensor in sensory neurons. The effects of the TRPM2 KO on the proportion of warmth sensing neurons are very subtle, and TRPM2 may also play a role in the behavioral assay through its expression in thermoregulatory processes in the brain. Future behavioral experiments on sensory-neuron specific TRPM2 knockout animals will be required to clarify this important point.

Reviewer #2 (Public review):

Summary:

This manuscript reports analyses of fMRI data from infants and toddlers watching naturalistic movies. Visual areas in the infant brain show distinct functions, consistent with previous studies using resting state and awake task-based infant fMRI. The pattern of activity in visual regions contains some features predicted by the regions' retinotopic responses. The revised version of the manuscript provides additional validation of the methodology, and clarifies the claims. As a result, the data provide clear support for the claims.

Strengths:

The authors have collected a unique dataset: the same individual infants both watched naturalistic animations and a specific retinotopy task. Using these data position the authors show that activity evoked by movies, in infants' visual areas, is correlated with the regions' retinopic response. The revised manuscript validates this methodology, using adult data. The revised manuscript also shows that an infant's movie watching data is not sufficient or optimal to predict their visual areas' retinotopic responses; anatomical alignment with a group of previous participants provides more accurate prediction of a new participant's retinotopic response.

Weaknesses:

A key step in the analysis of the movie-watching data is the selection of independent components of the movie evoked response that resemble retinotopic spatial patterns. While the trained researcher was unlikely to be biased by this infant's own retinotopy, he/she was actively looking for ICs that resemble average patterns of retinotopic response. To show that these ICs didn't arise by chance (i.e. in noise), the authors proposed an additional analysis in the revised manuscript, by misaligning the functional and anatomical data for a subset of participants. This only partially confirms the reliability of the original components, since when the (new) coder tried to be conservative to avoid false components, he/she identified just over half of the 'true' components (13 vs 22 estimated over the group of 6 infants).

Reviewer #2 (Public review):

Summary:

In this paper, the authors investigated the admixture history of domestic cattle since they were introduced into Iberia, by studying genomic data from 24 ancient samples dated to ~2000-8000 years ago and comparing them to modern breeds. They aimed to (1) characterize genomic variation of skeletal remains and concordance (or discordance) with morphological features; (2) test for hybridization between wild aurochs and domestic cattle; (3) test for correlation between genetic ancestry and stable isotope levels (which are indicative of ecological niche); and (4) test for previously hypothesized higher aurochs ancestry in a modern breed of fighting bulls.

Strengths:

Overall, this study collects valuable new data and tests several important hypotheses regarding the evolutionary history and genomic variation of domestic cattle in Iberia, such as admixture between domestic and wild populations, and correlation between genome-wide aurochs ancestry and aggressiveness.

Weaknesses:

Most conclusions are well supported by the data presented, with the strengths and caveats of each analysis clearly explained. The presence of admixed individuals in prehistorical periods strongly support hybridization between wild and domestic populations, although the evidence for sex-biased introgression and ecological niche sharing is relatively weak. Lastly, the authors presented convincing evidence for relatively constant aurochs ancestry across all modern breeds, including the Lidia breed that has been bred for aggressiveness for centuries.

Major comments:

As the authors pointed out, a major limitation of this study is uncertainty in the "population identity" for most sampled individuals (i.e., whether an individual belonged to the domesticated or wild herd when they were alive). Based on chronology, morphology and genetic data, it is clear the Mesolithic samples from the Artusia and Mendandia sites are bona fide aurochs, but the "population identities" of individuals from the other two sites are much less certain. Indeed, archeological and morphological evidence from El Portalon supports the presence of both domestic animals and wild aurochs, which is echoed by the inter-individual heterogeneity in genetic ancestry. Despite the strong evidence of hybridization, it is unclear whether these admixed individuals were raised in the domestic population or lived in the wild population and hunted, limiting the authors' ability to draw conclusions regarding the direction of gene flow.

In general, detecting sex-bias admixture is an inherently challenging problem, especially given limited data. The differential ancestry proportions (estimated by f4 ratios) on autosomes and X chromosome are indicative of sex-biased hybridization and consistent with previous mtDNA results and other non-genetic data. However, as shown in Fig 3, the confidence intervals of X and autosomal estimates overlap for all but a couple of individuals, despite the overall trend of the point estimates. Moreover, even if there is significant difference, it only suggests existence of sex-bias but does not speak to the extent (unless further quantitative argument is made). Statements such as "it was mostly aurochs males who contributed wild ancestry to domestic herds" is too strong and may be interpreted as extreme bias. The authors did a good job noting the caveats of this analysis and down-toned the statement in the main text, but claims regarding sex-bias hybridization that use the phrase "mostly" in the abstract and discussion need to be further weakened.

The stable isotope analysis is very under-powered, due to issues of categorization of wild vs domestic Bos, as discussed by the authors. Although the considerable overlap in stable isotope values between domestic and wild groups is consistent with shared ecological niche, but the absence of evidence (ie significant difference between groups) is not evidence of absence. Two alternative, non-mutually exclusive scenarios are (1) prevalent errors in classification of wild vs domestic individuals; (2) different ecological niches share similar isotope profiles. Thus, the claim "suggesting that wild and domesticated groups often did not occupy different niches in Iberia" is still too strong.

Reviewer #1 (Public review):

Summary:<br /> The authors set out to explore the role of upstream open reading frames (uORFs) in stabilizing protein levels during Drosophila development and evolution. By utilizing a modified ICIER model for ribosome translation simulations and conducting experimental validations in Drosophila species, the study investigates how uORFs buffer translational variability of downstream coding sequences. The findings reveal that uORFs significantly reduce translational variability, which contributes to gene expression stability across different biological contexts and evolutionary timeframes.

Strengths:<br /> (1) The study introduces a sophisticated adaptation of the ICIER model, enabling detailed simulation of ribosomal traffic and its implications for translation efficiency.<br /> (2) The integration of computational predictions with empirical data through knockout experiments and translatome analysis in Drosophila provides a compelling validation of the model's predictions.<br /> (3) By demonstrating the evolutionary conservation of uORFs' buffering effects, the study provides insights that are likely applicable to a wide range of eukaryotes.

Weaknesses:<br /> (1) Although the study is technically sound, it does not clearly articulate the mechanisms through which uORFs buffer translational variability. A clearer hypothesis detailing the potential molecular interactions or regulatory pathways by which uORFs influence translational stability would enhance the comprehension and impact of the findings.<br /> (2) The study could be further improved by a discussion regarding the evolutionary selection of uORFs. Specifically, it would be beneficial to explore whether uORFs are favored evolutionarily primarily for their role in reducing translation efficiency or for their capability to stabilize translation variability. Such a discussion would provide deeper insights into the evolutionary dynamics and functional significance of uORFs in genetic regulation.

Reviewer #1 (Public review):

Summary:

Cook et al. have presented an important study on the transcriptomic and epigenomic signature underlying craniofacial development in marsupials. Given the lack of a dunnart genome, the authors also prepared long and short-read sequence datasets to assemble and annotate a novel genome to allow for the mapping of RNAseq and ChIPseq data against H3K4me3 and H3K27ac, which allowed for the identification of putative promoter and enhancer sites in dunnart. They found that genes proximal to these regulatory loci were enriched for functions related to bone, skin, muscle and embryonic development, highlighting the precocious state of newborn dunnart facial tissue. When compared with mouse, the authors found a much higher proportion of promoter regions aligned between species than for enhancer regions, and subsequent profiling identified regulatory elements conserved across species and are important for mammalian craniofacial development. In contrast, the identification of dunnart-specific enhancers and patterns of RNA expression further confirm the precocious state of muscle development, as well as for sensory system development, in dunnart suggesting that early formation of these features are critical for neonate marsupials likely to assist with detecting and responding to cues that direct the joeys to the mother's teat after birth. This is one of the few epigenomic studies performed in marsupials (of any organ) and the first performed in fat-tailed dunnart (also of any organ). Marsupials are emerging as an important model for studying mammalian development and evolution and the authors have performed a novel and thorough analysis, impressively including the assembly of a new marsupial reference genome that will benefit many future studies.

Strengths:

The study provides multiple pieces of evidence supporting the important role enhancer elements play in mammalian phenotypic evolution, namely the finding of a lower proportion of peaks present in both dunnart and mouse for enhancers than for promoters, and dunnart showing more genes uniquely associated with it's active enhancers than any other combination of mouse and dunnart samples, whereas this pattern was less pronounced than for promoter-associated genes. In addition, rigorous parameters were used for the cross-species analyses to identify the conserved regulatory elements and the dunnart-specific enhancers. For example, for the results presented in Figure 1, I agree that it is a little surprising that the average promoter-TSS distance is greater than that for enhancers, but that this could be related to the possible presence of unannotated transcripts between genes. The authors addressed this well by examining the distribution of promoter-TSS distances and using proximal promoters (cluster #1) as high confidence promoters for downstream analyses.

The genome assembly method was thorough, using two different long read methods (Pacbio and ONT) to generate the long reads for contig and scaffold construction, increasing the quality of the final assembled genome.

Weaknesses:

Biological replicates of facial tissue were collected at a single developmental time point of the fat-tailed dunnart within the first postnatal day (P0), and analysed this in the context of similar mouse facial samples from the ENCODE consortium at six developmental time points, where previous work from the authors have shown that the younger mouse samples (E11.5-12.5) approximately corresponds to the dunnart developmental stage (Cook et al. 2021). However, it would be useful to have samples from at least one older dunnart time point, for example, at a developmental stage equivalent to mouse E15.5. This would provide additional insight into the extent of accelerated face development in dunnart relative to mouse, i.e. how long do the regulatory elements that activated early in dunnart remain active for and does their function later influence other aspects of craniofacial development?

The authors refer to the development of the CNS being delayed in marsupials relative to placental mammals, however, evidence shows how development of the dunnart brain (whole brain or cortex) is protracted compared to mouse, by a factor of at least 2 times, rather than delayed per se (Workman et al. 2013; Paolino et al. 2023). In addition, there is evidence that cortical formation and cell birth may begin at approximately the same stage across species equivalent to the neonate period in dunnart (E10.5 in mouse), and that shortly after this at the stage equivalent to mouse E12.5, the dunnart cortex shows signs of advanced neurogenesis followed by a protracted phase of neuronal maturation (Paolino et al. 2023). Therefore, it is possible that marsupial CNS development appears delayed relative to mouse but instead begins at the same stage and then proceeds to develop on a different timing scale.

Reviewer #1 (Public review):

Summary:

This study by Howe and colleagues investigates the role of the posterolateral cortical amygdala (plCoA) in mediating innate responses to odors, specifically attraction and aversion. By combining optogenetic stimulation, single-cell RNA sequencing, and spatial analysis, the authors identify a topographically organized circuit within plCoA that governs these behaviors. They show that specific glutamatergic neurons in the anterior and posterior regions of plCoA are responsible for driving attraction and avoidance, respectively, and that these neurons project to distinct downstream regions, including the medial amygdala and nucleus accumbens, to control these responses.

Strengths:

The major strength of the study is the thoroughness of the experimental approach, which combines advanced techniques in neural manipulation and mapping with high-resolution molecular profiling. The identification of a topographically organized circuit in plCoA and the connection between molecularly defined populations and distinct behaviors is a notable contribution to understanding the neural basis of innate motivational responses. Additionally, the use of functional manipulations adds depth to the findings, offering valuable insights into the functionality of specific neuronal populations.

Weaknesses:

There are some weaknesses in the study's methods and interpretation. The lack of clarity regarding the behavior of the mice during head-fixed imaging experiments raises the possibility that restricted behavior could explain the absence of valence encoding at the population level. Furthermore, while the authors employ chemogenetic inhibition of specific pathways, the rationale for this choice over optogenetic inhibition is not fully addressed, and this could potentially affect the interpretation of the results. Additionally, the choice of the mplCoA for manipulation, rather than the more directly implicated anterior and posterior subregions, is not well-explained, which could undermine the conclusions drawn about the topographic organization of plCoA.

Despite these concerns, the work provides significant insights into the neural circuits underlying innate behaviors and opens new avenues for further research. The findings are particularly relevant for understanding the neural basis of motivational behaviors in response to sensory stimuli, and the methods used could be valuable for researchers studying similar circuits in other brain regions. If the authors address the methodological issues raised, this work could have a substantial impact on the field, contributing to both basic neuroscience and translational research on the neural control of behavior.

Reviewer #1 (Public review):

Summary:

MHC (Major Histocompatibility Complex) genes have long been mentioned as cases of trans-species polymorphism (TSP), where alleles might have their most recent common ancestor with alleles in a different species, rather than other alleles in the same species (e.g., a human MHC allele might coalesce with a chimp MHC allele, more recently than the two coalesce with other alleles in either species). This paper provides a more complete estimate of the extent and ages of TSP in primate MHC loci. The data clearly support deep TSP linking alleles in humans to (in some cases) old world monkeys, but the amount of TSP varies between loci.

Strengths:

The authors use publicly available datasets to build phylogenetic trees of MHC alleles and loci. From these trees they are able to estimate whether there is compelling support for Trans-species polymorphisms (TSPs) using Bayes Factor tests comparing different alternative hypotheses for tree shape. The phylogenetic methods are state-of-the-art and appropriate to the task.

The authors supplement their analyses of TSP with estimates of selection (e.g., dN/dS ratios) on motifs within the MHC protein. They confirm what one would suspect: classical MHC genes exhibit stronger selection at amino acid residues that are part of the peptide binding region, and non-classical MHC exhibit less evidence of selection. The selected sites are associated with various diseases in GWAS studies.

Weaknesses:

An implication drawn from this paper (and previous literature) is that MHC has atypically high rates of TSP. However, rates of TSP are not estimated for other genes or gene families, so readers have no basis of comparison. No framework to know whether the depth and frequency of TSP is unusual for MHC family genes, relative to other random genes in the genome, or immune genes in particular. I expect (from previous work on the topic), that MHC is indeed exceptional in this regard, but some direct comparison would provide greater confidence in this conclusion.

Given the companion paper's evidence of genic gain/loss, it seems like there is a real risk that the present study under-estimates TSP, if cases of TSP have been obscured by the loss of the TSP-carrying gene paralog from some lineages needed to detect the TSP. Are the present analyses simply calculating rates of TSP of observed alleles, or are you able to infer TSP rates conditional on rates of gene gain/loss?

Figure 5 (and 6) provide regression model fits (red lines in panel C) relating evolutionary rates (y axis not labeled) to site distance from the peptide binding groove, on the protein product. This is a nice result. I wonder, however, whether a linear model (as opposed to non-linear) is the most biologically reasonable choice, and whether non-linear functions have been evaluated. The authors might consider generalized additive models (GAMs) as an alternative that relaxes linearity assumptions.

The connection between rapidly evolving sites, and disease associations (lines 382-3) is very interesting. However, this is not being presented as a statistical test of association. The authors note that fast-evolving amino acids all have at least one association: but is this really more disease-association than a random amino acid in the MHC? Or, a randomly chosen polymorphic amino acid in MHC? A statistical test confirming an excess of disease associations would strengthen this claim.

Reviewer #1 (Public review):

Summary:

Meissner et al describe an update on the collection of split-GAL4 lines generated by a consortium led by Janelia Research Campus. This follows the same experimental pipeline described before and presents as a significant increment to the present collection. This will strengthen the usefulness and relevance of "splits" as a standard tool for labs that already use this tool and attract more labs and researchers to use it.

Strengths:

This manuscript presents a solid step to establish Split-GAL4 lines as a relevant tool in the powerful Drosophila toolkit. Not only the raw number of available lines contribute to the relevance of this tool in the "technical landscape" of genetic tools, but additional features of this effort contribute to the successful adoption. These include:

(1) A description of expression patterns in the adult and larvae, expanding the "audience" for these tools<br /> (2) A classification of line combination according to quality levels, which provides a relevant criterion while deciding to use a particular set of "splits".<br /> (3) Discrimination between male and female expression patterns, providing hints regarding the potential role of these gender-specific circuits.<br /> (4) The search engine seems to be user-friendly, facilitating the retrieval of useful information.<br /> (5) An acknowledgement of the caveats and challenges that splits (like any other genetic tool) can carry.<br /> Overall, the authors employed a pipeline that maximizes the potential of the Split-GAL4 collection to the scientific community.

Weaknesses:

My concerns were resolved regarding the existence of caveats while using these tools that researchers should be aware of, particularly those using them for the first time.

Reviewer #1 (Public review):

Summary:

This study provides convincing data showing that expression of the PIK3R1(deltaExon11) dominant negative mutation in Activated PI3K Delta Syndrome 1/2 (APDS1/2) patient-derived cells reduces AKT activation and p110δ protein levels. Using a 3T3-L1 model cell system, the authors show that overexpressed p85α(deltaExon 11) displays reduced association with the p110α catalytic subunit but strongly interacts with Irs1/2. Overexpression of PIK3R1 dominant negative mutants inhibit AKT phosphorylation and reduce cellular differentiation of preadipocytes. The experimental design, interpretation, and quantification broadly support the authors' conclusions, which establishes a new paradigm that warrants future studies.

Strengths:

The strength of this study is the clear results derived from Western blots analysis of cell signaling markers (e.g. pAKT1), and co-immunoprecipitation of PI3K holoenzyme complexes and associated regulatory factors (e.g. Irs1/2). The authors analyze a variety of PIK3R1 mutants (i.e. deltaExon11, E489K, R649W, and Y657X), which reveals a range of phenotypes that support the proposed model for dominant negative activity. The use of clonal cell lines with doxycycline induced expression of the PIK3R1 mutants (deltaExon 11, R649W, and Y657X) provides convincing experimental data concerning the relationship between p85α mutant expression and AKT phosphorylation in vivo. This approach for overexpression is excellent and should be utilized more broadly by cell biologists. The authors convincingly show that p85α(deltaExon11, R649W, or Y657X) is unable to associate with p110α but instead more strongly associates with Irs1/2 compared to wild type p85α. Overall, this article does a great job of motivating future studies of SHORT and APDS2 PIK3R1 mutants expressed from their endogenous loci (e.g. knock-in mice).

Weaknesses:

The limitations for this study lie in the complexity of the cell signaling pathway under investigation, rather than a lack of rigor by the authors. Future experimentation will help reconcile the cell type specific differences (e.g. APDS2 patient derived cells vs. the 3T3-L1 cell model system) in PIK3R1 mutant behavior reported by the authors. This is also intimately linked to variable expression of PIK3R1 mutants and cell-type specific regulatory factors. Although beyond the scope of this work, an unbiased proteomic study that broadly evaluates the cell signaling landscape could provide a more holistic understanding of the APDS2 and SHORT mutants compared to a candidate-based approach. Additional structural biochemistry of the p110α/p85α(deltaExon 11) complex is needed to explain why PIK3R1 mutant regulatory subunits do not strongly associate with the p110 catalytic subunit. A more comprehensive biochemical analysis of p110α/p85α, p110β/p85α, and p110δ/p85α mutant protein complexes will also be necessary to explain various cell signaling phenotypes. A minor limitation of this study is the use of single end point assays to measure PI3K lipid kinase activity in the presence of one regulatory input (i.e. RTK-derived pY peptide). An expanded biochemical analysis of purified mutant PI3K complexes across the canonical membrane signaling landscape will be important for deciphering how competition between wild-type and mutant regulatory subunits are regulated in different cell signaling contexts.

Reviewer #1 (Public review):

This study presents an investigation into the physiological functions of RIPK1 within the context of liver physiology, particularly during short-term fasting. Through the use of hepatocyte-specific Ripk1-deficient mice (Ripk1Δhep), the authors embarked on an examination of the consequences of Ripk1 deficiency in hepatocytes under fasting conditions. They discovered that the absence of RIPK1 sensitized the liver to acute injury and hepatocyte apoptosis during fasting, a finding of significant interest given the crucial role of the liver in metabolic adaptation. Employing a combination of transcriptomic profiling and single-cell RNA sequencing techniques, the authors uncovered intricate molecular mechanisms underlying the exacerbated proinflammatory response observed in Ripk1Δhep mice during fasting. While the investigation offers valuable insights into the consequences of Ripk1 deficiency in hepatocytes during fasting conditions, there appears to be a primarily descriptive nature to the study with a lack of clear connection between the experiments. Thus, a stronger focus is warranted, particularly on understanding the dialogue between hepatocytes and macrophages. Moreover, the data would benefit from reinforcement through additional experiments such as Western blotting, flow cytometry, and rescue experiments, which would offer a more quantitative aspect to the findings. By incorporating these enhancements, the study could achieve a more comprehensive understanding of the underlying mechanisms and ultimately strengthen the overall impact of the research.

Comments on revision:

The authors have addressed my comments accordingly.

Reviewer #1 (Public Review):

Summary:

The emergence of Drosophila EM connectomes has revealed numerous neurons within the associative learning circuit. However, these neurons are inaccessible for functional assessment or genetic manipulation in the absence of cell-type-specific drivers. Addressing this knowledge gap, Shuai et al. have screened over 4000 split-GAL4 drivers and correlated them with identified neuron types from the "Hemibrain" EM connectome by matching light microscopy images to neuronal shapes defined by EM. They successfully generated over 800 split-GAL4 drivers and 22 split-LexA drivers covering a substantial number of neuron types across layers of the mushroom body associative learning circuit. They provide new labeling tools for olfactory and non-olfactory sensory inputs to the mushroom body; interneurons connected with dopaminergic neurons and/or mushroom body output neurons; potential reinforcement sensory neurons; and expanded coverage of intrinsic mushroom body neurons. Furthermore, the authors have optimized the GR64f-GAL4 driver into a sugar sensory neuron-specific split-GAL4 driver and functionally validated it as providing a robust optogenetic substitute for sugar reward. Additionally, a driver for putative nociceptive ascending neurons, potentially serving as optogenetic negative reinforcement, is characterized by optogenetic avoidance behavior. The authors also use their very large dataset of neuronal anatomies, covering many example neurons from many brains, to identify neuron instances with atypical morphology. They find many examples of mushroom body neurons with altered neuronal numbers or mistargeting of dendrites or axons and estimate that 1-3% of neurons in each brain may have anatomic peculiarities or malformations. Significantly, the study systematically assesses the individualized existence of MBON08 for the first time. This neuron is a variant shape that sometimes occurs instead of one of two copies of MBON09, and this variation is more common than that in other neuronal classes: 75% of hemispheres have two MBON09's, and 25% have one MBON09 and one MBON08. These newly developed drivers not only expand the repertoire for genetic manipulation of mushroom body-related neurons but also empower researchers to investigate the functions of circuit motifs identified from the connectomes. The authors generously make these flies available to the public. In the foreseeable future, the tools generated in this study will allow important advances in the understanding of learning and memory in Drosophila.

Strengths:

(1) After decades of dedicated research on the mushroom body, a consensus has been established that the release of dopamine from DANs modulates the weights of connections between KCs and MBONs. This process updates the association between sensory information and behavioral responses. However, understanding how the unconditioned stimulus is conveyed from sensory neurons to DANs, and the interactions of MBON outputs with innate responses to sensory context remains less clear due to the developmental and anatomic diversity of MBONs and DANs. Additionally, the recurrent connections between MBONs and DANs are reported to be critical for learning. The characterization of split-GAL4 drivers for 30 major interneurons connected with DANs and/or MBONs in this study will significantly contribute to our understanding of recurrent connections in mushroom body function.

(2) Optogenetic substitutes for real unconditioned stimuli (such as sugar taste or electric shock) are sometimes easier to implement in behavioral assays due to the spatial and temporal specificity with which optogenetic activation can be induced. GR64f-GAL4 has been widely used in the field to activate sugar sensory neurons and mimic sugar reward. However, the authors demonstrate that GR64f-GAL4 drives expression in other neurons not necessary for sugar reward, and the potential activation of these neurons could introduce confounds into training, impairing training efficiency. To address this issue, the authors have elaborated on a series of intersectional drivers with GR64f-GAL4 to dissect subsets of labeled neurons. This approach successfully identified a more specific sugar sensory neuron driver, SS87269, which consistently exhibited optimal training performance and triggered ethologically relevant local searching behaviors. This newly characterized line could serve as an optimized optogenetic tool for sugar reward in future studies.

(3) MBON08 was first reported by Aso et al. 2014, exhibiting dendritic arborization into both ipsilateral and contralateral γ3 compartments. However, this neuron could not be identified in the previously published Drosophila brain connectomes. In the present study, the existence of MBON08 is confirmed, occurring in one hemisphere of 35% of imaged flies. In brains where MBON08 is present, its dendrite arborization disjointly shares contralateral γ3 compartments with MBON09. This remarkable phenotype potentially serves as a valuable resource for understanding the stochasticity of neurodevelopment and the molecular mechanisms underlying mushroom body lobe compartment formation.

Reviewer #1 (Public review):

Summary:

Knudstrup et al. use two-photon calcium imaging to measure neural responses in the mouse primary visual cortex (V1) in response to image sequences. The authors presented mice with many repetitions of the same four-image sequence (ABCD) for four days. Then on the fifth day, they presented unexpected stimulus orderings where one stimulus was either omitted (ABBD) or substituted (ACBD). After analyzing trial-averaged responses of neurons pooled across multiple mice, they observed that stimulus omission (ABBD) caused a small, but significant, strengthening of neural responses but observed no significant change in the response to stimulus substitution (ACBD). Next, they performed population analyses of this dataset. They showed that there were changes in the correlation structure of activity and that many features about sequence ordering could be reliably decoded. This second set of analyses is interesting and exhibited larger effect sizes than the first results about predictive coding. However, concerns about the design of the experiment temper my enthusiasm.

The most recent version of this manuscript makes a few helpful changes (entirely in supplemental figures--the main text figures are unchanged). It does not resolve any of the larger weaknesses of the experimental design, or even perform single-neuron tracking in the one case where it was possible (between similar FOVs shown in Supplemental Figure 1).

Strengths:

(1) The topic of predictive coding in the visual cortex is exciting, and this task builds on previous important work by the senior author (Gavornik and Bear 2014) where unexpectedly shuffling sequence order caused changes in LFPs recorded from visual cortex.

(2) Deconvolved calcium responses were used appropriately here to look at the timing of the neural responses.

(3) Neural decoding results showing that the context of the stimuli could be reliably decoded from trial-averaged responses were interesting. But I have concerns about how the data was formatted for performing these analyses.

Weaknesses:

(1) All analyses were performed on trial-averaged neural responses that were pooled across mice (except for Supplementary Figure 6, see below). Owing to differences between subjects in behavior, experimental preparation quality, and biological variability, it seems important to perform most analyses on individual datasets to assess how behavioral training might differently affect each animal.

In the most recent draft, a single-mouse analysis was added for Figure 4C (Supplementary Figure 6). This effect of "representational drift" was not statistically quantified in either the single-mouse results or in the main text figure panel. Moreover, the apparent correlational drift could be accounted for by a reduction in SNR as a consequence of photobleaching.

(2) The correlation analyses presented in Figure 3 (labeled the second Figure 2 in the text) should be conducted on a single-animal basis. Studying population codes constructed by pooling across mice, particularly when there is no behavioral readout to assess whether learning has had similar effects on all animals, appears inappropriate to me. If the results in Figure 3 hold up on single animals, I think that is definitely an interesting result.

In the most recent draft, this analysis was still not performed on single mice. I was referring to the "decorrelation of responses" analysis in Figure 3, not the "representational drift" analysis in Figure 4. See my comments on Supplementary Figure 6 above.

(3) On Day 0 and Day 5, the reordered stimuli are presented in trial blocks where each image sequence is shown 100 times. Why wasn't the trial ordering randomized as was done in previous studies (e.g. Gavornik and Bear 2014)? Given this lack of reordering, did neurons show reduced predictive responses because the unexpected sequence was shown so many times in quick succession? This might change the results seen in Figure 2, as well as the decoder results where there is a neural encoding of sequence order (Figure 4). It would be interesting if the Figure 4 decoder stopped working when the higher order block structure of the task were disrupted.

In the rebuttal letter for the most recent draft, the authors refer to recent work in press (Hosmane et al. 2024) suggesting that because sleep may be important for plastic changes between sessions, they do not expect much change to be apparent within a session. However, they admit that this current study is too underpowered to know for sure--and do not cite or mention this yet unpublished work in the manuscript itself.

As a control, I would be interested to at least know how much variance in neural responses is observed between intermediate "training" sessions with identical stimuli, e.g. between Day 1 and Day 4, but this is not possible as imaging was not performed on these days.

Despite being referred to as "similar" I do not think early and late responses are clearly shown--aside from the histograms comparing "early traces" to "all traces" which include early traces in Figure 5B and Figure 6A. Showing variance in single-cell responses would be helpful to add in Supplementary Figure 3 and Supplementary Figure 4.

(4) A primary advantage of using two-photon calcium imaging over other techniques like extracellular electrophysiology is that the same neurons can be tracked over many days. This is a standard approach that can be accomplished by using many software packages-including Suite2P (Pachitariu et al. 2017), which is what the authors already used for the rest of their data preprocessing. The authors of this paper did not appear to do this. Instead, it appears that different neurons were imaged on Day 0 (baseline) and Day 5 (test). This is a significant weakness of the current dataset.

In the most recent draft, this concern has not been mitigated. Despite Supplementary Figure 1 showing similar FOVs, mostly different neurons were still extracted. In all other sessions, it is not reported how far apart the other recorded FOVs were from each other.

The rebuttal comment that the PE statistic is computed on an individual cell within-session basis is reasonable. Moreover, the bootstrapped version of the PE analysis in Supplementary Figure 8 is an improvement of the main analysis in the paper. As a control, it would have been helpful to compute the stability of the PE ratio statistics between training days (e.g. between day 1 and day 4). How much change would have been observed when none is expected? Unfortunately, imaging was not performed on these training days so this analysis will not be readily possible to perform. Moreover, the PE statistic requires averaging across cells and trials and is therefore very likely to wash out many interesting effects. Even if it is the population response that is changing, why would it be the arithmetic mean that changes in particular vs. some other projection of the population activity? The experimental and analysis design of the paper here remains weak in my mind.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors provide a method aiming to accurately reflect the individual deviation of longitudinal/temporal change compared to the normal temporal change characterized based on pre-trained population normative model (i.e., a Bayesian linear regression normative model), which was built based on cross-sectional data. This manuscript aims at solving a recently identified problem of using normative models based on cross-sectional data to make inferences about longitudinal change.

Strengths:

The efforts of this work make a good contribution to addressing an important question of normative modeling. With the greater availability of cross-sectional studies for normative modeling than longitudinal studies, and the inappropriateness of making inferences about longitudinal subject-specific changes using these cross-sectional data-based normative models, it's meaningful to try to address this gap from the aspect of methodological development.

Reviewer #1 (Public review):

Summary:

This work aims at understanding the role of thalamus POm in dorsal lateral striatum (DLS) projection in learning a sensorimotor associative task. The authors first confirm that POm forms "en passant" synapses with some of the DLS neuronal subtypes. They then perform a go/no-go associative task that consists of the mouse learning to discriminate between two different textures and to associate one of them with an action. During this task they either record the activity of the POm to DLS axons using endoscopy or silence their activity. They report that POm axons in the DLS are activated around the sensory stimulus but that the activity is not modulated by the reward. Last, they showed that silencing the POm axons at the level of DLS slows down learning the task.

The authors show convincing evidence of projections from POm to DLS and that POm inputs to DLS code for whisking whatever the outcome of the task is. However, their results do not allow to conclude if more neurones are recruited during the learning process or if the already activated fibres get activated more strongly. Last, because POm fibres in the DLS are also projecting to S1, silencing the POm fibres in the DLS could have affected inputs in S1 as well and therefore, the slowdown in acquiring the task is not necessarily specific to the POm to DLS pathway.

Strengths:

One of the main strengths of the paper is to go from slice electrophysiology to behaviour to get an in-depth characterization of one pathway. The authors did a comprehensive description of the POm projections to the DLS using transgenic mice to unambiguously identify the DLS neuronal population. They also used a carefully designed sensorimotor association task, and they exploited the results in depth.

It is a very nice effort to have measured the activity of the axons in the DLS not only after the mice have learned the task but throughout the learning process. It shows the progressive increase of activity of POm axons in the DLS, which could imply that there is a progressive strengthening of the pathway. The results show convincingly that POm axons in the DLS are not activated by the outcome of the task but by the whisker activity, and that this activity in average increases with learning.

Weaknesses:

One of the main targets of the striatum from thalamic input are the cholinergic neurons that weren't investigated here, is there information that could be provided?

It is interesting to know that the POm projects to all neuronal types in the DLS, but this information is not used further down the manuscript so the only take-home message of Figure 1 is that the axons that they image or silence in the DLS are indeed connected to DLS neurons and not just passing fibres. In this line, are these axons the same as the ones projecting to S1? If this is the case, why would we expect a different behaviour of the axon activity at the DLS level compared to S1?

The authors used endoscopy to measure the POm axons in the DLS activity, which makes it impossible to know if the progressive increase of POm response is due to an increase of activity from each individual neurons or if new neurons are progressively recruited in the process.

The picture presented in Figure 4 of the stimulation site is slightly concerning as there are hardly any fibres in neocortical layer 1 while there seems to be quite a lot of them in layer 4, suggesting that the animal here was injected in the VB. This is especially striking as the implantation and projection sites presented in Figure 1 and 2 are very clean and consistent with POm injection.

Comment after review: The weaknesses remain as concerns have not been addressed. The dataset is interesting but the interpretation, due partly to the lack of control (especially relative to VPM contamination), is difficult.

Reviewer #1 (Public review):

Summary:

In this series of studies, Locantore et al. investigated the role of SST-expressing neurons in the entopeduncular nucleus (EPNSst+) in probabilistic switching tasks, a paradigm that requires continued learning to guide future actions. In prior work, this group had demonstrated EPNSst+ neurons co-release both glutamate and GABA and project to the lateral habenula (LHb), and LHb activity is also necessary for outcome evaluation necessary for performance in probabilistic decision-making tasks. Previous slice physiology works have shown that the balance of glutamate/GABA co-release is plastic, altering the net effect of EPN on downstream brain areas and neural circuit function. The authors used a combination of in vivo calcium monitoring with fiber photometry and computational modelling to demonstrate that EPNSst+ neural activity represents movement, choice direction and reward outcomes in their behavioral task. However, viral-genetic manipulations to synaptically silence these neurons or selectively eliminate glutamate release had no effect on behavioral performance in well-trained animals. The authors conclude that despite their representation of task variables, EPN Sst+ neuron synaptic output is dispensable for task performance.

Strengths and Weaknesses:

Overall, the manuscript is exceptionally scholarly, with a clear articulation of the scientific question and a discussion of the findings and their limitations. The analyses and interpretations are careful and rigorous. This review appreciates the thorough explanation of the behavioral modelling and GLM for deconvolving the photometry signal around behavioral events, and the transparency and thoroughness of the analyses in the supplemental figures. This extra care has the result of increasing the accessibility for non-experts, and bolsters confidence in the results. To bolster a reader's understanding of results, we suggest it would be interesting to see the same mouse represented across panels (i.e. Fig 1 F-J, Supp 1 F,K etc i.e via inclusion of faint hash lines connecting individual data points across variables. Additionally, Fig 3E demonstrates that eliminating the 'reward' and 'choice and reward' terms from the GLM significantly worsens model performance; to demonstrate the magnitude of this effect, it would be interesting to include a reconstruction of the photometry signal after holding out of both or one of these terms, alongside the 'original' and 'reconstructed' photometry traces in panel D. This would help give context for how the model performance degrades by exclusion of those key terms. Finally, the authors claimed calcium activity increased following ipsilateral movements. However, figure 3C clearly shows that both SXcontra and SXisi increase beta coefficients. Instead, the choice direction may be represented in these neurons, given that beta coefficients increase following CXipsi and before SEipsi, presumably when animals make executive decisions. Could the authors clarify their interpretation on this point? Also, it is not clear if there is a photometry response related to motor parameters (i.e. head direction or locomotion, licking), which could change the interpretation of the reward outcome if it is related to a motor response; could the authors show photometry signal from representative 'high licking' or 'low licking' reward trials, or from spontaneous periods of high. Vs low locomotor speeds (if the sessions are recorded) to otherwise clarify this point?

There are a few limitations with the design and timing of the synaptic manipulations that would improve the manuscript if discussed or clarified. The authors take care to validate the intersectional genetic strategies: Tetanus Toxin virus (which eliminates synaptic vesicle fusion) or CRISPR editing of Slc17a6, which prevents glutamate loading into synaptic vesicles. The magnitude of effect in the slice physiology results are striking. However, this relies on co-infection of a second AAV to express channelrhodopsin for the purposes of validation, and it is surely the case that there will not be 100% overlap between the proportion of cells infected. Alternative means of glutamate packaging (other VGluT isoforms, other transporters etc) could also compensate for the partial absence of VGluT2, which should be discussed. The authors do not perform a complimentary experiment to delete GABA release (i.e. via VGAT editing), which is understandable, given the absence of an effect with the pan-synaptic manipulation. A more significant concern is the timing of these manipulations as the authors acknowledge. The manipulations are all done in well-trained animals, who continue to perform during the length of viral expression. Moreover, after carefully showing that mice use different strategies on the 70/30 version vs the 90/10 version of the task, only performance on the 90/10 version is assessed after the manipulation. Together, the observation that EPNsst activity does not alter performance on a well learned, 90/10 switching task decreases the impact of the findings, as this population may play a larger role during task acquisition or under more dynamic task conditions. Additional experiments could be done to strengthen the current evidence, although the limitations is transparently discussed by the authors.

Finally, intersectional strategies target LHb-projecting neurons, although in the original characterization it is not entirely clear that the LHb is the only projection target of EPNsst neurons. A projection map would help clarify this point.

Overall, the authors used a pertinent experimental paradigm and common cell-specific approaches to address a major gap in the field, which is the functional role of glutamate/GABA co-release from the major basal ganglia output nucleus in action selection and evaluation. The study is carefully conducted, their analyses are thorough, and the data are often convincing and thought-provoking. However, the limitations of their synaptic manipulations with respect to the behavioral assays reduces generalizability and to some extent the impact of their findings.

Comments on the latest version:

Specifically, they have included more thorough analyses to address several concerns related to interpreting activity patterns of EPSst+ neurons. The authors clearly point out that calcium activity increased during ipsilateral movements, and the increase was statistically larger during the choice phase (Figure 2 supplement 1F-G), indicating that these neurons may represent movement and additional factors (e.g. executive decision-making). Correspondingly, we appreciate the thorough explanation of using a GLM model to determine which behavioural variables contribute to observed physiological signals and adding the example reconstructed signal with direction and reward variables omitted in Figure 3 supplements 1 and 2.

Although no new manipulation experiment is added to the manuscript, the authors respond to common critiques related to testing the behavioural effect after the manipulations in well-trained mice. The discussion related to technical limitations, possible compensatory mechanisms and alternative interpretations is thorough and overall satisfying. Based on the behaviour modeling results, the authors speculate that animals need to integrate more evidence from the past to guide choice in a more uncertain environment (70/30 version), instead of adopting a 'win-stay, lose-shift' strategy in the more deterministic 90/10 version. The authors expand the discussion, but the possibility that EPNSst+ neurons contribute to task performance in well-trained animals under uncertainty is not directly tested. Along with other alternative explanations discussed in the manuscript, we think the paper is valuable literature for future studies to understand the basal ganglia circuits in learning and decision-making.

Reviewer #2 (Public review):

This study by Bell et al. focuses on understanding the roles of two alternatively spliced exons in the single Drosophila Cav2 gene cac. The authors generate a series of cac alleles in which one or the other mutually exclusive exons are deleted to determine the functional consequences at the neuromuscular junction. They find alternative splicing at one exon encoding part of the voltage sensor impacts the activation voltage as well as localization to the active zone. In contrast, splicing at the second exon pair does not impact Cav2 channel localization, but it appears to determine the abundance of the channel at active zones. Together, the authors propose that alternative splicing at the Cac locus enables diversity in Cav2 function generated through isoform diversity generated at the single Cav2 alpha subunit gene encoded in Drosophila.

Overall this is an excellent, rigorously validated study that defines unanticipated functions for alternative splicing in Cav2 channels. The authors have generated an important toolkit of mutually exclusive Cac splice isoforms that will be of broad utility for the field, and show convincing evidence for distinct consequences of alternative splicing of this single Cav2 channel at synapses. Importantly, the authors use electrophysiology and quantitative live sptPALM imaging to determine the impacts of Cac alternative splicing on synaptic function. There remain some questions regarding the mechanisms underlying the changes in Cac localization to somatodendritic compartments. Nonetheless, this is a compelling investigation of alternative splicing in Cav2 channels that should be of interest to many researchers.

Reviewer #1 (Public review):

Summary:

Authors benchmarked 5 IBD detection methods (hmmIBD, isoRelate, hap-IBD, phasedIBD, and Refined IBD) in Plasmodium falciparum using simulated and empirical data. Plasmodium falciparum has a mutation rate similar to humans but a much higher recombination rate and lower SNP density. Thus, the authors evaluated how recombination rate and marker density affect IBD segment detection. Next, they performed parameter optimization for Plasmodium falciparum and benchmarked the robustness of downstream analyses (selection detection and NE inference) using IBD detected by each of the methods. They also tracked the computational efficiency of these methods. The authors work is valuable for the tested species and the analyses presented appear to support their claim that users should be cautious calling IBD when SNP density is low and recombination rate is high.

Strengths:

The study design was solid. The authors set up their reasoning for using P. falciparum very well. The high recombination rate and similar mutation rate to human is indeed an interesting case. Further, they chose methods that were developed explicitly for each species. This was a strength of the work, as well as incorporating both simulated and empirical data to support their goal that IBD detection should be benchmarked in P. falciparum.

Weaknesses:

The scope of the optimization and application of results from the work are narrow, in that everything is fine-tuned for Plasmodium. Some of the results were not entirely unexpected for users of any of the tested software that was developed for humans. For example, it is known that Refined IBD is not going to do well with the combination of short IBD segments and low SNP density. Lastly, it appears the authors only did one large-scale simulation (there are no reported SDs).

Reviewer #1 (Public review):

Summary:

This Tanzanian study focused on the relationship between human genetic ancestry, Mycobacterium tuberculosis complex (MTBC) diversity, and tuberculosis (TB) disease severity. The authors analyzed the genetic ancestry of 1,444 TB patients and genotyped the corresponding MTBC strains isolated from the same individuals. They found that the study participants predominantly possess Bantu-speaking genetic ancestry, with minimal European and Asian ancestry. The MTBC strains identified were diverse and largely resulted from introductions from South or Central Asia. Unfortunately, no associations were identified between human genetic ancestry, the MTBC strains, or TB severity. The authors suggest that social and environmental factors are more likely to contribute to TB severity in this setting.

Strengths:

In comparison to other studies investigating the role of human genetics in TB phenotypes, this study is relatively large, with more than 1,400 participants.

The matched human-MTBC strain collection is valuable and offers the opportunity to address questions about human-bacterium co-evolution.

Weaknesses:

Although the authors had genome-wide genotyping and whole genome sequencing data, they only compared the associations between human ancestry and MTBC strains. Given the large sample size, they had the opportunity to conduct a genome-wide association study similar to that of Muller et al. (https://doi.org/10.1016/j.ygeno.2021.04.024).

The authors tested whether human genetic ancestry is associated with TB severity. However, the basis for this hypothesis is unclear. The studies cited as examples all focused on progression to active TB (from a latent infection state), which should not be conflated with disease severity. It is difficult to ascertain whether the role of genetic ancestry in disease severity would be detectable through this study design, as some participants might simply have been sicker for longer before being diagnosed (despite the inquiry about cough duration). This delay in diagnosis would not be influenced solely by human genetics, which is the conclusion of the study.

Additionally, the study only included participants who attended the TB clinic.

Including healthy controls from the general population would have provided an interesting comparison to see if ancestry proportions differ.

Although the authors suggest that social and environmental factors contribute to TB severity, only age, smoking, and HIV status were characterised in the study.

Reviewer #1 (Public review):

Summary:

This work introduces the differentiable Gillespie algorithm, DGA, which is a differentiable variant of the celebrated (and exact) Gillespie algorithm commonly used to perform stochastic simulations across numerous fields, notably in the life sciences. The proposed DGA approximates the exact Gillespie algorithm using smooth functions yielding a suitable approximate differentiable stochastic system as a proxy for the underlying discrete stochastic system, where DGA stochastic reactions have continuous reaction index and the species abundances. To illustrate their methodology, the authors specifically consider in detail the case of a well-studied two-state promoter gene regulation system that they analyze using a machine learning approach, and by combining simulation data with analytical results. For the two-state promoter gene system, the DGA is benchmarked by accurately reproducing the results of the exact Gillespie algorithm. For this same simple system, the authors also show that how the DGA can be used for estimating kinetic parameters of both simulated and real noisy experimental data. This let them argue convincingly that the DGA can become a powerful computation tool for applications in quantitative and synthetic biology. In order to argue that the DGA can be employed to design circuits with ad-hoc input-output relations, these considerations are then extended to a more complex four-state promoter model of gene regulation.

Strengths:

The main strength of the paper is its clarity and its pedagogical presentation of the simulation methods.

Weaknesses:

It would have been useful to have a brief discussion, based on a concrete example, of what can be achieved with the DGA and is totally beyond the reach of the Gillespie algorithm and the numerous existing stochastic simulation methods. A more comprehensive and quantitative analysis of the limitations of the DGA, e.g. for rare events, would have also been helpful.

Reviewer #1 (Public review):

Summary:

The authors provide a resource to the systems neuroscience community, by offering their Python-based CLoPy platform for closed-loop feedback training. In addition to using neural feedback, as is common in these experiments, they include a capability to use real-time movement extracted from DeepLabCut as the control signal. The methods and repository are detailed for those who wish to use this resource. Furthermore, they demonstrate the efficacy of their system through a series of mesoscale calcium imaging experiments. These experiments use a large number of cortical regions for the control signal in the neural feedback setup, while the movement feedback experiments are analyzed more extensively.

Strengths:

The primary strength of the paper is the availability of their CLoPy platform. Currently, most closed-loop operant conditioning experiments are custom built by each lab and carry a relatively large startup cost to get running. This platform lowers the barrier to entry for closed-loop operant conditioning experiments, in addition to making the experiments more accessible to those with less technical expertise.

Another strength of the paper is the use of many different cortical regions as control signals for the neurofeedback experiments. Rodent operant conditioning experiments typically record from the motor cortex and maybe one other region. Here, the authors demonstrate that mice can volitionally control many different cortical regions not limited to those previously studied, recording across many regions in the same experiment. This demonstrates the relative flexibility of modulating neural dynamics, including in non-motor regions.

Finally, adapting the closed-loop platform to use real-time movement as a control signal is a nice addition. Incorporating movement kinematics into operant conditioning experiments has been a challenge due to the increased technical difficulties of extracting real-time kinematic data from video data at a latency where it can be used as a control signal for operant conditioning. In this paper they demonstrate that the mice can learn the task using their forelimb position, at a rate that is quicker than the neurofeedback experiments.

Weaknesses:

There are several weaknesses in the paper that diminish the impact of its strengths. First, the value of the CLoPy platform is not clearly articulated to the systems neuroscience community. Similarly, the resource could be better positioned within the context of the broader open-source neuroscience community. For an example of how to better frame this resource in these contexts, I recommend consulting the pyControl paper. Improving this framing will likely increase the accessibility and interest of this paper to a less technical neuroscience audience, for instance by highlighting the types of experimental questions CLoPy can enable.

While the dataset contains an impressive amount of animals and cortical regions for the neurofeedback experiment, and an analysis of the movement-feedback experiments, my excitement for these experiments is tempered by the relative incompleteness of the dataset, as well as its description and analysis in the text. For instance, in the neurofeedback experiment, many of these regions only have data from a single mouse, limiting the conclusions that can be drawn. Additionally, there is a lack of reporting of the quantitative results in the text of the document, which is needed to better understand the degree of the results. Finally, the writing of the results section could use some work, as it currently reads more like a methods section.

Suggestions for improved or additional experiments, data or analyses:

Not necessary for this paper, but it would be interesting to see if the CLNF group could learn without auditory feedback.

There are no quantitative results in the results section. I would add important results to help the reader better interpret the data. For example, in: "Our results indicated that both training paradigms were able to lead mice to obtain a significantly larger number of rewards over time," You could show a number, with an appropriate comparison or statistical test, to demonstrate that learning was observed.

For: "Performing this analysis indicated that the Raspberry Pi system could provide reliable graded feedback within ~63 {plus minus} 15 ms for CLNF experiments." The LED test shows the sending of the signal, but the actual delay for the audio generation might be longer. This is also longer than the 50 ms mentioned in the abstract.

It could be helpful to visualize an individual trial for each experiment type, for instance how the audio frequency changes as movement speed / calcium activity changes.

The sample sizes are small (n=1) for a few groups. I am excited by the variety of regions recorded, so it could be beneficial for the authors to collect a few more animals to beef up the sample sizes.

I am curious as to why 60 trials sessions were used. Was it mostly for the convenience of a 30 min session, or were the animals getting satiated? If the former, would learning have occurred more rapidly with longer sessions?

Figure 4 E is interesting, it seems like the changes in the distribution of deltaF was in both positive and negative directions, instead of just positive. I'd be curious as to the author's thoughts as to why this is the case. Relatedly, I don't see Figure 4E, and a few other subplots, mentioned in the text. As a general comment, I would address each subplot in the text.

For: "In general, all ROIs assessed that encompassed sensory, pre-motor, and motor areas were capable of supporting increased reward rates over time," I would provide a visual summary showing the learning curves for the different types of regions.

Relatedly, I would further explain the fast vs slow learners, and if they mapped onto certain regions.

Also I would make the labels for these plots (e.g. Supp Fig3) more intuitive, versus the acronyms currently used.

The CLMF animals showed a decrease in latency across learning, what about the CLNF animals? There is currently no mention in the text or figures.

Reviewer #1 (Public review):

Summary:

Fluorescence imaging has become an increasingly popular technique for monitoring neuronal activity and neurotransmitter concentrations in the living brain. However, factors such as brain motion and changes in blood flow and oxygenation can introduce significant artifacts, particularly when activity-dependent signals are small. Yogesh et al. quantified these effects using GFP, an activity-independent marker, under two-photon and wide-field imaging conditions in awake behaving mice. They report significant GFP responses across various brain regions, layers, and behavioral contexts, with magnitudes comparable to those of commonly used activity sensors. These data highlight the need for robust control strategies and careful interpretation of fluorescence functional imaging data.

Strengths:

The effect of hemodynamic occlusion in two-photon imaging has been previously demonstrated in sparsely labeled neurons in V1 of anesthetized animals (see Shen and Kara et al., Nature Methods, 2012). The present study builds on these findings by imaging a substantially larger population of neurons in awake, behaving mice across multiple cortical regions, layers, and stimulus conditions. The experiments are extensive, the statistical analyses are rigorous, and the results convincingly demonstrate significant GFP responses that must be accounted for in functional imaging experiments. However, whether these GFP responses are driven by hemodynamic occlusion remains less clear, given the complexities associated with awake imaging and GFP's properties (see below).

Weaknesses:

(1) The authors primarily attribute the observed GFP responses to hemodynamic occlusion. While this explanation is plausible, other factors may also contribute to the observed signals. These include uncompensated brain movement (e.g., axial-direction movements), leakage of visual stimulation light into the microscope, and GFP's sensitivity to changes in intracellular pH (see e.g., Kneen and Verkman, 1998, Biophysical Journal). Although the correlation between GFP signals and blood vessel diameters supports a hemodynamic contribution, it does not rule out significant contributions from these (or other) factors. Consequently, whether GFP fluorescence can reliably quantify hemodynamic occlusion in two-photon microscopy remains uncertain.

(2) Regardless of the underlying mechanisms driving the GFP responses, these activity-independent signals must be accounted for in functional imaging experiments. However, the present manuscript does not explore potential strategies to mitigate these effects. Exploring and demonstrating even partial mitigation strategies could have significant implications for the field.

(3) Several methodology details are missing from the Methods section. These include: (a) signal extraction methods for two-photon imaging data (b) neuropil subtraction methods (whether they are performed and, if so, how) (c) methods used to prevent visual stimulation light from being detected by the two-photon imaging system (d) methods to measure blood vessel diameter/area in each frame. The authors should provide more details in their revision.

Reviewer #1 (Public review):

Summary:

This study aims to investigate the links between social behaviors observed in free-moving situations and behavioral performances measured in well-controlled, laboratory settings. The authors assessed general social tendencies and dyadic relationships among four monkeys in a group by scoring agonistic (aggression) and affiliative (grooming and proximity) behaviors in each pair. By measuring the saccadic reaction time in a classic social interference task, the authors reported that the monkeys with higher SEIs (i.e., more social individuals) were less distracted by the faces of other monkeys. These effects were enhanced when the distractors were out-group monkey faces rather than in-group ones. Lastly, oxytocin administration increased the impact of the out-group monkey faces in the social interference task, while reducing the magnitude of general social tendencies measured with SEI.

Strengths:

(1) The combination of behavioral data obtained in a colony room and in a laboratory environment is rare and important.<br /> (2) The evaluation of social interactions were successfully performed based on an automated target detection algorithm. The resulting multi-dimensional, complicated social interactions were summarized into simple indices (SEI and IEI). These indices provide a good measure for the social tendencies of each monkey.<br /> (3) Well-designed and robust experiments in the laboratory environment that are linked nicely with the general social tendencies observed in spontaneous behaviors.

Weaknesses:

(1) While the overall results are interesting, I am somewhat left confused about how to interpret the difference in the scores derived from different conditions. For example, the authors stated "Comparing the weights for in-group and out-group distractors, the effect of proximity was larger than that of aggression and grooming" in p.8. Does this mean that the proximity is indeed the type of behavior most affected in the out-group condition compared to the in-group condition? The out-group effects are difficult to examine with actual behavioral data, but some in-group effects such as those involving OT can be tested, which possibly provides good insights into interpreting the differences of the weights observed across the experimental conditions.

(2) I think it is important to provide how variable spontaneous social interactions were across sessions and how impactful the variability of the interactions is on the SEI and IEI, as it helps to understand how meaningful the differences of weights are across the conditions, but such data are missing. In line with this point, although the conclusions still hold as those data were obtained during the same experimental periods, shouldn't the weights in Fig. 3f and Figs. 4g and 4h (saline) be expected to be similar, if not the same?

Reviewer #1 (Public review):

Summary:

In this manuscript, Kondo et al. developed a method to suppress somatic action potentials while recording spine calcium signals using two-photon imaging in the L2/3 visual cortex in response to visual stimuli. The authors identified different patterns of dendritic spine activation by visual stimuli and analyzed how the different patterns of spine responses may contribute to somatic visual responses. Their analysis results suggest that spines on dendrites with a clustered arrangement can potentially generate sharply tuned output.

Strengths:

This is an interesting study addressing a standing question of how previously reported pepper-and-salt-like distributed sensory inputs on individual spines may give rise to somatic sensory selectivity. The method of somatic inhibition to prevent bAPs appears new and effective. The measurements of spine activity are carefully done. The finding that a small number of spines located in the same branch with similar tuning properties would predict the somatic tuning is consistent with local dendritic nonlinear integration mechanisms.

Weaknesses:

(1) The demonstration of the effectiveness of soma-specific inhibition is inadequate. Figure 1 only provides a single example trace showing the inhibition of somatic visual responses. The authors should provide statistical analysis over grouped data. For the effect of soma-specific inhibition on spine activity, the authors provided mostly negative results, lacking effects on spine responses for both soma inhibition and bAP subtraction. This is confusing. One possible explanation is that bAPs normally have little influence on spine activity. However, this would conflict with the known fact that somatic APs can easily invade spines in L2/3 neurons (e.g., Chen et al., Nature 2011). Another possibility is that under the current experimental conditions, somatic APs were rarely evoked by the visual stimulus. The authors should also rule out the possibility that the spines they imaged are from different neurons than the ones with somatic inhibition. The authors may consider identifying those cases where somatic APs have a significant impact on spine activity or spine tuning and show how bAP inhibition influences the dendritic and spine responses.

(2) Figure 4 shows that the proportion of spines with a preferred orientation similar to the soma (ΔOri {less than or equal to} 30{degree sign}) was 60%, which is surprisingly high. It is intriguing that without somatic AP invasion, there could be such a high degree of similarity between spine activity and somatic tuning. What is the ratio without soma inhibition? One could reason that with bAP invasion, there should be even more spines showing visual responses similar to those of the soma. Moreover, with such a high proportion of spines showing similar sensory tuning to the soma, it is inevitable that many branches contain more spines with similar tuning as the soma, exhibiting an apparent branch-specific clustering. While such apparent clustering may well predict somatic tuning, it primarily reflects a correlational relationship rather than a causal synaptic integration mechanism.

(3) There has been extensive work studying how the integration of spine activity or sub-branch activity gives rise to somatic output. The proposed main contribution of this study is to use an improved method to inhibit somatic activity in order to more confidently measure spine-specific activity and examine the integration mechanisms. However, the results showed that the measured spine-specific activity under soma inhibition was not significantly different from that measured under normal conditions (see point 1). It becomes unclear how this new method contributes to obtaining new insights into the synaptic integration mechanism.

(4) Figure 6 shows how the tuning similarity between spines depends on the distance between them. It is unclear what new information was acquired regarding the functional clustering of spines. This result can be largely explained by the overall higher proportion of similarly tuned spines (60%) compared to the soma's preferred orientations. Moreover, the authors did not demonstrate how such clustering may contribute to nonlinear synaptic integration.

(5) The results shown in Figure 7 can again be largely explained by the static property of a higher proportion of spines tuned similarly to the soma. These results do not reveal any active dendritic integration mechanisms.

Reviewer #1 (Public review):

In this manuscript, Chang et al. investigated the cell type-specific role of the integrin activator Shv in activity-dependent synaptic remodeling. Using the Drosophila larval neuromuscular junction as a model, they show that glial-secreted Shv modulates synaptic plasticity by maintaining the extracellular balance of neuronal Shv proteins and regulating ambient extracellular glutamate concentrations, which in turn affects postsynaptic glutamate receptor abundance. Furthermore, they report that genetic perturbation of glial morphogenesis phenocopies the defects observed with the loss of glial Shv. Altogether, their findings propose a role for glia in activity-induced synaptic remodeling through Shv secretion. While the conclusions are intriguing, several issues related to experimental design and data interpretation merit further discussion.

Reviewer #1 (Public review):

Summary:

As TDP-43 mislocalization is a hallmark of multiple neurodegenerative diseases, the authors seek to identify pathways that modulate TDP-43 levels. To do this, they use a FACS based genome wide CRISPR KD screen in a Halo tagged TDP-43 KI iPSC line. Their screen identifies a number of genetic modulators of TDP-43 expression including BORC which plays a role in lysosome transport.

Strengths:

Genome wide CRISPR based screen identifies a number of modulators of TDP-43 expression to generate hypotheses regarding RNA BP regulation and perhaps insights into disease.

Weaknesses:

It is unclear how altering TDP-43 levels may relate to disease where TDP-43 is not altered in expression but mislocalized. This is a solid cell biology study, but the relation to disease is not clear without providing evidence of BORC alterations in disease or manipulation of BORC reversing TDP-43 pathology in disease.

The mechanisms by which BORC and lysosome transport modulate TDP-43 expression are unclear. Presumably, this may be through altered degradation of TDP protein but this is not addressed.

Previous studies have demonstrated that TDP-43 levels can be modulated by altering lysosomal degradation so the identification of lysosomal pathways is not particularly novel.

It is unclear whether this finding is specific to TDP-43 levels or whether lysosome localization may more broadly impact proteostasis in particular of other RNA BPs linked to disease.

Unclear whether BORC depletion alters lysosome function or simply localization.

Reviewer #1 (Public review):

Summary of what is achieved: This manuscript validates and extends upon the sigh generating circuit between the NMB/GRP+ RTN/parafacial neurons and the NMBR/GRPR+ preBötC neurons established in Li et al., 2016. The authors generate multiple transgenic lines that enable selective targeting of these various sub-populations of cells and demonstrate the sufficiency of each type in generating a sigh breath. Additionally, they show that NMBR and GPRP preBötC neurons are glutamatergic, have overlapping and distinct expression, and do not express SST. Beyond this validation, the authors show that ectopic stimulation of SST neurons is sufficient to evoke sighs and that they are necessary for NMB/GRP induced sighing. This data is the first time that preBötC neurons downstream of NMBR/GRPR neurons have been identified that transform a eupneic breath into a sign breath. The five conclusions stated at the end of the introduction are supported by the data.

Summary of a primary weakness: A strong emphasis throughout the manuscript is the identification of an unsubstantiated slow sigh rhythm that is produced by NMBR/GRPR neurons. It is even suggested that this is an intrinsic property of these neurons. However, to make such a novel (and quite surprising) claim requires many more studies and the conclusion is dependent on how the authors have defined a sigh. Moreover, some data within the paper conflicts with this idea. The resubmitted manuscript does not contain any revisions and the rebuttal does not sufficiently address the critiques.

In summary, the optogenetic and chemogenetic characterization of the neuropeptide pathway transgenic lines nicely aligns with and provides important validation of the previous study by Li et. al., 2016 and the SST neuron studies provide a new mechanism for the transformation of NMBR/GRPR neuropeptide activation into a sigh. These are important findings, and they should be the points emphasized. The proposal of a slow sigh rhythm should be more rigorously established with new experiments and analysis or should be more carefully described and discussed.

Reviewer #1 (Public review):

Summary:

Dong et al here have studied the impact of the small Ras-like GTPase Rab10 on the exocytosis of dense core vesicles (DVC), which are important mediators of neuropeptide signaling in brain. They use optical imaging to show that lentiviral depletion of Rab10 in mouse hippocampal neurons in culture independent of the established defects in neurite outgrowth hamper DCV exocytosis. They further demonstrate that such defects are paralleled by changes in ER morphology and defective ER-based calcium buffering as well as reduced ribosomal protein expression in Rab10-depleted neurons. Re-expression of Rab10 or supplementation of exogenous L-leucine to restore defective neuronal protein synthesis rescues impaired DCV secretion. Based on these results they propose that Rab10 regulates DCV release by maintaining ER calcium homeostasis and neuronal protein synthesis.

Strengths:

This work provides interesting and potentially important new insights into the connection between ER function and the regulated secretion of neuropeptides via DCVs. The authors combine advanced optical imaging with light and electron microscopy, biochemistry and proteomics approaches to thoroughly assess the effects of Rab10 knockdown at the cellular level in primary neurons. The proteomic dataset provided may be valuable in facilitating future studies regarding Rab10 function. This work will thus be of interest to neuroscientists and cell biologists.

Weaknesses:

Whether and how the phenotypes of Rab10 reported in this study are linked remains an open question. Likewise, a possible role of Rab10 in exocytosis cannot be excluded at this stage.

Comments on revisions:

My previous questions and concerns have been satisfactorily addressed by the authors.

Reviewer #2 (Public review):

This work aggregates data across 5 openly available stopping studies (3 at 7 tesla and 2 at 3 tesla) to evaluate activity patterns across the common contrasts of Failed Stop (FS) > Go, FS > stop success (SS), and SS > Go. Previous work has implicated a set of regions that tend to be positively active in one or more of these contrasts, including the bilateral inferior frontal gyrus, preSMA, and multiple basal ganglia structures. However, the authors argue that upon closer examination, many previous papers have not found subcortical structures to be more active on SS than FS trials, bringing into question whether they play an essential role in (successful) inhibition. In order to evaluate this with more data and power, the authors aggregate across five datasets and find many areas that are *more* active for FS than SS, including bilateral preSMA, GPE, thalamus, and VTA. They argue that this brings into question the role of these areas in inhibition, based upon the assumption that areas involved in inhibition should be more active on successful stop than failed stop trials, not the opposite as they observed.

Comments on revisions:

The authors have been responsive to the feedback of both reviewers and they have significantly improved the manuscript. I now judge the work as valuable and solid. The authors have achieved their aims to characterize subcortical BOLD activation in the stop-signal paradigm.

Reviewer #1 (Public review):

The authors set out to define the molecular basis for LP as the origin of BRCA1-deficient breast cancers. They showed that LPs have the highest level of replicative stress, and hypothesise that this may account for their tendency to transform. They went on to identify ELF3 as a candidate driver of LP transformation and showed that ELF3 expression is up-regulated in response to replicative stress as well as BRCA1 deficiency. They went on to show that ELF3 inactivation led to a higher level of DNA damage, which may result from compromised replicative stress responses.

While the manuscript supports the interesting idea wherein ELF3 may fuel LP cell transformation, it remains obscure how ELF3 promotes cell tolerance to DNA damage. Interestingly the authors proposed that ELF3 suppresses excessive genomic instability, but in my opinion, I do not see any evidence that supports this claim. In fact, one might think that genomic instability is key to cell transformation.

Comments on revisions:

The authors have addressed most of my concerns.

This being said, the one major criticism raised by both Reviewers is the lack of evidence to support ELF3 as a driver of transformation of and in LP cells. The authors appear to have invested much resource and time but were not successful in isolating LP cells for experimentations. I would therefore suggest that the authors tone down their claims throughout the manuscript.

Reviewer #1 (Public review):

Summary:

This study analyzed biomarker data from 28 subjects with geographic atrophy (GA) in a Phase I/II clinical trial of PPY988, a subretinal AAV2 complement factor I (CFI) gene therapy, to evaluate pharmacokinetics and pharmacodynamics. Post-treatment, a 2-fold increase in vitreous humor (VH) FI was observed, correlating with a reduction in FB breakdown product Ba but minimal changes in other complement factors. The aqueous humor (AH) was found to be an unreliable proxy for VH in assessing complement activation. In vitro assays showed that the increase in FI had a minor effect on the complement amplification loop compared to the more potent C3 inhibitor pegcetacoplan. These findings suggest that PPY988 may not provide enough FI protein to effectively modulate complement activation and slow GA progression, highlighting the need for thorough biomarker review to determine optimal dosing in future studies.

Strengths:

This manuscript provides critical data on the efficacy of gene therapy for the eye, specifically introducing complement FI expression. It presents the results from a halted clinical trial, making the publication of this data essential for understanding the outcomes of this gene therapy approach. The findings offer valuable insights and lessons for future gene therapy attempts in similar contexts.

Weaknesses:

No particular weaknesses. The study was carefully performed and limitations are discussed.

I have just some concerns about the methodology used. The authors use the MILLIPLEX assays, which allow for multiplexed detection of complement proteins and they mention extensive validation. How are the measurements with this assay correlating with gold standard methods? Is the specificity and the expected normal ranges preserved with this assay? This also stands for the Olink assay. Some of the proteins are measured by both assay and/or by standard ELISA. How do these measurements correlate?

Comments on revisions:

The authors answered part of my comments. Only one remained - please provide a comparison between ELISA/Multiplex and Olink data to judge the robustness of the Olinkl assay for complement.

Reviewer #1 (Public review):

Summary:

Chlamydia spp. has a biphasic developmental cycle consisting of an extracellular, infectious form called an elementary body (EB) and an intracellular, replicative form known as a reticular body (RB). The structural stability of EBs is maintained by extensive cross linking of outer membrane proteins while the outer membrane proteins of RBs are in a reduced state. The overall redox state of EBs is more oxidized than RBs. The authors propose that redox state may be a controlling factor in the developmental cycle. To test this, alkyl hydroperoxide reductase subunit C (ahpC) was overexpressed or knocked down to examine effects on developmental gene expression. KD of ahpC induced increased expression of EB-specific genes and accelerated EB production. Conversely, overexpression of phpC delayed differentiation to EBs. The results suggest that chlamydial redox state may play a role in differentiation.

Strengths:

Uses modern genetic tools to explore the difficult area of temporal gene expression throughout the chlamydial developmental cycle.

Weaknesses:

The environmental signals triggering ahpC expression/activity are not determined.

Comments on revisions:

I am satisfied with the modifications made to the manuscript.

Reviewer #2 (Public review):

Summary:

In this manuscript the authors evaluate the attenuation, immunogenicity, and protection efficacy of a live-attenuated SARS-CoV-2 vaccine candidate (BK2102) against SARS-CoV-2.

Strengths:

The authors demonstrate that intranasal inoculation of BK2102 is safe and able to induce humoral and cellular immune responses in hamsters, without apparent signs of damage in the lungs, that protects against homologous SARS-CoV-2 and Omicron BA.5 challenge. Safety of BK2102 was further confirmed in a new hACE2 transgenic mouse model generated by the authors.

Weaknesses:

The authors have addressed my previous comments on the first submission of the document.

Reviewer #1 (Public Review):

Summary:

The present study's main aim is to investigate the mechanism of how VirR controls the magnitude of MEV release in Mtb. The authors used various techniques, including genetics, transcriptomics, proteomics, and ultrastructural and biochemical methods. Several observations were made to link VirR-mediated vesiculogenesis with PG metabolism, lipid metabolism, and cell wall permeability. Finally, the authors presented evidence of a direct physical interaction of VirR with the LCP proteins involved in linking PG with AG, providing clues that VirR might act as a scaffold for LCP proteins and remodel the cell wall of Mtb. Since the Mtb cell wall provides a formidable anatomical barrier for the entry of antibiotics, targeting VirR might weaken the permeability of the pathogen along with the stimulation of the immune system due to enhanced vesiculogenesis. Therefore, VirR could be an excellent drug target. Overall, the study is an essential area of TB biology.

Strengths:

The authors have done a commendable job of comprehensively examining the phenotypes associated with the VirR mutant using various techniques. Application of Cryo-EM technology confirmed increased thickness and altered arrangement of CM-L1 layer. The authors also confirmed that increased vesicle release in the mutant was not due to cell lysis, which contrasts with studies in other bacterial species.

Another strength of the manuscript is that biochemical experiments show altered permeability and PG turnover in the mutant, which fits with later experiments where authors provide evidence of a direct physical interaction of VirR with LCP proteins.

Transcriptomics and proteomics data were helpful in making connections with lipid metabolism, which the authors confirmed by analyzing the lipids and metabolites of the mutant.

Lastly, using three approaches, the authors confirm that VirR interacts with LCP proteins in Mtb via the LytR_C terminal domain.

Altogether, the work is comprehensive, experiments are designed well, and conclusions were made based on the data generated after verification using multiple complementary approaches.

Weaknesses:

The major weakness is that the mechanism of VirR-mediated EV release remains enigmatic. Most of the findings are observational and only associate enhanced vesiculogenesis observed in the VirR mutant with cell wall permeability and PG metabolism. Authors suggest that EV release occurs during cell division when PG is most fragile. However, this has yet to be tested in the manuscript-the AFM of the VirR mutant, which produces thicker PG with more pore density, displays enhanced vesiculogenesis. No evidence was presented to show that the PG of the mutant is fragile, and there are differences in cell division to explain increased vesiculogenesis. These observations, counterintuitive to the authors' hypothesis, need detailed experimental verification.

Transcriptomic data only adds a little substantial. Transcriptomic data do not correlate with the proteomics data. It remains unclear how VirR deregulates transcription. TLCs of lipids are not quantitative. For example, the TLC image of PDIM is poor; quantitative estimation needs metabolic labeling of lipids with radioactive precursors. Further, change in PDIMs is likely to affect other lipids (SL-1, PAT/DAT) that share a common precursor (propionyl- CoA).

The connection of cholesterol with cell wall permeability is tenuous. Cholesterol will serve as a carbon source and contribute to the biosynthesis of methyl-branched lipids such as PDIM, SL-1, and PAD/DAT. Carbon sources also affect other aspects of physiology (redox, respiration, ATP), which can directly affect permeability and import/export of drugs. Authors should investigate whether restoration of the normal level of permeability and EV release is not due to the maintenance of cell wall lipid balance upon cholesterol exposure of the VirR mutant.

Finally, protein interaction data is based on experiments done once without statistical analysis. If the interaction between VirR and LCP protein is expected on the mycobacterial membrane, how SPLIT_GFP system expressed in the cytoplasm is physiologically relevant. No explanation was provided as to why VirR interacts with the truncated version of LCP proteins and not with the full-length proteins.

Comments on revisions:

The authors have addressed my comments. I have no further issues.

Reviewer #1 (Public review):

Summary:

In this preprint, the authors systematically and rigorously investigate how specific classes of residue mutations alter the critical temperature as a proxy for the driving forces for phase separation. The work is well executed, the manuscript well-written, and the results reasonable and insightful.

Strengths:

The introductory material does an excellent job of being precise in language and ideas while summarizing the state of the art. The simulation design, execution, and analysis are exceptional and set the standard for large-scale simulation studies. The results, interpretations, and Discussion are largely nuanced, clear, and well-motivated, and the pedagogical nature with which sampling convergence is discussed is greatly appreciated. Finally, the underlying data are shared in a clear and accessible manner. Overall, the manuscript is a model

Weaknesses:

The simplicity of a one-bead-per-residue model parameterized to capture UCST-type phase behavior does perhaps impact some aspects of the generality of this work. That said, the authors carefully acknowledge these limitations, and overall, this is not seen as a major weakness of the conclusions drawn or the manuscript, given those conclusions are appropriately couched.

Joint Public Review:

This manuscript by Tao et al. reports on an effort to better specify the underlying interactions driving the effects of biodiversity on productivity in biodiversity experiments. The authors are especially concerned with the potential for competitive interactions to drive positive biodiversity-ecosystem functioning relationships by driving down the biomass of subdominant species. The authors suggest a new partitioning schema that utilizes a suite of partial density treatments to capture so-called competitive ability.

Readers are encouraged to consider the original reviews in full, which outline the strengths and weaknesses of the work:

First version: https://elifesciences.org/reviewed-preprints/98073v1/reviews

Second version: https://elifesciences.org/reviewed-preprints/98073v2/reviews

There are no further reviews for this version because the authors declined to make further improvements to their manuscript.

Reviewer #1 (Public review):

Summary

In this human neuroimaging and electrophysiology study, the authors aimed to characterise effects of a period of visual deprivation in the sensitive period on excitatory and inhibitory balance in the visual cortex. They attempted to do so by comparing neurochemistry conditions ('eyes open', 'eyes closed') and resting state, and visually evoked EEG activity between ten congenital cataract patients with recovered sight (CC), and ten age-matched control participants (SC) with normal sight.

First, they used magnetic resonance spectroscopy to measure in vivo neurochemistry from two locations, the primary location of interest in the visual cortex, and a control location in the frontal cortex. Such voxels are used to provide a control for the spatial specificity of any effects, because the single-voxel MRS method provides a single sampling location. Using MR-visible proxies of excitatory and inhibitory neurotransmission, Glx and GABA+ respectively, the authors report no group effects in GABA+ or Glx, no difference in the functional conditions 'eyes closed' and 'eyes open'. They found an effect of group in the ratio of Glx/GABA+ and no similar effect in the control voxel location. They then perform multiple exploratory correlations between MRS measures and visual acuity, and report a weak positive correlation between the 'eyes open' condition and visual acuity in CC participants.

The same participants then took part in an EEG experiment. The authors selected two electrodes placed in the visual cortex for analysis and report a group difference in an EEG index of neural activity, the aperiodic intercept, as well as the aperiodic slope, considered a proxy for cortical inhibition. Control electrodes in the frontal region did not present with the same pattern. They report an exploratory correlation between the aperiodic intercept and Glx in one out of three EEG conditions.

The authors report the difference in E/I ratio, and interpret the lower E/I ratio as representing an adaptation to visual deprivation, which would have initially caused a higher E/I ratio. Although intriguing, the strength of evidence in support of this view is not strong. Amongst the limitations are the low sample size, a critical control cohort that could provide evidence for higher E/I ratio in CC patients without recovered sight for example, and lower data quality in the control voxel. Nevertheless, the study provides a rare and valuable insight into experience-dependent plasticity in the human brain.

Strengths of study

How sensitive period experience shapes the developing brain is an enduring and important question in neuroscience. This question has been particularly difficult to investigate in humans. The authors recruited a small number of sight-recovered participants with bilateral congenital cataracts to investigate the effect of sensitive period deprivation on the balance of excitation and inhibition in the visual brain using measures of brain chemistry and brain electrophysiology. The research is novel, and the paper was interesting and well written.

Limitations

Low sample size. Ten for CC and ten for SC, and further two SC participants were rejected due to lack of frontal control voxel data. The sample size limits the statistical power of the dataset and increases the likelihood of effect inflation.

In the updated manuscript, the authors have provided justification for their sample size by pointing to prior studies and the inherent difficulties in recruiting individuals with bilateral congenital cataracts. Importantly, this highlights the value the study brings to the field while also acknowledging the need to replicate the effects in a larger cohort.

Lack of specific control cohort. The control cohort has normal vision. The control cohort is not specific enough to distinguish between people with sight loss due to different causes and patients with congenital cataracts with co-morbidities. Further data from a more specific populations, such as patients whose cataracts have not been removed, with developmental cataracts, or congenitally blind participants, would greatly improve the interpretability of the main finding. The lack of a more specific control cohort is a major caveat that limits a conclusive interpretation of the results.

In the updated version, the authors have indicated that future studies can pursue comparisons between congenital cataract participants and cohorts with later sight loss.

MRS data quality differences. Data quality in the control voxel appears worse than in the visual cortex voxel. The frontal cortex MRS spectrum shows far broader linewidth than the visual cortex (Supplementary Figures). Compared to the visual voxel, the frontal cortex voxel has less defined Glx and GABA+ peaks; lower GABA+ and Glx concentrations, lower NAA SNR values; lower NAA concentrations. If the data quality is a lot worse in the FC, then small effects may not be detectable.

In the updated version, the authors have added more information that informs the reader of the MRS quality differences between voxel locations. This increases the transparency of their reporting and enhances the assessment of the results.

Because of the direction of the difference in E/I, the authors interpret their findings as representing signatures of sight improvement after surgery without further evidence, either within the study or from the literature. However, the literature suggests that plasticity and visual deprivation drives the E/I index up rather than down. Decreasing GABA+ is thought to facilitate experience dependent remodelling. What evidence is there that cortical inhibition increases in response to a visual cortex that is over-sensitised to due congenital cataracts? Without further experimental or literature support this interpretation remains very speculative.

The updated manuscript contains key reference from non-human work to justify their interpretation.

Heterogeneity in patient group. Congenital cataract (CC) patients experienced a variety of duration of visual impairment and were of different ages. They presented with co-morbidities (absorbed lens, strabismus, nystagmus). Strabismus has been associated with abnormalities in GABAergic inhibition in the visual cortex. The possible interactions with residual vision and confounds of co-morbidities are not experimentally controlled for in the correlations, and not discussed.

The updated document has addressed this caveat.

Multiple exploratory correlations were performed to relate MRS measures to visual acuity (shown in Supplementary Materials), and only specific ones shown in the main document. The authors describe the analysis as exploratory in the 'Methods' section. Furthermore, the correlation between visual acuity and E/I metric is weak, not corrected for multiple comparisons. The results should be presented as preliminary, as no strong conclusions can be made from them. They can provide a hypothesis to test in a future study.

This has now been done throughout the document and increases the transparency of the reporting.

P.16 Given the correlation of the aperiodic intercept with age ("Age negatively correlated with the aperiodic intercept across CC and SC individuals, that is, a flattening of the intercept was observed with age"), age needs to be controlled for in the correlation between neurochemistry and the aperiodic intercept. Glx has also been shown to negatively correlates with age.

This caveat has been addressed in the revised manuscript.

Multiple exploratory correlations were performed to relate MRS to EEG measures (shown in Supplementary Materials), and only specific ones shown in the main document. Given the multiple measures from the MRS, the correlations with the EEG measures were exploratory, as stated in the text, p.16, and in Fig.4. yet the introduction said that there was a prior hypothesis "We further hypothesized that neurotransmitter changes would relate to changes in the slope and intercept of the EEG aperiodic activity in the same subjects." It would be great if the text could be revised for consistency and the analysis described as exploratory.

This has been done throughout the document and increases the transparency of the reporting.

The analysis for the EEG needs to take more advantage of the available data. As far as I understand, only two electrodes were used, yet far more were available as seen in their previous study (Ossandon et al., 2023). The spatial specificity is not established. The authors could use the frontal cortex electrode (FP1, FP2) signals as a control for spatial specificity in the group effects, or even better, all available electrodes and correct for multiple comparisons. Furthermore, they could use the aperiodic intercept vs Glx in SC to evaluate the specificity of the correlation to CC.

This caveat has been addressed. The authors have added frontal electrodes to their analysis, providing an essential regional control for the visual cortex location.

Comments on the latest version:

The authors have made reasonable adjustments to their manuscript that addressed most of my comments by adding further justification for their methodology, essential literature support, pointing out exploratory analyses, limitations and adding key control analyses. Their revised manuscript has overall improved, providing valuable information, though the evidence that supports their claims is still incomplete.

Reviewer #1 (Public review):

Contractile Injection Systems (CIS) are versatile machines that can form pores in membranes or deliver effectors. They can act extra or intracellularly. When intracellular they are positioned to face the exterior of the cell and hence should be anchored to the cell envelope. The authors previously reported the characterization of a CIS in Streptomyces coelicolor, including significant information on the architecture of the apparatus. However, how the tubular structure is attached to the envelope was not investigated. Here they provide a wealth of evidence to demonstrate that a specific gene within the CIS gene cluster, cisA, encodes a membrane protein that anchors the CIS to the envelope. More specifically, they show that:

- CisA is not required for assembly of the structure but is important for proper contraction and CIS-mediated cell death<br /> - CisA is associated to the membrane (fluorescence microscopy, cell fractionation) through a transmembrane segment (lacZ-phoA topology fusions in E. coli)<br /> - Structural prediction of interaction between CisA and a CIS baseplate component<br /> - In addition they provide a high-resolution model structure of the >750-polypeptide Streptomyces CIS in its extended conformation, revealing new details of this fascinating machine, notably in the baseplate and cap complexes.

All the experiments are well controlled including trans-complemented of all tested phenotypes.

One important information we miss is the oligomeric state of CisA.

While it would have been great to test the interaction between CisA and Cis11, to perform cryo-electron microscopy assays of detergent-extracted CIS structures to maintain the interaction with CisA, I believe that the toxicity of CisA upon overexpression or upon expression in E. coli render these studies difficult and will require a significant amount of time and optimization to be performed. It is worth mentioning that this study is of significant novelty in the CIS field because, except for Type VI secretion systems, very few membrane proteins or complexes responsible for CIS attachment have been identified and studied.

Reviewer #1 (Public review):

Summary:

Carter et al. present the eduWOSM imaging platform, a promising development in open-source microscopy for educational purposes. The paper outlines the construction and setup of this versatile microscope, demonstrating its capabilities through three key examples: single fluorophore tracking of tubulin heterodimers in gliding microtubules, 4D deconvolution imaging and tracking of chromosome movements in dividing human cells, and automated single-particle tracking in vitro and in live cells, with motion classified into sub-diffusive, diffusive, and super-diffusive behaviors.

The paper is well-written and could be strengthened by providing more empirical data on its performance, addressing potential limitations, and offering detailed insights into its educational impact. The project holds great potential and more discussion on long-term support and broader applications would provide a more comprehensive view of its relevance in different contexts.

Strengths:

(1) The eduWOSM addresses a crucial need in education, providing research-quality imaging at a lower cost (<$10k). The fact that it is open-source adds significant value, enabling broad accessibility even in under resourced areas.<br /> (2) There is availability of extensive resources, including a dedicated website, YouTube channel, and comprehensive tutorial guides to help users replicate the microscope.<br /> (3) The compact, portable, and stable design makes it easy to build multiple systems for use in diverse environments, including crowded labs and classrooms. This is further enhanced by the fact multiple kind of imaging experiments can be run on the system, from live imaging to super-resolution imaging.<br /> (4) The paper highlights the user-friendly nature of the platform, with the imaging examples in the paper being acquired by undergrad students.

Weaknesses:

(1) The paper mentions the microscope is suitable not just for education but even for research purposes. This claim needs validation through quantitative comparison to existing research-grade microscopes in terms of resolution, signal-to-noise ratio, and other key metrics. Adding more rigorous comparisons would solidify its credibility for research use, which would immensely increase the potential of the microscope.<br /> (2) The open-source microscope field is crowded with various options catering to hobby, educational, and research purposes (e.g., openFLexure, Flamingo, Octopi, etc.). The paper would benefit from discussing whether any aspects set the eduWOSM platform apart or fulfill specific roles that other microscopes do not.<br /> (3) While the eduWOSM platform is designed to be user-friendly, the paper would benefit from discussing whether the microscope can be successfully built and operated by users without direct help from the authors. It's important to know if someone with basic technical knowledge, relying solely on the provided resources (website, YouTube tutorials, and documentation), can independently assemble, calibrate, and operate the eduWOSM.<br /> (4) Ensuring long-term support and maintenance of the platform is crucial. The paper would benefit from addressing how the eduWOSM developers plan to support updates, improvements, or troubleshooting.

Reviewer #1 (Public review):

Summary:

The authors show that early life experience of juvenile bats shape their outdoor foraging behaviors. They achieve this by raising juvenile bats either in an impoverished or enriched environment. They subsequently test the behavior of bats indoors and outdoors. The authors show that behavioral measures outdoors were more reliable in delineating the effect of early life experiences as the bats raised in enriched environments were more bold, active and exhibit higher exploratory tendencies.

Strengths:

The major strength of the study is providing a quantitative study of animal "personality" and how it is likely shaped by innate and environmental conditions. The other major strength is the ability to do reliable long term recording of bats in the outdoors giving researchers the opportunity to study bats in their natural habitat. To this point, the study also shows that the behavioral variables measured indoors do not correlate to that measured outdoors, thus providing a key insight into the importance of testing animal behaviors in their natural habitat.

Weaknesses:

It is not clear from the analysis presented in the paper how persistent those environmentally induced changes, do they remain with the bats till the end of their lives.

Reviewer #1 (Public review):

Summary:

The manuscript aims to elucidate the impact of a prophage within the genome of Shewanella fidelis on its interaction with the marine tunicate Ciona robusta. The authors made a deletion mutant of S. fidelis that lacks one of its two prophages. This mutant exhibited an enhanced biofilm phenotype, as assessed through crystal violet staining, and showed reduced motility. The authors examined the effect of prophage deletion on several genes that could modulate cyclic-diGMP levels. While no significant changes were observed under in vitro conditions, the gene for one protein potentially involved in cyclic-diGMP hydrolysis was overexpressed during microbe-host interactions. The mutant was retained more effectively within a one-hour timeframe, whereas the wild-type (WT) strain became more abundant after 24 hours. Fluorescence microscopy was used to visualize the localization patterns of the two strains, which appeared to differ. Additionally, a significant difference in the expression of one immune protein was noted after one hour, but this difference was not evident after 23 hours. An effect of VCBC-C addition on the expression of one prophage gene was also observed.

Strengths:

I appreciate how the authors integrate diverse expertise and methods to address questions regarding the impact of prophages on gut microbiome-host interactions. The chosen model system is appropriate, as it allows for high-throughput experimentation and the application of simple imaging techniques.

Weaknesses:

My primary concern is that the manuscript primarily describes observations without providing insight into the molecular mechanisms underlying the observed differences. It is particularly unclear how the presence of the prophage leads to the phenotypic changes related to bacterial physiology and host-microbe interactions. Which specific prophage genes are critical, or is the insertion at a specific site in the bacterial genome the key factor? While significant effects on bacterial physiology are reported under in vitro conditions, there is no clear attribution to particular enzymes or proteins. In contrast, when the system is expanded to include the tunicate, differences in the expression of a cyclic-diGMP hydrolase become apparent. Why do we not observe such differences under in vitro conditions, despite noting variations in biofilm formation and motility? Furthermore, given that the bacterial strain possesses two prophages, I am curious as to why the authors chose to target only one and not both.

Regarding the microbe-host interaction, it is not clear why the increased retention ability of the prophage deletion strain did not lead to greater cell retention after 24 hours, especially since no differences in the immune response were observed at that time point.

Concerning the methodological approach, I am puzzled as to why the authors opted for qPCR instead of transcriptomics or proteomics. The latter approaches could have provided a broader understanding of the prophage's impact on both the microbe and the host.

Reviewer #1 (Public review):

Summary:

The authors showed the presence of Mtb in human liver biopsy samples of TB patients and reported that chronic infection of Mtb causes immune-metabolic dysregulation. Authors showed that Mtb replicates in hepatocytes in a lipid rich environment created by up regulating transcription factor PPARγ. Authors also reported that Mtb protects itself from anti-TB drugs by inducing drug metabolising enzymes.

Strengths:

It has been shown that Mtb induces storage of triacylglycerol in macrophages by induction of WNT6/ACC2 which helps in its replication and intracellular survival, however, creation of favorable replicative niche in hepatocytes by Mtb is not reported. It is known that Mtb infects macrophages and induces formation of lipid-laden foamy macrophages which eventually causes tissue destruction in TB patients. In a recent article it has been reported that "A terpene nucleoside from M. tuberculosis induces lysosomal lipid storage in foamy macrophages" that shows how Mtb manipulates host defense mechanisms for its survival. In this manuscript, authors reported the enhancement of lipid droplets in Mtb infected hepatocytes and convincingly showed that fatty acid synthesis and triacylglycerol formation is important for growth of Mtb in hepatocytes. The authors also showed the molecular mechanism for accumulation of lipid and showed that the transcription factor associated with lipid biogenesis, PPARγ and adipogenic genes were upregulated in Mtb infected cells.

The comparison of gene expression data between macrophages and hepatocytes by authors is important which indicates that Mtb modulates different pathways in different cell type as in macrophages it is related to immune response whereas, in hepatocytes it is related to metabolic pathways.

Authors also reported that Mtb residing in hepatocytes showed drug tolerance phenotype due to up regulation of enzymes involved in drug metabolism and showed that cytochrome P450 monooxygenase that metabolize rifampicin and NAT2 gene responsible for N-acetylation of isoniazid were up regulated in Mtb infected cells.

Weaknesses:

There are reports of hepatic tuberculosis in pulmonary TB patients especially in immune-compromised patients, therefore finding granuloma in human liver biopsy samples is not surprising.<br /> Mtb infected hepatic cells showed induced DME and NAT and this could lead to enhanced metabolism of drug by hepatic cells as a result Mtb in side HepG2 cells get exposed to reduced drug concentration and show higher tolerance to drug. The authors mentioned that " hepatocyte resident Mtb may display higher tolerance to rifampicin". In my opinion higher tolerance to drugs is possible only when DME of Mtb inside is up regulated or the target is modified. Although, in the end authors mentioned that drug tolerance phenotype can be better attributed to host intrinsic factors rather than Mtb efflux pumps. It may be better if the Drug tolerant phenotype section can be rewritten to clarify the facts.

Reviewer #1 (Public review):

Summary:

This is an interesting theoretical study examining the viability of Virtual Circular Genome (VCG) model, a recently proposed scenario of prebiotic replication in which a relatively long sequence is stored as a collection of its shorter subsequences (and their compliments). It was previously pointed out that VCG model is prone to so-called sequence scrambling which limits the overall length of such a genome. In the present paper, additional limitations are identified. Specifically, it is shown that VCG is well replicated when the oligomers are elongated by sufficiently short chains from "feedstock" pool. However, ligation of oligomers from VCG itself results in a high error rate. I believe the research is of high quality and well written. However, the presentation could be improved and the key messages could be clarified.

(1) It is not clear from the paper whether the observed error has the same nature as sequence scrambling<br /> (2) The authors introduce two important lengths LS1 and LS2 only in the conclusions and do not explain enough which each of them is important. It would make sense to discuss this early in the manuscript.<br /> (3) It is not entirely clear why specific length distribution for VCG oligomers has to be assumed rather than emerged from simulations.<br /> (4) Furthermore, the problem has another important length, L0 that is never introduced or discussed: a minimal hybridization length with a lifetime longer than the ligation time. From the parameters given, it appears that L0 is sufficiently long (~10 bases). In other words, it appears that the study is done is a somewhat suboptimal regime: most hybridization events do not lead to a ligation. Am I right in this assessment? If that is the case, the authors might want to explore another regime, L0<br /> Strengths:

High-quality theoretical modeling of an important problem is implemented.

Weaknesses:

The conclusions are somewhat convoluted and could be presented better.

Reviewer #1 (Public review):

Summary:

Persistence is a phenomenon by which genetically susceptible cells are able to survive exposure to high concentrations of antibiotics. This is especially a major problem when treating infections caused by slow growing mycobacteria such as M. tuberculosis and M. abscessus. Studies on the mechanisms adopted by the persisting bacteria to survive and evade antibiotic killing can potentially lead to faster and more effective treatment strategies.

To address this, in this study, the authors have used a transposon mutagenesis based sequencing approach to identify the genetic determinants of antibiotic persistence in M. abscessus. To enrich for persisters they employed conditions, that have been reported previously to increase persister frequency - nutrient starvation, to facilitate genetic screening for this phenotype. M.abs transposon library was grown in nutrient rich or nutrient depleted conditions and exposed to TIG/LZD for 6 days, following which Tn-seq was carried out to identify genes involved in spontaneous (nutrient rich) or starvation-induced conditions. About 60% of the persistence hits were required in both the conditions. Pathway analysis revealed enrichment for genes involved in detoxification of nitrosative, oxidative, DNA damage and proteostasis stress. The authors then decided to validate the findings by constructing deletions of 5 different targets (pafA, katG, recR, blaR, Mab_1456c) and tested the persistence phenotype of these strains. Rather surprisingly only 2 of the 5 hits (katG and pafA) exhibited a persistence defect when compared to wild type upon exposure to TIG/LZD and this was complemented using an integrative construct. The authors then investigated the specificity of delta-katG susceptibility against different antibiotic classes and demonstrated increased killing by rifabutin. The katG phenotype was shown to be mediated through the production of oxidative stress which was reverted when the bacterial cells were cultured under hypoxic conditions. Interestingly, when testing the role of katG in other clinical strains of Mab, the phenotype was observed only in one of the clinical strains demonstrating that there might be alternative anti-oxidative stress defense mechanisms operating in some clinical strains.

Strengths:

While the role of ROS in antibiotic mediated killing of mycobacterial cells have been studied to some extent, this paper presents some new findings with regards to genetic analysis of M. abscessus susceptibility, especially against clinically used antibiotics, which makes it useful. Also, the attempts to validate their observations in clinical isolates is appreciated.

Weaknesses:

- Fig. 3 - 5 of the hits from the transposon screen were reconstructed as clean deletion strains and tested for persistence. However, only 1 (katG) gave a strong and 1 (Mab_1456c) exhibited a minor defect. Two of the clones did not show any persistence phenotype (blaR and recR) and one (pafA) showed a minor phenotype, however it was not clear if this difference was really relevant as the mutant exhibited differences at Day 0, prior to the addition of antibiotics. Considering these results from the validation, the conclusion would be that the Tn-seq approach to screen persistence defects is not reliable and is more likely to result in misses than hits.

- Fig 3 - Why is there such a huge difference in the extent of killing of the control strain in media, when exposed to TIG/LZD, when compared to Fig. 1C and Fig. 4. In Fig. 1C, M. abs grown in media decreases by >1 log by Day 3 and >4 log by Day 6, whereas in Fig. 3, the bacterial load decreases by <1 log by Day 3 and <2 log by Day 6. This needs to be clarified, if the experimental conditions were different, because if comparing to Fig. 1C data then the katG mutant strain phenotype is not very different.