Reviewer #3 (Public Review):

This study highlights the functional consequences of combined genomic losses of CIC and ERF which results in the activation of ETV1, in the absence of the canonical fusion event involving TMPRSS2 in a subset of prostate cancer. ETV1 is an oncogenic driver of cell growth and metastatic behaviour in many cancer types including prostate cancer. The experiments performed provided tantalizing evidence on the biological and functional consequences of combined losses of CIC and ERF and appeared to support the findings of the mined publicly available cancer genomic datasets.

The manuscript could be improved by providing evidence of proteomic interactions between CIC and ERF proteins in the form of immune-precipitation and Western protein blots. The authors had provided predominantly genomic, transcriptomic, and functional data. In most parts, the manuscript is logical and thorough and leveraged available genomic data. This is followed by genomic-functional experimentations. Given the postulate of co-operativity between CIC and ERF, it would be logical to investigate their potential proteomic interactions.

Reviewer #1 (Public Review):

The current study by Sakabe et al identifies an adrenergic signaling mechanism controlling cardiac regenerative capacity in mice. Using pharmacological and genetic loss-of-function studies, the authors demonstrate that inhibition of beta adrenergic signaling prolongs the cardiac regenerative window in neonatal mice. The study mechanistically connects several signaling pathways that are known to control cardiomyocyte proliferation including adrenergic signaling, G-proteins and the Hippo/Yap pathway. The results are potentially clinically significant given the widespread use of beta blockers in heart failure management.

Strengths:<br /> This is an impressive body of work that addresses an important and largely unresolved question in the field regarding signaling mechanisms controlling cardiac regeneration in the postnatal period in mammals. Through pharmacological and conditional genetic loss-of-function studies the authors provide several lines of evidence implicating the beta adrenergic signaling and the Hippo/Yap pathway in cardiomyocyte proliferation. The conditional genetic loss-of-function studies are a particular strength of the manuscript and provide strong support for the Gas/Yap-dependent nature of the cardiomyocyte proliferative response to beta adrenergic blockade.

Weaknesses:<br /> Although the study clearly implicates beta adrenergic signaling in the developmental regulation of cardiomyocyte proliferative potential, it is unclear whether the protective effects observed following myocardial infarction are due to cardiac regeneration or alternative mechanisms (e.g. immunomodulation, inhibition of cell death, angiogenesis, reduced contractile loading, improved coronary flow, etc). Induction of cardiomyocyte proliferation following administration of metoprolol in neonatal mice is fairly modest (~0.3% pH3-positive cardiomyocytes) and it seems unlikely that such a small number of proliferating cardiomyocytes could mediate such marked effects on cardiac function and fibrosis post-MI. In the absence of definitive data demonstrating that improvements in cardiac function are due to induction of cardiomyocyte proliferation (and by inference cardiac regeneration), such conclusions should be tempered. In addition, it is unclear why beta blocker studies were not conducted in adult mice (rather than P7/P14 mice) to determine whether inhibition of this pathway is sufficient to induce adult cardiomyocyte cell cycle re-entry and regeneration post-MI.

Reviewer #3 (Public Review):

The authors study mammalian heart regeneration and study the connection between Yap and β-adrenergic receptor (β-AR) blockade. Interestingly, metoprolol robustly enhanced cardiomyocyte proliferation and promoted cardiac regeneration post myocardial infarction, resulting in reduced scar formation and improved cardiac function. The conclusion was also supported by genetic deletion of Gnas. CMs had an immature cell state with enhanced activity of Hippo-effector YAP. They also find that increased YAP activity is modulated by RhoA.

Overall, the data are supportive of the conclusions and this may provide new insight into treating heart disease. The final mechanisms connecting Hippo signaling to Rho activity remain incompletely defined.

Reviewer #1 (Public Review):

This study reveals for the first time the involvement of Prdx5 in the differentiation, of osteoclast and osteoblast, which are key cellular components of bone remodelling. This novel function of Prdx5 was shown to be mediated by specific binding and regulating the function of hnRNPK, which acts as a transcription factor in bone remodelling. Moreover, Prdx5 was shown to be colocalized with hnRNPK during osteoblast differentiation. In support of this findings, the authors also reported that mice deficient for Prdx5 expression (Prdx6KO) showed osteoporosis-like phenotypes, which was associated with reduced osteoblast and increased osteoclast differentiation.

It is important to emphasise that the osteoporotic phenotypes were only demonstrated in males, but not in female mice. The observed phenotypes were not hormone-dependent, as no significant differences in examined bone parameters were observed between wild type and Prdx5KO female mice in an ovariectomy-induced osteoporosis model. However, women over 50 have a four times higher rate of osteoporosis compared with men, and the role of testosterone in the development of osteoporosis in Prdx5KO mice should be investigated. It is known that the osteoporosis is increased in men with low level of testosterone.

It is misleading for authors to state throughout the manuscript that osteoporotic phenotypes are observed in Prdx5KO mice, while it is only observed in male mice.

Reviewer #1 (Public Review):

In this manuscript, Williams et al. present a mainly bioinformatic analysis of HERV-K(HML-2) related retroviruses in Old World Monkey species. HML-2 viruses represent the youngest clade of human retroviruses and have also been shown in other primates. This manuscript is focused on identifying and characterizing HML-2-like proviruses in rhesus macaques. A majority of the identified proviruses (106/145) contain sequences that the authors determine were derived from ancient recombination with a HERV-K(HML-8)-related virus (likely after the OWM/great ape split). They further identify similar sequences in white-cheeked gibbons, which they conclude suggest interspecies transmission. Several of the viruses have deletions that disrupt the Rec-Response-Element (RcRE), which interacts with the viral Rec protein.This has previously been shown to be essential to overcome the host cell restrictions to nucleo-cytoplasmic export of unspliced/partially spliced mRNA. Export of this kind of RNA is essential for viral structural protein expression and genome packaging. The authors suggest that sequences derived from HML-8 contain a Constitutive Transport Element (CTE) that replaced the Rec-RcRE export system in the recombinant proviruses.

Strengths:<br /> This manuscript presents a comprehensive bioinformatic analysis of HML-2 like HERV-K proviruses present in rhesus macaques. Several of the authors are well-recognized experts in this kind of analysis and have published extensively in this area. They present convincing evidence (based on analysis of age, intact Open Reading Frames (ORFs), as well as insertional polymorphism) for recent and maybe even ongoing infectious activity of these viruses in rhesus macaques. The identification of the ancient recombination with HML-8 sequences is interesting and allows the authors to reasonably suggest that this event happened after the split of old world monkeys and great apes. The analysis led to the discovery of LTR deletions in recombinant proviruses. These were verified using PCR on genomic DNA and since the deletions included parts of the RcRE, this would indeed suggest that Rec/RcRE function is absent. This was also supported by the lack of function in conjunction with Rec, using a previously described reporter system, where GFP expression suggests the export of unspliced mRNA. However, when sequences from the 3' LTR (derived from HML-8) in the recombinant proviruses were tested in this reporter, GFP protein expression was observed in the absence of Rec. Based on this, the authors suggest that these sequences contain a Constitutive Transport Element (CTE). This is a potentially very interesting finding.

Weaknesses:

With the exception of the PCR analysis and the reporter assays, the manuscript does not contain any experiments or attempts to analyze current expression from any of the identified proviruses. No long-read RNASeq or other RNA analysis on cytoplasmic RNA was performed, nor any experiments to show that proteins are indeed expressed. The findings of a potential CTE are interesting, but the sequences that were appended to the reporter construct are much longer than previously identified CTEs. No data were presented to indicate whether this sequence show similarity to previously identified CTEs and no experiments to show whether this sequence functionally interacts with Nxf1, the protein shown to interact with previously identified bona fide CTEs. Also, since nucleo-cytoplasmic export was not directly analyzed, it remains possible that the sequences that were inserted into the reporter contained splice sites that would allow the RNA to be spliced "downstream" of the GFP gene, allowing the export of a "spliced" GFP mRNA.

Reviewer #1 (Public Review):

In their manuscript, the authors compared the CHC quality in around 50 different, including distantly related fruit fly species that occupy diverse habitats. Based on correlative data (produced by appropriate statistical analyses), they hypothesise that methyl-branched alkanes (mbCHC) might be decisive in resistance against desiccation in these species. They confirm previously published results that rather than the amount of CHC their quality plays an important role in desiccation resistance. Moreover, their results indicate that desiccation resistance increases with longer mbCHCs. In mbCHC coating experiments, the authors tentatively support their hypothesis.

Overall, the data are impressive, and their statistical analyses (Pearson's method, etc) are performed accurately yielding convincing results. These results, however, are not as strong as the statement in the abstract that longer mbCHCs are a "key determinant" of desiccation resistance. Admittedly, the authors' wording is very and appropriately careful about this issue.

One major weakness of this work is the coating experiments. The authors simply incubate Drosophila melanogaster flies with mbCHC and observe that they become more resistant to drought. Along with this "adding" experiment, "replacement" experiments should be carried out, ie add the mbCHC to flies without CHCs. Moreover, control experiments with CHCs with the same chain lengths but without branched methyl groups should be added. This point is crucial as the authors tempt with it to render their correlative data causative.

A second point that needs more attention regarding the discrepancies between males and females (e.g Fig. 3 & 5). These data are not discussed.

Reviewer #3 (Public Review):

This study utilizes 46 species of Drosophila and 4 closely related species to try and determine the relative role of specific hydrocarbons on desiccation resistance. The use of many species of Drosophila that have variations in hydrocarbon profiles and variations in natural desiccation resistances allowed the researchers to draw conclusions about the relative role of specific hydrocarbons contributing to preventing water loss through the cuticle. By using a statistical package they were able to conclude that methyl-branched hydrocarbons are the most important in those species that were more desiccation resistant. This is not surprising since a previous study has shown that 2 methyl-branched hydrocarbons have the highest melting temperatures. In addition, it seems that desiccation resistance also involves other factors since some species that had lower desiccation rates had similar amounts of methyl branched hydrocarbons. It is also difficult to extrapolate to other insects that have a variety of lipids on their cuticular surface. Probably most insects will have hydrocarbons but some have a variety of other lipids on the cuticular surface that will contribute to preventing desiccation. The use of Drosophila species in this study is fortuitous because apparently only hydrocarbons are found on the cuticular surface.

Reviewer #1 (Public Review):

According to the space-time wiring hypothesis proposed by (Kim, Greene et al. 2014), the BC-off SAC circuit mimics the structure of a Reichardt detector; BCs closer to SAC soma have slower dynamics (they can be more sustained, have a delay in activation or slower rise time), while BCs further away are more transient. Later studies confirmed the connectivity and expanded the model on SACs (Ding, Smith et al. 2016, Greene, Kim et al. 2016). However, physiological studies that used somatic recordings to assess the BC properties at different dendritic distances were inconclusive (Stincic, Smith et al. 2016, Fransen and Borghuis 2017). Here, the authors used iGluSnFR, a glutamate sensor to measure the signals impinging on SAC dendrites. Their experimental findings align with the space-time wiring hypothesis, revealing sustained responses closer to SAC soma (mediated by prolonged release from type 7 BCs, and only slightly affected by amacrine cells), which according to their simulated SAC should produce a substantial increase in direction selectivity (DS).

I find the work to be clear and well presented. However, I do have some reservations with the findings:

Main points:<br /> 1. Very low number of cells examined in the key experiment presented in the first figure. The authors used a viral approach to express flex- iGluSnFR in SACs in Chat-Cre mice. Sometimes (apparently twice) the construct was expressed in individual SACs - this is a very underpowered experiment! The low number of successes precludes adequately judging the validity of the findings.<br /> 2. The model doesn't represent key known properties of BC-SACs and the interactions within SAC dendrites. First, the authors decided to construct a ball and stick model that doesn't consider the dendritic morphology of the starburst cell. A stimulus moving over a SAC is expected to engage multiple dendrites with complex spatiotemporal patterns that are expected to have a substantial effect on the voltages recorded on the investigated dendrite (Koren, Grove et al. 2017). For example, the dendrites in the orthogonal orientation will be activated at about the same time as the proximal dendrites; how such strong input will affect dendritic integration is unclear but should be taken into account in the model. Second, the authors assume a similar peak BC drive between proximal and distal inputs. However, a recent study found an enhanced glutamate release from proximal BCs, mediated by cholinergic SAC drive ((Hellmer, Hall et al. 2021); not cited). How different release amplitude would affect the conclusions of the model?<br /> 3. Another reason for including an accurate dendritic morphology is in the differences in the number of BCs that target a cell. Because SAC dendrites cover the entire receptive field area, type 7 BCs, which occupy the proximal third of the dendrites (Ding, Smith et al. 2016, Greene, Kim et al. 2016), are expected to cover only 11% of the area covered by SAC dendrites (1/3 x 1/3 = 1/9) and correspondingly mediate just 11% of the BC drive. A non-bifurcating model presented here would dramatically overrepresent their contribution to SAC responses.<br /> 4. (Fransen and Borghuis 2017) found that off-SACs have a more pronounced distinction in the time to peak than on-SACs. I found it surprising that given the large body of work demonstrating the effectivity of the viral approach in expressing iGluSnFR in off BC (Borghuis, Marvin et al. 2013, Franke, Berens et al. 2017, Szatko, Korympidou et al. 2020, Gaynes, Budoff et al. 2021, Strauss, Korympidou et al. 2021), that the authors did not compare between on and off SAC populations.<br /> 5. Recent work (Gaynes, Budoff et al. 2021) suggests that BCs' responses to motion and to static flashes have distinct dynamics. However, the current manuscript tests responses to flashed stationary stimuli experimentally, and then combines them in a simulation modeling a moving stimulus. This potential limitation of the study should at least be discussed.

Reviewer #3 (Public Review):

In the study "Spatiotemporal properties of glutamate input support direction selectivity in the dendrites of retinal starburst amacrine cells", Srivastava, deRosenroll, and colleagues study the role of excitatory inputs in generating direction selectivity in the mouse retina. Computational and anatomical studies have suggested that the "space-time-wiring" model contributes to direction-selective responses in the mammalian retina. This model relies on temporally distinct excitatory inputs that are offset in space, thereby yielding stronger responses for motion in one versus the other direction. Conceptually, this is similar to the Reichardt detector of motion detection proposed many decades ago. So far, however, there is little functional evidence for the implementation of the space-time-wiring model.

Here, Srivastava, deRosenroll and colleagues use local glutamate imaging in the ex-vivo mouse retina combined with biophysical modeling to test whether temporally distinct and spatially offset excitatory inputs might generate direction-selective responses in starburst amacrine cells (SACs). Consistent with the space-time-wiring model, they find that glutamatergic inputs at proximal SAC dendrites are more sustained than inputs at distal dendrites. This finding was consistent across different sizes of stationary, flashed stimuli. They further linked the sustained input component to the genetically identified type 7 bipolar cell and showed that the difference in temporal responses across proximal and distal inputs was independent of inhibition, but rather relied on excitatory interactions. By estimating vesicle release rates and building a simple biophysical model, the authors suggest that next to already established mechanisms like asymmetric inhibition, excitatory inputs with distinct kinetics contribute to direction-selective responses in SACs for slow and relatively large stimuli.

In general, this study is well-written, the data is clearly presented and the conclusion that (i) the temporal kinetics of excitatory inputs varies along SAC dendrites and that (ii) this might then contribute to direction selectivity is supported by the data. The study addresses the important question of how excitation contributes to the generation of direction-selective responses. There have been several other studies published on this topic recently, and I believe that the results will be of great interest to the visual neuroscience community.

However, the authors should address the following concerns:<br /> - They should demonstrate that differences in response kinetics between proximal and distal dendrites are unrelated to differences in signal-to-noise ratio.<br /> - To demonstrate consistency across recordings/mice, the authors should indicate data points from different recordings (e.g. Fig. 2C).<br /> - The authors mention in the introduction that the space-time-wiring model is conceptually similar to other correlation-type motion detectors that have been experimentally verified in different species. It would be great to expand on the similarity and differences of the different mechanisms in the Discussion, especially focusing on Drosophila where experimental evidence at the synaptic level exists.<br /> - The authors use stationary spot stimuli of different sizes to characterize the response kinetics of excitatory inputs to SACs. I suggest the authors add an explanation for choosing only stationary stimuli for studying the role of excitatory inputs in direction selectivity/motion processing. In addition, the authors use simulated moving edges to stimulate the model bipolar cells. They should provide details about the size of the stimulus and the rationale behind using this size, given their previous results.<br /> - Using the biophysical model, the authors show that converting sustained bipolar cell inputs to transient ones reduces direction selectivity in SACs. I suggest the authors also do the opposite manipulation/flip the proximal and distal inputs or provide a rationale why they performed this specific manipulation.<br /> - In each figure, the authors should note whether traces show single trial responses or mean across how many trials. If the mean is presented (e.g. Suppl. Fig. 2a), the authors should include a measure of variability - either show single ROIs in addition and/or add an s.d. shading to the mean traces.

Reviewer #1 (Public Review):

The manuscript "Interplay between PML NBs and HIRA for H3.3 dynamics following type I interferon stimulus" by Kleijwegt and colleagues describes a study that's set out to explore the details of the PML-HIRA axis in H3.3 deposition at ISGs upon IFN-I stimulation. First, the authors establish that HIRA colocalized at PML NBs upon TNFa and TNFb treatment. This process is SUMO-dependent and facilitated by at least one of the identified SIM domains of HIRA. Next, the authors set out to determine whether interferon responsive genes (ISGs) are dependent on HIRA or PML. By knocking-down either HIRA or PML, only an effect on ISGs was observed when PML was knocked down. In fact, immune-FISH showed that PML NBs are in close proximity of ISGs upon TNFb treatment. To address the histone chaperone function of HIRA, the deposition of the replication-independent H3.3 on ISGs is tested. In specific, the enrichment of H3.3 across the ISG gene body. ChIP-seq data (Fig 5B) showed an enrichment around the TES, whereas qPCR (Fig 5A) showed less convincing enrichment (for details see below). When either HIRA or PML are knocked down, a mild loss of H3.3 enrichment was observed (Fig 5E). Interestingly, when HIRA is sequestered away from PML NBs by Sp100, an increased enrichment of H3.3 was observed. To understand the interplay between H3.3 deposition and HIRA's role in this process in the presence of PML NBs, H3.3 was overexpressed. Two population of cells were observed: low or high levels of H3.3. In the former, HIRA formed foci and the latter, HIRA did not form foci. Surprisingly, when HIRA is overexpressed, PML NBs form in the absence of TNFb. Finally, a two-sided model is proposed, where PML NBs is required for ISG transcription promoting H3.3 loading. The second side is that PML NBs function as a "storage center" for HIRA to regulate its availability.

Overall, it the model is intriguing, but the data presented seems insufficient to support the current claims.

Major concerns:<br /> - The suggested function of HIRA at the PML NBs as storage is interesting. Ideally, this would be tested by real-time single molecule tracking.

- The link between PML NBs containing HIRA and H3.3 deposition is very intriguing and indeed the ChIP-seq data shown in Figure 5B shows a clear increase in the H3.3 signal around the TES. This distribution is very intriguing as recent work (Fang et al 2018 Nat Comm) showed that H3.3 deposition across the gene body was diverse and dynamic. Ideally, the qPCR of some select ISGs would confirm the ChIP-seq data. Here a more complex picture emerges. Just as with the ChIP-seq, a modest decrease of H3.3 at the TSS was observed, but only in 2 of the 3 genes shown is H3.3 enriched at the TES and only in 1 gene (ISG54) is H3.3 enriched at the gene body. As qPCR is later used in the manuscript (Fig 5E and 5G), it is essential that the results of two different techniques give similar results. With regards to Fig 5E and 5G, it is unclear why certain gene regions are shown, but not others.<br /> Overall, the link between HIRA and PML in H3.3 loading is only mildly affected (Fig 5E and 5F). The conclusion that HIRA and PML are essential (Page 12, line 8) is not represented by the presented data. The authors propose that DAXX could play a role. Indeed, work on another H3 variant, CENP-A, showed that non-centromeric localization is dependent on both HIRA and DAXX (Nye et al 2018 PLoS ONE). It would be interesting to learn if a double knock-down of HIRA and DAXX can prevent the enrichment of H3.3 at TES of ISGs upon TNFb treatment.

- In Figure 6B, two versions of HIRA are overexpressed and the authors conclude that the number of PML NBs goes up. Earlier in the manuscript, the authors showed that PML NB formation upon IFNb exposure brings HIRA into the PML NBs via a SUMO-dependent mechanism. Is overexpression of HIRA and its accumulation in PML NBs also SUMO-dependent or SUMO-independent? Overexpressing the SIM mutants from Figure 3F would address this question. In addition, the link between the proposed HIRA being stored at PML NBs could be strengthened by overexpressing HIRA and see at both short and late time points whether H3.3 is enriched on ISG genes.

- BJ cells are known to senesce rather easily. Did the authors double-check what fraction of their cells were in senescence and whether this correlated with the high or low expression of ectopic H3.3?

- In Figure 6 - figure supplement D, it appears that the levels of HIRA go up upon TSA and IFNb treatment. Rather than relying on visual inspection, ideally, all Western blots should be quantified to confirm the assessment that protein levels are not affected by different experimental procedures.

Reviewer #1 (Public Review):

Neural circuits of the fruit fly mushroom body provide an interesting system to study molecular processes underlying learning and formation of memories since the input-output relationship of the circuit is quite well characterized and - importantly- genetic tools to manipulate specific circuit components are available. The current manuscript focuses on the role of different subunits of the nicotinergic Acetylcholine receptors.

The authors use a series of state-of-the-art techniques and several elegant - and partly innovative, explorative - approaches to address a specific set of neurons as models (the MBONs M4/M6), that are relevant for appetitive memories. The logic of the manuscript is overall well developed, and the experiments provided are widely in support of the model the authors propose. They first corroborate that synaptic transmission of M4/M6 is critical for appetitive memories and subsequently test different subunits of the nicotinergic Acetylcholine receptors using RNAi. Interestingly, only the alpha-5 subunit shows learning defects (acquisition or induction), while alpha-1,2, and 5 (and Dlg) show defects after 3 hours (what the authors call "memory expression"). This result indicated the differential requirement for acquisition versus that expression.

The subsequent and particularly elegant and intriguing set of experiments aims to recapitulate the processes while circumventing a direct synaptic transmission from the KCs. The authors therefore optogenetically activate DANs, while blocking KCs neurotransmitter release and measure neuronal activity in the MBONs. Only when ACh is locally applied an effect of synaptic facilitation can be observed. While this experiment is not particularly critical in the context of the current manuscript it provides a very different, complementary support for the proposed overall model.

In order to gain insight into the function of the alpha-5 and alpha-2 subunits, the authors next investigated the expression of these genes and report non-uniform patterns between the lobe systems, presumably by using GFP fusion reporters. A weakness in this section is that the technical details are not well described and thus the impact of these results remains a bit elusive. The results indicate that alpha-2 is somehow genetically "downstream" of alpha-5 and Dlg. While many parts of the manuscript are of great impact and clear, this notion - even though extremely interesting - may actually be one of the weakest parts, since no explanation for the phenomenon is provided. One would assume that dopamine signaling and coincidence detection may be involved. It is however true that the authors conceptually take this up to some degree in the discussion, particularly the point that alpha-2 may be a molecular central point to switch.

Using Calcium imaging in vivo the authors show that the physiological formation of a memory trace in M4/6 shows the expected dynamics in an alpha-2-dependent fashion and similarly that alpha-2 itself (by photobleaching) shows the expected expression/localization dynamics.

The final section is indeed an important extension and addresses the generality of the alpha-5 to alpha-2 transition by investigating familiarity rather than associative learning. The results provided are in line that this mechanism appears to be general, a point also taken up in the discussion. What I felt was especially refreshing in the discussion section is the global comparison of NMDA/AMPA as a concept and possibilities of how this task may be resolved in other systems using other transmitters, again maybe not at the molecular depth that may have added an explorative touch.

Reviewer #3 (Public Review):

In invertebrates, learning-dependent plasticity was reported to take place predominantly in presynaptic neurons. In Drosophila appetitive olfactory learning, cholinergic synapses between presynaptic Kenyon cells and postsynaptic MBONs undergo behaviourally relevant associative plasticity, and it was shown to reside largely in Kenyon cell output sites. This study provided several lines of evidence for postsynaptic plasticity in MBONs. The authors nicely showed the requirement of Kenyon cell output during training, strongly suggesting that behaviourally relevant associative plasticity also resides downstream of Kenyon cell output. This is further supported by impaired appetitive memory by downregulating nAChR subunits (a2, a5) and scaffold protein Dlg in specific MBONs. Live imaging experiments demonstrated that the learning-dependent depression in M4-MBON was reduced upon knocking down the a2 nAChR subunit. Using in-vivo FRAP experiments, the authors showed recovery rates of nAChR-a2::GFP were altered by the co-application of olfactory stimulation and DA. All these lines of evidence point to the significance of nAChR subunits in MBONs for postsynaptic plasticity.

On the technical side, this study achieved a very high standard, such as the measurement of low-expressed receptor mobility by in-vivo FRAP. The authors conducted a wide array of experiments for collecting data supporting postsynaptic mechanisms. The downside of this multitude is somewhat compromised coherence. To give an example, the authors duplicated many behaviour and imaging experiments in different MBONs for non-associative learning (Fig. 7 and 8), which is primarily out of the scope of this paper (cf. title).

Reviewer #1 (Public Review):

This paper describes an "ERAD-like" pathway for the turnover of the SUN2 protein. In this pathway, ubiquitylation of SUN2 in the nucleoplasm by the SCFbTRCP ubiquitin ligase leads to extraction of the membrane protein by p97 for delivery to the proteasome. This process involves phosphorylation of non-canonical degrons on SUN2 by CK2, which was identified using a genome-wide crispr screening approach. The CTDNEP1 phosphatase acts to reverse phosphorylation and stabilize SUN2. Non-degradable forms of SUN2 promote altered nuclear architecture and a delay in double-strand break repair. The conclusions are based on strong biochemical and cell biological data. The paper sets the stage for further analysis of how defects in SUN2 degradation alter additional nuclear processes.

Reviewer #3 (Public Review):

The manuscript by Krshnan et al. reports a cellular mechanism akin to the endoplasmic reticulum-associated degradation (ERAD) that degrades SUN2, a nuclear inner membrane protein. The authors previously identified the Asi ubiquitin ligase complex that mediates the degradation of inner nuclear membrane proteins in budding yeast. In this manuscript, they identified the SCF β TrCP, and SCF as another ligase that regulates the ubiquitination and degradation of SUN2 in mammalian cells. The key findings include the identification of a substrate recognition motif that appears to undergo casein kinase (CK) dependent phosphorylation. Mutagenesis studies show that mutants defective in phosphorylation are stabilized while a phosphor-mimetic mutant is more unstable. They further show that the degradation of SUN2 requires the AAA ATPase p97, which allows them to draw the analogy between SUN2 degradation and Vpu-induced degradation of CD4, which occurs on the ER membrane via the ERAD pathway. Lastly, they show that the stability of endogenous SUN2 is regulated by a phosphatase and that over-expression of a non-degradable SUN2 variant disrupts nuclear envelope morphology, cell cycle kinetics, and DNA repair efficiency. Overall, the study dissects another example of inner nuclear envelope protein turnover and the involvement of a pair of kinase and phosphatase in this regulation. The data are of extremely high quality and the manuscript is clearly written. That being said, the following questions should be addressed to improve the robustness of the conclusions and to avoid potential misinterpretation of the data.

1. Since SUN2 is normally incorporated into a SUN2-SYNE2-KASH2 LINC heterohexamer complex, the authors should be cautious with the use of over-expressed SUN2 in this study. Over-expressed SUN2 is expected to stay mostly as unassembled molecules and thus is likely degraded by a protein quality control mechanism that targets unassembled proteins. Consistent with this possibility, CK2 has been implicated in the regulated turnover of aggregation-prone proteins (Watabe, M. et al., JCS 2011). This mechanism would be potentially distinct from the one proposed for endogenous SUN2 degradation.<br /> 2. Certain conclusions appear to be an overstatement. This is particularly the case for the title, which implies that SUN2 is a protein that undergoes regulated turnover (under certain physiological conditions). Given that CK2 is a constitutive kinase and that the authors have not identified the conditions under which the activity of CTDNEP1 is regulated, it is premature to make such a conclusion.<br /> 3. Likewise, the demonstration of the impact of SUN2 accumulation on different cellular pathways mainly relies on the over-expression of a non-degradable SUN2 mutant. Whether similar defects could be seen when the degradation of endogenous SUN2 is blocked remains an open question.

Reviewer #1 (Public Review):

In this study, the authors use a powerful mix of histone mutations, biochemistry, and proteomics to show that histones H3 and H4 are imported into the nucleus as monomers by Imp5 where they are transferred to NASP and HAT1-RBBP7 respectively. This challenges the standing paradigm that H3 and H4 are imported into the nucleus as heterodimers.

Reviewer #3 (Public Review):

The goal of this paper is to describe how newly synthesized histones are imported into the nucleus.

Prior biochemical purifications suggest that H3-H4 dimers fold in the cytoplasm, are regulated by the sNASP histone chaperone, and translocate to the nucleus in association with the ASF1 histone chaperone and the importin-4 (Imp4) karyopherin. However, using an imaging-based approach, the authors previously showed that histones H3 and H4 can be imported into the nucleus as monomers.

Here, the authors show that new, cytoplasmic H3.1 and H4 monomers are bound by HSPA8 and importin-5 (Imp5). Imp5 then translocates monomeric histones into the nucleus and transfers H3.1 to sNASP. They further propose that the previously observed cytosolic H3-H4 dimers are not new histones but rather old nucleosomal histones that diffuse into the cytoplasm, which are then re-imported via Imp4. Therefore, folding of H3-H4 dimers exclusively occurs in the nucleus.

The authors certainly provide compelling evidence that monomeric histones are imported into the nucleus via Imp5. Constitutively monomeric histone mutants co-purified with Imp5 and the association was recapitulated in vitro. A wide range of exciting techniques is used to address how monomeric histones are handled in cells (i.e., biochemical, FRAP, imaging of cytoplasmic tethered and released histones, proximity-dependent protein labeling, etc). The aim of finding how monomeric histones are imported into the nucleus is certainly attained. More data could however support some of the conclusions regarding the association of histones to ASF1 and Imp4 and whether they truly exclusively represent evicted nucleosomal histones that diffused out of the nucleus.

Otherwise, the data shown here is certainly important for the field, as it provides an explanation of how monomeric histones are handled in the cytoplasm.

Reviewer #1 (Public Review):

Wang et al. suggested that the most prominent gene downregulated in postmenopausal osteoporosis patients is miR-27a. Data suggested that removing miR27a resulted in considerable bone loss due to disturbed remodeling. Their findings further imply that miR-27a is unnecessary for osteoblast differentiation and bone formation. This work presents good data to show that miR-27a is necessary for controlling bone resorption. The deletion of miR-27a in mice increases the number of osteoclast cells as well as key parameters for bone resorption. As a result, directing miR-27a to bone resorption surfaces using synthetic drugs such as bisphosphonates or osteoclast-targeted molecules such as acid octapeptides containing aspartic acid can be crucial therapeutics for future use and miRNAs can very well be the next wave of future therapeutics.

Reviewer #3 (Public Review):

The authors show miR-23a and miR-27a as an important regulator of bone homeostasis. They observed that miR 23a and miR27a regulates osteoclast function and loss of miR 23a and miR27a causes severe osteopenia conditions in mice without affecting osteoblast function. It has been already reported that miR27a regulates osteoclast function and inhibits osteoclast mediated bone resorption and F action formation (Guo L, et al). But the novelty of this manuscript is that single deletion of miR27a causes severe osteoporosis without affecting cortical bone. Reports suggest that p62 is an important regulator of osteoclastogenesis and deficiency of p62 impaired osteoclast differentiation. In paper, authors established a link between miR27a and p62 in osteoclast cells which could be a potential target for treatment of bone related disorders. Importantly, the mechanism of miR27a-p62 is not well explored in osteoclast cells.

Reviewer #1 (Public Review):

RPG was identified as a mutant with poor nodulation in 2008, but its activity was unclear. Interest in its role was rejuvenated by a phylogenetic approach where RPG was one of only three genes that have been consistently lost in non-nodulating species in the Fagales/Fabales/Cucurbitales/Rosales families. In this careful genetic and imaging-based analysis of the role of the RPG gene in nodulation, the authors use transient and stable genetic transformation in nodulating Medicago roots to clarify the subcellular localization of RPG and its relationship to infection thread maintenance along with two other better characterized symbiosis proteins VAPYRIN (VPY) and LUMPY INFECTION (LIN). Detailed phenotypic analysis of two RPG mutants indicates that RPG is required to sustain polarization of the membrane at the advancing tip of the IT, and without RPG, the plant microtubule cytoskeleton organization, nuclear position, and localization of VPY and the cell wall modifying enzyme NODULE PECTATE LYASE all differ from normal.

The model that emerges and that is well supported by the presented data is that RPG behaves as a scaffold to convert/customize the cellular machinery that organizes cytoskeleton and membrane into something that facilitates infection thread polarity. This work established a hierarchy of "infectosome" assembly using genetics and localization of components.

The strengths are the use of new imaging modalities to define the location and functional dependencies of complex components. This paper makes numerous significant advances in the understanding of infection thread maintenance and assigning a cellular activity for RPG. It leaves open still questions about how RPG is localized to discrete puncta and how it recruits other proteins to these still symbiosis-related structures.

This is an exciting addition to the literature detailing the mechanisms by which host plant cells make environments hospitable to symbionts, the strong correlation with the presence/absence of RPG and the ability/failure to support nodulation in a variety of plant species makes RPG an attractive candidate for engineering nodulation in crop plants that currently lack it.

Reviewer #1 (Public Review):

The objective of mobile phone (mHealth) interventions in African countries is to cost-effectively increase access to care and improve health. Due to resource constraints on the healthcare systems in many African countries, inaccessibility to healthcare is more noticeable in rural areas. While there is an increase in mHealth interventions in many African countries, it is salient to examine inequity in the distribution of smartphones that enable these interventions.

Investigators used the 2017-2018 Afrobarometer data from 33 countries to estimate the percentage of the population with a mobile phone (smartphone or otherwise). The analyses were conducted at different levels: (1) among all 33 countries; (2) at the country level; and (3) at the sub-national level (within each country).

The study is well designed, and the manuscript is clearly written. The findings are important from a policy and intervention perspective. This study shows that there are substantial inequities in smartphone ownership between and within African countries. These results have important implications for designing and rolling out mHealth interventions in African countries. This study shows that people who live in rural areas are less likely to own a smartphone and less likely to live close to a healthcare center. For mHealth intervention to work, individuals who are in high need of mHealth interventions would need to own mobile phones.

Reviewer #3 (Public Review):

The work is of general interest to audiences of public policy and public health. The data found some evidence that mobile health interventions may be affected by the type of mobile used but failed to substantiate the claim conclusively on how the lack of mobile ownership may hinder their rollout process. The claim about gender or geographic inequality must be elaborated in detail and many countries in developing countries are now connecting more users in rural areas through unconventional methods such as village phones instead of just mobile ownership.

Strengths:

The main strength of this paper is the usage of the cross-sectional data from the R7 Afrobarometer survey which is a newly available dataset and contains comprehensive data from more than 50 African countries. The usage of the Bayesian Logistic Regression (BLR) model produced some useful findings.

Weakness:

1) The authors have generalized a lot of things in a very simple manner. For example, they have assumed if participants have access to the internet means they own a smartphone and if they don't then they are basic phone users. It is possible a lot of smartphone owners do not have subscriptions to the internet due to the high cost of internet in African countries.

2) They have consistently talked about inequalities in gender, and rural-urban geographic regions based on the odds ratio derived from the BLR. A regression decomposition technique can quantify these differences more elaborately in detail.

3) They failed to explain why a lot of poor people own smartphones. This could be due to the usage of village phones (first implemented by Grameen Phone in Bangladesh). This has expanded in African countries as well where multiple users communicate through a community phone connecting more users in rural areas.

4) Basic phones may also be effective for mobile health interventions through voice-enabled systems and disseminating important messages to communities. (For e.g. there is extensive literature on how community-level messages, such as instructions on personal hygiene and usage of masks, were transmitted through basic phones during the beginning of covid19 in developing parts of Asia).

5) Further clarification of why lack of ownership of a mobile phone may propagate inequalities in health is needed beyond just simple associations. A latent factor may also cause these differences.

Reviewer #1 (Public Review):

In this study, the authors overexpress GFP-tagged NMN-D, a prokaryotic enzyme that consumes NMN, and find that it is strongly protective in several in vivo injury paradigms in flies. This is an important finding that clarifies previously published work, which found that an untagged NMN-D construct provided only weak axon protection (Hsu et al., 2021). The authors of the current manuscript argue convincingly that the previous result stemmed from the low stability of the untagged variant. Llobet Rossell et al. also use a very nice grooming assay for synaptic connectivity following axotomy to demonstrate that NMN-D overexpression maintains synaptic connectivity. Further pointing to NMN as a crucial regulator of dSARM activation, they show that increasing NMN levels by increasing NMN synthesis through mNAMPT overexpression accelerates injury-induced axon degeneration. They provide the support that NMN-D and mNAMPT overexpression are having the expected effect on NAD+ metabolic flux via LC-MS/MS. Finally, they provide evidence that a dSARM variant that cannot bind NMN does not rescue the dSARM LOF phenotype indicating that NMN exerts its effects through dSARM.

This study is carefully done and the evidence that NMN levels are a critical determinant of axon degeneration is strong. The combination of in vivo degeneration assays, metabolomics, and behavioral assays provides confidence in the results. For the most part, the conclusions of the study are well-supported by the results. The claim that the protection afforded by low NMN levels is as strong as the loss of SARM is justified. The authors repeatedly state that protection provided by low NMN is "even stronger" than that of essential mediators of axon degeneration, which doesn't really make sense given that all their data support the hypothesis that low NMN protects because it blocks dSARM activation. However, the primary weakness of this study is that the novelty of this work comes almost entirely from showing that the NMN/NAD+ ratio is determinative for axon degeneration in Drosophila; it has already been shown both in mammalian cultured neurons and in vivo.

Reviewer #3 (Public Review):

Llobet-Rosell et al. use Drosophila to decipher the relationships between factors in the Wallerian degeneration pathway and the metabolite NMN, an activator of the central pathway enzyme dSarm. NMN had previously been proposed to be a crucial regulator of Sarm, but there was a shortage of good in vivo evidence, especially in the crucial Drosophila system. The authors addressed this here by generating optimized fly lines, including a strongly expressing transgenic line for the NMN-consuming enzyme NMN-deamidase (NMNd). This variant conferred extremely strong protection against degeneration both in morphological and functional studies, thus confirming the key role of NMN as an activator of the degeneration pathway. They also confirm that NMNd alters NMN/NAD metabolism using mass spec of Drosophila heads, and then use Drosophila genetics to show that dSarm is the crucial NMN target. In a reverse experiment, the authors also use overexpression of murine NAMPT, an NMN-producing enzyme, to speed up degeneration. As in mammals, NMNd delays degeneration induced by loss of Nmnat.

A clear strength of the fly system is the degree of rescue conferred by the optimized NMN-D reagent which essentially establishes NMN as a crucial regulator in the pathway. The rigor of experimentation is also very high. Essentially all reagents are optimized, and most conclusions are backed by complementary analyses. The manuscript also nicely describes a metabolomic analysis of NAD biosynthetic pathways from fly heads.

Reviewer #1 (Public Review):

The monogamous vole provides unique opportunities to study the neural basis of pair bonding and this study exploits that opportunity in a novel way. Focusing on the nucleus accumbens, the authors conduct RNA-Seq to characterize the transcriptome in same-sex and opposite-sex pairs when bonded, when separated for a short time and when separated for a long time at which point the literature has in the past demonstrated the willingness to form a new bond. They determine that the transcriptome of pair bonding includes a preponderance of glial-associated gene changes and that it degrades with long-term separation. To the latter point, they then conduct a neuron enriching trap schema to find those genes subject to change specifically in neurons.

The strength of the report is the clever experimental design, the unusual animal model, and the comparisons of same-sex and opposite-sex pairs and long-term and short-term separations.

The weakness is that the behavioral changes observed are not what was expected based on prior work and are relatively modest, providing a disconnect between the outcome and the more dramatic transcriptional changes. A second weakness is the focus on the nucleus accumbens which is a brain region most closely associated with reward. While pair bonding may be rewarding, that component may be independent of the memory of a partner or the willingness to partner anew. Lastly, there is no clear connection between the identified transcriptome and either the formation or degradation of the pair bond.

Reviewer #3 (Public Review):

In this manuscript, the authors investigate the behavioral and brain transcriptional alterations associated with short- and long-term partner separation in the monogamous male prairie vole. Male prairie voles continue to show affiliative behavior after short- (2 days) and long-term (4-weeks) partner separation, with similar effects for same and opposite-sex pairs. However, the transcriptional signature in the nucleus accumbens exhibits marked alterations after long-term separation.

Strengths:<br /> 1) A key strength of this manuscript is its use of the monogamous prairie vole to investigate transcriptional alterations associated with pair bonding and subsequent pair separation. This sort of behavior cannot be investigated in typical rodent model systems (e.g., mice, rats), and the choice of using prairie voles allows for dissection of potential mechanisms of social bonding with relevance to partner loss in humans.<br /> 2) Investigation of behavioral measures and transcriptional alterations at both short- and long-term time points after pairing and separation is a strength of the manuscript. These time points were selected based on previous studies in laboratory and wild prairie voles related to the time it takes to form a pair bond and for the male prairie vole to leave the nest after the loss of the female pair. The datasets generated will be of great use to the scientific community.<br /> 3) The authors investigate the behavior and transcriptional profiles after same-sex as well as opposite-sex pairing. This is considered a thoughtful decision on the authors' part which allows them to tease apart the effects of same vs. opposite sex.<br /> 4) The use of numerous behavioral measures to assess both affiliative and aggressive behaviors is a strength of the approach.<br /> 5) The authors use many biostatistical approaches (e.g., RRHO, WGCNA, Enrichr) to probe the transcriptomics data. These approaches allow the authors to move beyond simply assessing transcriptional profiles separately, but to look for patterns that are similar or different across datasets.<br /> 6) The authors use rigorous statistical methods to assess behavioral measures.<br /> 7) The TRAP approach in prairie voles is novel and will provide a great resource to the research community.

Weaknesses:<br /> 1) The methods state that prairie voles were treated differently in the behavioral and transcriptomics studies. Specifically, for the separation in the behavioral studies, prairie voles were separated by sight, but not necessarily by the smell from partners (i.e., partners were kept ~1 foot apart). However, prairie voles in the transcriptomics studies were separated by both sight and smell (i.e., partners were sacrificed after separation). Thus, it is possible that the lack of degradation of pair bond-related behavior after long-term separation might be due to these prairie voles being able to smell their partners after separation. This is considered a moderate flaw in the design of the studies which limits the integration of results between behavior and transcriptomics. This might be why the authors do not see a strong behavioral degradation of pair bond-related behavior after long-term separation but do see a strong transcriptional signature.<br /> 2) While RRHO is helpful to assess overall patterns of transcriptional signatures across datasets, its utility for determining the exact transcripts is limited. This is because of how RRHO determines the overlapping transcripts for its Venn diagram feature (by taking the point where the p-value is most significant and taking the list to the outside corner of that quadrant).<br /> 3) TRAP expression was verified in only one animal. Thus, the approach has not been appropriately confirmed.

Reviewer #1 (Public Review):

In this paper the authors present variations in carbon oxidation state and hydration state in proteomes available in RefSeq. Then they use this information to predict community level proteomes, and their corresponding carbon oxidation states and hydration states, based on available 16S rRNA gene sequences from selected previously published datasets. When combining this with information about the environmental setting of the individual samples analyzed, the authors are able to demonstrate connections between redox conditions and proteomic carbon oxidation state and hydration state. Furthermore, they explore how individual taxonomic groups at different taxonomic levels contribute to forming these connections.

A weakness with the study is that the described environmental proteomes are inferred from 16S rRNA gene sequence data and not observed directly. However, there is good reason to believe that the conclusions drawn in the paper are valid.

The study sheds light on microbial adaptations on the genome level that so far have received relatively little attention. The paper is also interesting from an ecological perspective regarding the general question of how microbial communities are shaped by environmental settings.

Reviewer #1 (Public Review):

In this manuscript, Dodd et al. study the uptake and cytosolic release of tau fibrils. Based on a Crispr knock-out screen which identified that vacuolar H+ ATPase factors were required for tau fibril uptake, the authors focused on the role of endocytosis and endocytic acidification. Using genetic and pharmacological approaches, the authors made the surprising observation that endocytosis inhibition reduced tau fibril uptake but strongly increased fibril-induced tau seeding in the cells. Authors demonstrate that giant plasma membrane vesicles unable to undergo endocytosis still take up tau fibrils. Dodd and colleagues conclude that tau fibrils likely directly penetrate the lipid bilayer, which represents a novel entry pathway involved in tau seeding

The authors present some perplexing divergent effects of genetic or pharmacological inhibition of the endo-lysosomal system on tau fibril uptake and seeding. Surprisingly, inhibition of endo-lysosomal acidification inhibited fibril uptake but increased seeding in biosensor cells. Likewise, inhibition of Rab5a, a GTPase required for early endosome trafficking strongly impaired fibril uptake, but highly elevated tau seeding in biosensors. Low temperature during fibril incubation inhibited fibril uptake but increased seeding when cells were subsequently shifted to a normal growth temperature. Authors conclude that these results most likely show that endocytosis is not required for seeing tau. Instead, tau fibrils might directly translocate through the membrane to induce seeding.

Clearly, and rightfully stressed by the authors, the experiments demonstrate that the amount of internalized tau fibrils does not correlate with the tau seeding, arguing that minute (and potentially undetectable) amounts of tau seeds can very efficiently seed tau aggregation in biosensor cells. The massive amounts of labelled fibrils taken up by basically 100 % of cells might obscure the actual relevant tau seeds taken up by alternative routes that result in cytosolic tau seeding. This could apply to tau fibrils translocating through the membrane, as suggested by the authors, this could also apply to minute amounts of fibrils that still manage to enter the endo-lysosomal pathway even when this pathway is compromised pharmacologically or genetically. Any impairment of the endo-lysosomal system, however, likely also reduces the lysosomal degradative capacity. As a consequence, tau seeds in the endo-lysosomal system will have a higher propensity to escape to the cytosol to induce seeding. While the findings presented here are indeed very interesting and the Crispr screen is very elegant and informative, experiments are not sufficient to explain the cellular mechanisms involved.

Reviewer #3 (Public Review):

In this manuscript, Dodd et al. measure the internalization of exogenous fluorescently-labelled tau by cultured HEK cells and iPSC-derived neurons, as well as the aggregation of fluorescent fusion proteins of the repeat domain of tau with the P301S mutation (tau RD) expressed in these cells. They find that inhibition or reduction of V-ATPases and Rab5A reduces tau internalization and increases tau RD aggregation, as does culturing the cells at cold temperatures. The authors also find that exogenous fluorescently-labelled tau is internalized by HEK cell-derived GPMVs. All conditions are dependent on HSPGs, which presumably act as cell-surface attachment factors, similar to their role in the attachment of viruses to the cell surface. Based on the involvement of V-ATPases and Rab5A in endocytosis, the authors conclude that endocytosis of tau does not contribute to the aggregation of expressed tau. In addition, based on the lack of endocytosis in GPMVs, the authors conclude that tau can translocate across membranes and that this contributes to the aggregation of expressed tau.

The observation that conditions that decrease the overall internalization of exogenous tau can increase the aggregation of expressed tau suggests that multiple internalization routes exist, some of which are non-productive for the aggregation of expressed tau. This has important consequences for therapeutic strategies aiming to limit the internalization of tau. However, the conclusions that tau can translocate across membranes and that this contributes to the aggregation of expressed tau, whereas endocytosis of tau is non-productive for the aggregation of expressed tau, are not fully supported by the data.

Major comments:<br /> 1. There appear to be several alternative interpretations other than a reduction of endocytosis for the effects of perturbing V-ATPase and Rab5A function and culturing cells at cold temperatures. First, internalized tau was measured 4 h after the addition of exogenous tau to the cells. This seems like a long time for the study of endocytosis, which occurs in minutes. By 4 h, degradation of tau may have an effect on the amount of measurable internalized tau. This is important because, in addition to their roles in endocytosis, V-ATPases and Rab5A also have roles in protein degradation via the endolysosomal system. Similarly, culturing cells at cold temperatures for 4 h is expected to have many effects beyond the inhibition of endocytosis. In addition, the authors do not control for humidity and CO2 concentration, which could also affect their measurements. Perturbation of V-ATPases and Rab5A could also be exerting their effects by reducing the translocation of tau across endolysosomal membranes, instead of endocytosis. The authors found that the expression of dominant-negative dynamin increased the amount of internalized tau. Is this unexpected, given that dynamin is required for most forms of endocytosis and has been previously reported to be required for tau endocytosis (Wu et al. 2013. J. Biol. Chem. 288, 1856-1870; Falcon et al. 2018. J. Biol. Chem. 293, 2438-2451; Evans et al. 2018. Cell Rep. 22, 3612¬-3624)?

2. It is difficult to draw parallels between the experiments using cells and those using GPMVs. The authors use 25 nM tau for cell experiments, but 500 uM tau for GPMV experiments. This is a huge difference in concentration. The authors should carry out the GPMV experiments using the same concentration of tau as in the cell experiments. 500 uM is also a very high concentration and raises the question of if the GMPVs are completely sealed. GMPVs have recently been reported to be permeable to hydrophilic macromolecules (Skinkle et al. 2020. Biophys. J. 118, 1292-1300). Tau and the TAT peptide are more hydrophilic than the two negative controls used, transferrin and albumin.

3. It is not clear which molecular species of tau (monomers, oligomers, or fibrils) are being studied. The authors refer to tau fibrils, but the species of recombinant tau they are using are never characterised. Incubation of tau with heparin can be expected to result in a mixture of fibrils, oligomers, and monomers. Sonication may also change the distribution of tau species by liberating oligomers and monomers from fibrils. Similarly, key details about the immunoprecipitation are lacking, including neuropathological characterization of the human cases, the brain region, the amount of brain tissue, the lysis buffer, the epitope of the Tau B antibody, the amount of Dynabeads, and analysis of the immunoprecipitated sample to show what species of tau are present.

Reviewer #1 (Public Review):

The manuscript presents an interesting study that uses the previously generated yeast strains harboring human disease-specific mutations modelled in the yeast ortholog of SEC53 (V238M & F126L variants). These variants are either controlled by a strong heterologous promoter (pACT1) or a less-efficient native promoter. In either scenario, the strains manifest growth defects. The current study uses an experimental evolution strategy to evolve the strains to identify genetic suppressors of the slow growth phenotype. The authors identify several mutations in evolved strains and find a significant number of the suppressors in phosphoglucomutase 1, PGM1 (congenital disorders of glycosylation, CDG type I human ortholog). The synthetic setup replicates the compensatory mutations, but the growth rescue did not match the primary suppressors with several other mutations suggesting synergistic effects. Furthermore, reconstituted strains harboring LOF of PGM1 also showed the growth rescue, yet none of the evolved strains possessed a LOF of PGM1. The authors identify the PGM1 suppressors to be dominant. Finally, the protein activity assays reveal that the mutations in PGM1 reduce the protein activity rather than eliminate it. Overall, the assays show the power of yeast genetics for discovering the potential therapeutic targets in human diseases such as Congenital Disorders of Glycosylation 1.

1. While the authors identify the suppressors in known genetic interactors (GIs) of the yeast SEC53, it is worth testing if the compensatory mutations are rewiring the GIs, thereby explaining the lack of comparable compensations observed in reconstituted strains. If altered GIs explain the suppression, then while yeast serves as an excellent tool to perform these assays, the human context of the disease may require a different set of genetic suppressors and, therefore, a different target than the yeast PGM1 ortholog. Also, are Sec53 and Pgm1 proteins directly interacting in yeast and whether these mutations are on the interaction interface?

2. Based on the data obtained between pACT1 and pSEC53-driven expression of the SEC53 mutant alleles, the pattern of suppressors appears to be different. Authors report that the variants expressed from strong pACT1 promoters show more suppressors than those driven by native promoters. Is this a general trend in experimental evolution that slower-growing strains tend to show lesser suppressors? For example, on Page 6, line 154, "compensating for Sec53-F126L dimerization defects are rare or not easily accessible". The statement suggests that the authors did obtain suppressors that compensate for the dimerization defect. At the same time, while rare (also, are authors suggesting suppression of dimerization defect as in better dimerization?), the rate of obtaining suppressors seems to be linked to the severity of the fitness defects of the strains. The lack of suppressors may be a limitation of the evolution experiments. Indeed later in the manuscript, the authors noticed that while PGM1 suppressors obtained in V238M can also suppress F126L alleles, the suppression was not as efficient. Could it be that evolution experiments in slower-growing strains predominantly enrich suppressors in other pathways (i.e., not in the CDG orthologs) that restore the growth better and compete out the relatively weaker suppressors in PGM1? In fact, the authors report similar effects on Page 7, lines 204-210. These two paragraphs are contradictory and should be explained further.

3. Authors report that the LOF of PGM1 compensates for the SEC53 mutations. However, the evolution experiments did not capture any LOFs in PGM1. The fitness comparisons in evolution experiments are different as many different genotypes compete in a mix. Therefore, the fitness assays in a clonal population may not represent these differences well. To test this argument, authors can try to mimic the evolution experiments by mixing two genotypes to check competitive fitness, like the co-culture of pgm1 suppressor obtained via evolution experiments with pgm1Δ.

Reviewer #3 (Public Review):

Vignogna et al. used yeast genetics, experimental evolution and biochemistry to tackle human congenital disorders of glycosylation (CDG), a disease mostly caused by mutations in PMM2. They took advantage of the observation that the budding yeast gene SEC53 is almost identical to human PMM2, and used experimental evolution to find interactors of SEC53/PMM2. They found an overrepresentation of mutations in genes corresponding to other human CDG genes, including PGM1. Genetic and biochemical characterizations of the pgm1 mutations were carried out. This work is solid, although authors did not reveal why reduction of pgm1 activity could compensate for defects of a particular mutant allele of sec53.

Out of curiosity, if the authors were to simply focus on the preexisting mutations, would they have gotten the materials for most of the experiments in this article? In other words, how important is the experimental evolution?<br /> A strain table with full genotypes is needed.

Reviewer #1 (Public Review):

The goals of this paper were to provide structural and biochemical insight into the role that Fip1 plays as a protein interaction scaffold with other members of the cleavage and polyadenylation machinery. To that end, the authors solve two crystal structures as the featured basis for the study: FIp1 and CPSF30; Fip1 and Cst77. In both cases, subdomains of proteins were used for crystallography. These two structures are of high quality and are validated with rigorous and clear biochemistry. These points represent the technical strengths of the manuscript. One weakness of the study is the overall findings with those previously reported by the Tong laboratory. While the authors present some potentially newer information in terms of the number of PAP proteins capable of interacting with FIp1/CPSF30, there are experimental caveats that would need to be addressed first.

The novelty of the Fip1/CstF77 structure represents the strongest aspect of the paper as well as the observation that CstF77 can inhibit polyadenylation. The in vitro data support this conclusion but additional experiments would strengthen this overall model.

Reviewer #3 (Public Review):

The authors report the structure of CPSF30 bound to 2 molecules of FIP1, as well as the structure of FIP1 bound to CSTF77. Their data supports a model in which two molecules of FIP1, are present in the mPSF subcomplex of CPSF, although only one PAP may be bound to this complex. TheCstF77 binding to Fip1, which likely inhibits polyadenylation since it interferes with PAP binding, would occur as the complete complex assembles on the substrate, and result in the active cleavage complex not containing PAP or active in polyadenylation, only becoming active in polyadenylation after cleavage and loss of CstF from the complex.

Reviewer #1 (Public Review):

In this work, Maxime R. and co-authors intended to investigate the consequence of dystrophin absence/alteration in myoblasts, the effector cells of muscle growth and regeneration, and the early role of such cells in the pathogenesis of the disease. They carried out a transcriptomic analysis, comparing transcripts expressed by dystrophic myoblasts isolated from two murine models of DMD (Dmdmdx and Dmdmdx-βgeo) and control healthy mice. The expression of a large number of genes, comprising key regulator of myogenic differentiation (Myod1, Myog, Pax3 etc.) resulted affected in comparison to control in both mouse lines. Authors highlighted similar gene expression modifications also in a myoblast cell line previously established from the mdx mouse. To assess the outcomes from the gene ontology analysis, which pointed on the alteration of muscle system and regulation of muscle system processes, authors evaluated the proliferative, chemotactic and differentiative capacities of dystrophic myoblasts. Myoblasts presented increased proliferation, reduced chemotaxis and quite surprisingly, improved differentiating capacity, if considering the transcriptomic data. Finally, Maxime R. and co-authors carried out a transcriptomic analysis in myoblasts from DMD human subjects. Even though the profile of altered gene expression resulted similar and the GO studies seemed to focus on the same pathway categories, a significative divergence was observed particularly at the level of gene expression.

Authors link transcriptomic abnormalities and functional changes in proliferation, chemotaxis and differentiation of the dystrophic myoblasts with the alterations (probably epigenetic changes) occurring in satellite cells of dystrophic mice, consequent to the absence of the dystrophin protein. Such modifications in gene expression are supposed to be inherited by pathological myoblasts due to the division of the SC that is no longer asymmetric as occurring in healthy tissue.

Strengths<br /> Transcriptomic data from samples of different sources are solid and rigorous statistical analyses have been carried out.<br /> Transcriptomic and functional data from primary proliferating myoblasts of the two mouse models and from the myoblast cell line are similar. This is a convincing evidence that the transcriptomic alterations observed in primary myoblasts are not influenced by the exposure to the niche environment present in the dystrophic muscle, but that are cell autonomous.<br /> Authors adopted a 3D culture for the functional analysis concerning myoblasts differentiations, in this way better mimicking the process occurring in vivo.

Weaknesses<br /> The mdx mouse phenotype is mild in comparison to the severe symptoms and the rapid disease progression experimented by most of the human DMD subjects. Mdx mice is characterized by cycle of degeneration/regeneration initiating around the age of 6 weeks and continuing for several weeks. It was expected that authors discussed this point in detail, also considering that the animals used in this study were 8 weeks old. Furthermore, transcriptomic analysis of the human DMD myoblasts highlighted many differences as well as similarities when compared to mouse samples. Why do not focus more on this aspect?<br /> According to the authors, dystrophic abnormalities in myoblasts manifest irrespective of differences in genetic backgrounds and across species. The last one is a strong statement that should have been supported at least by functional data regarding chemotaxis proliferation and differentiation of human DMD myoblasts.<br /> In the discussion, the authors suggest two possible mechanisms as responsible for alterations in the behavior of the SC that ultimately affect the functionality of myoblasts, an RNA-mediated pathological process or an alteration in epigenetic regulation. They consider the latter mechanism more likely. This is based in particular on transcriptomic data showing the downregulation of important genes involved in histone modifications, normally linked to transcriptional activation. They also reported from the literature that HDAC inhibitors upregulate MyoD, a gene that is effectively downregulated in this study. Since the authors postulate that the epigenetic dysregulation of Myod1 expression is responsible for the pathological cascade of gene downregulation, ultimately leading to the pathological phenotype, it would have been interesting to evaluate the impact of HDACi on this pathways or the overexpression of enzymes responsible for H3K4 methylation as Smid1 (downregulated in this study).

Reviewer #1 (Public Review):

The study presents the observation of the coexistence of slowly moving broad waves of high F-actin concentration and rapidly propagating planar F-actin pulses in giant Dictyostelium discoideum cells and focuses on their theoretical analysis on the basis of a 3 component activator-inhibitor reaction-diffusion model with globally conserved actin. The authors conclude that the pattern coexistence is generic in a system with mass conservation close to a primary codimension-2 T-point bifurcation. The observation of the wave patterns in Dictyostelium discoideum is interesting and adds new phenomena to intracellular self-organization and patterns. The theoretical analysis is very careful and provides deep insight into the pattern mechanism in terms of non-linear dynamics. A very interesting and careful study. Well written, excellent figures.

While the observation of the wave patterns in Dictyostelium discoideum is interesting, the focus is on the theoretical analysis - which indeed is excellent. Unfortunately, the authors did not use their experimental abilities to verify predictions of their theoretical analysis, and thus in the end the conclusions lean heavily to the theoretical side. There are little biological conclusions from the theoretical analysis. In particular, a clear discussion on whether the observations are restricted to the artificial giant cells or may also have meaning for normal-sized cells is missing.

Reviewer #1 (Public Review):

In this study, the authors use T1-weighted structural MRI and FDG-PET data from an open access cohort to estimate individual-level structural and metabolic covariance networks across the Alzheimer-continuum using well-established sites of Alzheimer's pathology as seeds, detecting clear differences between diagnostic groups. They proceed to show that the evolution of these networks along the disease continuum is associated with memory performance in a nonlinear manner, with different trajectories. The results provide insight into structural and metabolic covariance network contributions to memory performance throughout the disease course, which adds to the current knowledge about potential network-based biomarkers and might have relevance to evaluating these markers in a clinical setting.

Strengths:<br /> - The analyses were performed on a well-characterised cohort with an adequate sample size that contributes to the robustness of the results.<br /> - Taking into account the non-linearity of the association between network-based descriptors and memory performance conforms better to current hypothetical models of biomarker dynamics in Alzheimer's disease.

Weaknesses:<br /> - It is encouraging that the results were replicated in a validation dataset, however, based on the manuscript it seems it is not independent from the main analysed data. Since the individual network estimation step relies on back-projection from a group-level salience map, the generalisability of the results might be better assessed by keeping the validation dataset independent.<br /> - Along this line, the employed method estimates individual network scores that quantify the relative contribution of a certain participant to the group-level salience map. This could potentially sensitise the method to the relative imbalance of group sizes across diagnoses and/or A/T categories, or to outliers.

Reviewer #1 (Public Review):

In this well-written manuscript by Barber and colleagues from UCL in the UK, the authors seek to identify a new predictive biomarker for patients with recurrent/metastatic head and neck cancer who are treated with chemotherapy. The manuscript is clearly written. This is an impressive body of correlative research performed in the context of samples collected from patients enrolled on a phase II trial, with samples collected and analyzed for immune monitoring. There are several novel assays employed beyond the standard immune monitoring. The question is of moderate clinical significance. There are a number of critical statistical limitations.

The question is of moderate clinical significance to the field. It is correct that we have only modest predictive biomarkers for chemotherapy response in R/M HNSCC. For this study to be significant, one would want to see a marked improvement over current biomarkers, in a robust and generalizable population. Unfortunately, this study falls short in these respects. First, the authors do not adequately discuss the prior literature. Even a fairly crude and old-fashioned blood-based biomarker such as neutrophil:lymphocyte ratio has quite good predictive and prognostic capability in R/M HNSCC. It is not clear to me that there is a compelling need to do better -- given that existing predictive biomarkers based on clinical nomograms or NLR are actually used in practice.

To establish that this fairly labor-intensive and expensive assay would add value, a comparison to other existing biomarkers is necessary. It is not clear qualitatively that the biomarker presented here is an improvement beyond what is currently available. This comparison could easily be performed.

A large number (31 of 87) patients were not included due to lack of biomaterials. No analyses have been performed to examine the characteristics of these patients. It is unlikely that the collection of biomaterials has no correlation with disease characteristics, prognostic features, outcomes, or the analytes in this study. This exclusion -- akin to unequal censoring in clinical trials -- is likely to significant impact results. Given that the population enrolled in a phase II trial, and that sub-population of patients who survive long enough and are feeling well enough to submit to large volume blood draws on trial, would not necessarily represent the real world population of R/M HNSCC patients, a broader population is needed to justify conclusions about this assay having robust predictive value.

It is unclear why OS as a hard endpoint was not analyzed here. No explanation is provided, other than OS was not available, a statement that is difficult to understand, given that PFS was available, and overall survival is a component of PFS.

There is no validation set for the biomarker. The biomarker was trained and cross-validated using Bayesian techniques to reduce overfitting. This is a valid approach for training and cross-validation, but for the biomarker to be testable and interpretable, it requires assessment in an independent dataset. There is no statistical technique that I am aware of that generates informative biomarkers without an independent validation dataset, and the use of these techniques to minimize overfitting does not circumvent this limitation, if one's goal is to develop a clinically useful biomarker. The 2 articles cited to justify this approach are not germane to the question -- one is an article describing the FRET-FLIM technique, and the other article describes the effectiveness of this approach to minimize overfitting.

In the end, the degree of predictive value, as assessed by C-index and the spread in the PFS curves, is modest, and not clearly an improvement beyond currently available biomarkers. Given that this dataset is the training dataset -- with no validation dataset -- in a population that is unlikely to be representative of the R/M population, it is not clear that this expensive and labor-intensive immune monitoring approach has much to offer.

Reviewer #3 (Public Review):

The authors explored the net patterns of selection in cancers as measured from tumor:normal exome and whole genome sequencing data. They found that by stratifying tumors on total mutation load, tumors with a low mutation burden exhibited net diversifying selection on previously identified oncogenic driver genes and net purifying selection on non-driver genes. Somewhat counter-intuitively both of these patterns decayed with increasing total mutation burden to the point where for tumors with the highest mutation burden, no net selection signals were identifiable. These findings were replicated using two dN/dS based approaches (with distinct means of defining the null expectation) and also using structural rearrangements as an orthogonal approach. The findings seem well demonstrated.

The proposed explanation for these observations is that of Hill-Roberson interference, where the (almost) perfect linkage disequilibrium of the whole genome in a clonally expanding population of cells provides little opportunity to separate mutations of opposing fitness effects leading to the accumulation of deleterious mutations without opportunity for their removal by selection. An important implication of this conclusion is that tumors, particularly those with a high mutation load, carry a high burden of deleterious mutations.

The modelling of clonal evolution demonstrates that Hill-Robertson like processes can in principal explain the decay of selection signals wither a high mutation burden, though this modelling by the authors own admission has lax parameter constraints and are gross simplifications of reality. As a proof of principal this modelling seems sufficient, and the estimated fitness effects appropriately qualified as "highly provisional".

The authors present the up-regulation of heat-shock/chaperone/protein-degradation pathways as a plausible mechanism through which cancers could manage the accumulation of many deleterious mutations and provide correlative evidence for increased expression of such genes in tumors with higher mutation burdens (Fig 2G). By considering only one such scenario the authors are perhaps placing too much emphasis on that one mechanistic hypothesis for (amino acid changing) mutational tolerance. Other plausible mechanisms include suppression of epitope presentation (adaptive immune evasion), replication stress etc.

Understanding that tumors carry substantial deleterious mutation loads and some prelimiary quantitative estimates of that will be of broad interest to the cancer genomics and also wider fields. The preprint is already being cited and found to be useful. The work also raises an important question - what are the main mechanisms employed to tolerate that deleterious mutation load, if there are predominant mechanisms such as the proposed protein-misfolding response, they become interesting targets for therapeutic suppression in a broad spectrum of cancers.

Reviewer #1 (Public Review):

Tilk et al investigate why cancer genomes show weak negative selection. They set out to differentiate between two scenarios whether selective pressures are relaxed during the course of tumor progression or whether selection is inefficient because of evolution without recombination thus genome-wide linkage resulting in interference among mutations referred to as Hill-Robertson interference. They calculate dN/dS for driver and passenger mutations in 50 cancer types with different levels of genome-wide mutational burden and show that passenger mutations exhibit negative selection and driver mutations show positive selection in low mutational burden tumors. The strength of selection is weakened in tumors with increasing mutational burden. The findings that the selection for passenger mutations is weak in high mutational burden tumors is novel and has not been shown before. Authors show this to be true for somatic copy number aberrations containing drivers versus passengers. Clonal mutations showed stronger selection than sub-clonal mutations. The accumulation of deleterious passenger mutations is buffered by upregulation of expression of genes encoding chaperones and the proteasome. Authors conclude that Hill-Robertson Interference can largely explain the weakened selection in drivers and passengers, which is also supported by their evolutionary model and they predict that cancer cells obtain fitness advantage from drivers by 130% and fitness cost from passengers of 40% conferring on cancer cells a net fitness advantage of 90%. This is an elegant study and the manuscript is well-written and logical. However, some aspects of the analyses require clarification.

1. Figure panels should be called out sequentially. For example, Fig. 2G is called out before Fig. 2D. This happens throughout the text, including main and supplementary figures, and should be corrected.<br /> 2. Fig. 2G shows that mean gene expression of genes encoding chaperones and the proteasome increases with increasing mutational burden. What about protein abundance? Is this in agreement with gene expression?<br /> 3. Fig. 2 mentions error bars in the figure legend, but no panel displays error bars. This is also true for Fig. S13 and other figures. Authors should display the error bars to which they are referring to make their analysis more convincing.<br /> 4. Pg. 9 line 295 describes results of the analysis across genes belonging to different GO terms. However, Fig. S13 only shows 3 categories: chromosome segregation, transcription and translation. How were these categories chosen? What about other categories? Such cherry picking doesn't convincingly support the conclusions that no specific GO functions are enriched. Also, translational regulation shows higher dN/dS in low mutation tumors suggesting that there is positive selection for passengers in this category. Authors should discuss in their manuscript why this is the case.<br /> 5. Fig. S15 shows the attenuation in selection of CNAs across cancer subtypes and broad cancer groups. However, HNSC and kidney cancer appear to be the exceptions. Authors should provide an explanation for these observations in the main text.<br /> 6. Generally, copy number variations are considered to be > 50 bp. Is there a rationale as to why authors chose 100 kb to be their cut-off in Fig. 2C? If the size of CNA is an important parameter, then authors should explain why that is.<br /> 7. Non-allelic recombination and non-homologous recombination mechanisms involving replication accidents that lead to chromosome breakage occur with some frequency in somatic cells. How does the frequency of these events impact the selection efficiency in cancer as it relates to drivers and passengers? Can this also be incorporated in their evolutionary model?<br /> 8. Authors mentioned that haploinsufficiency was not used in the model. What about loss of heterozygosity which is extensive in cancer genomes? Can this parameter be included in the evolutionary model and how would it impact the results?

Reviewer #1 (Public Review):

Tarasov and colleagues provide data that extensively phenotypes TGAC8 mice, which exhibit a cAMP-mediated increase in cardiac workload prior to developing heart failure. The authors confirm data from prior studies, showing increased cardiac output mediated by changes in heart rate with similar ejection fraction. Interestingly, canonical markers of LV hypertrophy did not differ from wildtype mice at the time period studied. The LV demonstrated proliferation of small cardiomyocytes and a network of interstitial non-cardiac myocytes. Transcriptomic and proteomic analyses of bulk LV tissue in TGAC8 mice compared to wildtype found pathways involved in immune responses, ROS scavenging, proliferation, and apoptosis to be activated in TGAC8 mice. Similarly, metabolic profiles shifted from fatty acid oxidation to glycolysis.

The study is overall well-planned and the amount of data presented by the authors is impressive. The work nicely incorporates animal-level physiology (echocardiography data), tests for known canonical markers of hypertrophy, and then delves into an unbiased analysis of the transcriptome and proteome of LV tissue in bulk. The techniques and analyses in the study are adequately executed and within the realm of expertise of the Lakatta laboratory. This study is a necessary and crucial first step to extensively phenotype this mouse line and generate hypotheses for further work.

Reviewer #3 (Public Review):

Tarasov et al have undertaken a very extensive series of studies in a transgenic mouse model (cardiomyocyte-specific overexpression of adenylyl cyclase type 8) that apparently resists the chronic stress of excessive cAMP signaling for around a year or so without overt heart failure. Based on the extensive analyses, including RNAseq and proteomic screening, the authors have hunted for potential "adaptive" or "protective" pathways. There is a wealth of information in this study and the experiments appear to have been carefully performed from a technical viewpoint. Many interesting pathways are identified and there is plenty of information where additional experiments could be designed.

General comments<br /> 1. Ultimately, this is a descriptive and hypothesis-generating study rather than providing directly proven mechanistic insights. T<br /> -Given several prior studies reporting a detrimental effect of chronically increased cAMP signaling, what is it that is different in this model? Is it something specific about AC8? Is it to do with when (in life) the stress commences?<br /> - Is the information herein relevant to stress adaptation in general or is it just something interesting in this specific mouse model?

2. None of the pathways that are apparently activated were directly perturbed so their mechanistic role requires further study.

Specific<br /> 1. The strain of the mice and their sex needs to be stated as well as the exact age at which the various assays were performed.<br /> 2. The hearts of the Tg mice have more cardiomyocytes but which are smaller. Since there is no observed increase in proliferation of cardiomyocytes, how (or when) did this increase in cell number occur?<br /> 3. While the mice do not show an increased mortality up to 12 months of age, HR/CO/EF are poor indices of contractile function. Data on end-systolic elastance or perhaps echo-based LV strain indices which will be relatively load-independent would be useful.<br /> 4. Quite a lot of conclusions are made relating to metabolism. However, this is entirely based on gene expression or protein levels. Given the substantial role of allosteric regulation in metabolic control, as well as the interconnectedness of metabolic pathways, ultimately any robust conclusions need to be based on an assessment of activity of key pathways.

Reviewer #1 (Public Review):

The manuscript by Park et al. uses CLEM, cryo-milling, and cryo-ET to visualize the major morphological transitions that occur in the bacteria Coxiella burnetii as it infects mammalian cells. These bacteria move from a small cell variant (SCV) to a large cell variant (LCV) that can replicate in the host lysosome. The authors focus on the C. burnetii T4SS which is similar in composition to the legionella Dot/Icm T4SS. They generate a homology model from the sub-tomographic 3D reconstruction of a little over 7,000 T4SSs using alpha-fold and structure of the Lp T4SS. This is a very descriptive analysis of gross cellular changes that occur in this bacteria during infection. While the authors suggest models for the developmental steps they think are occurring, it doesn't appear that they have fully mined their data for a more quantitative analysis of what is happening during the transition - let alone a mechanistic model for T4SS assembly and regulation.

Major Suggestions:

1. While it seems a reasonable hypothesis that the Dot/Icm T4SS assembly is regulated during the SCV-to-LCV transition it is unclear from the data shown if this is actually the case? Can the authors quantify the number and assembly state of the T4SSs during the transition from SCV to LCV (like they quantified the number of ribosomes)? They should also show sub-tomographic averages of the various structures making clear what parts of the complexes are assembled early and late during this transition? This type of detailed analysis is essential since it is the main point of the paper.<br /> 2. "The results indicate that 38.6% of the Dot/Icm machines identified from LCV images displayed an assembled inner membrane complex. By contrast, none of the bacteria in the transitional phase displayed a Dot/Icm-associated inner membrane complex, indicating that the complete core complex assembles upon transition to the LCV form (Fig. 5)."

Related to the previous comment - Figure 5 is a cartoon model - the authors should actually show this quantification and show structures.

3. A more detailed structural comparison between the Legionella and Coxiella burnetii T4SSs should be included as a major figure.

4. The number of ribosomes is quantified between SCV and LCV cells - however, it is unclear whether the argument is that in the SCV they are poised to be transcribed or that there is no change in transcriptional activity of the ribosomes during the transition.

5. The authors should further explain how folding of the inner membrane alone would allow for rapid expansion - shouldn't the outer membrane also have to rapidly expand? Do they see any sort of protein structures within the folded membrane that explains its topology?

6. Can the authors explain more why the LCV cells appear to be further away from the lysosome membrane? I would have predicted the bacteria would be closer so that proteins translocated through the T4SS would be able to get through the lysosome membrane and into the host cell cytoplasm - otherwise wouldn't these bacterial effector proteins simply be degraded in the acidified lysosome?

7. More information about the methods used needs to be included. How was phase shift correction done? How were different symmetry mismatches dealt with in the 3D reconstructions (in methods it simply states 13-fold symmetry was applied).

Reviewer #1 (Public Review):

This paper addresses an important question: whether the conduction velocity in white matter tracts is related to individual differences in memory performance. The authors use novel MRI techniques to estimate the "g-ratio" in vivo in humans - the ratio of the inner axon relative to the inner axon plus its outer myelin sheath. They find that autobiographical recall is positively related to the g-ratio in a specific white matter tract (the parahippocampal cingulum bundle) in a population of 217 healthy adults. This main finding is extended by showing that better memory is associated with larger inner axon diameters and lower neurite dispersion, which suggests more coherently organised neurites. The authors also argue that their results show that the magnetic resonance (MR) g-ratio can reveal novel insights into individual differences in cognition and how the human brain processes information.

The study is exploratory in nature and the analyses were not pre-registered. The technique has not been used before to associate cognitive performance with MR estimates of conduction velocity in candidate white matter tracts. It is therefore unknown how strong any associations are likely to be and what sort of sample size might be needed to observe them. Nevertheless, if the technique proves to be reliable, then it certainly offers a valuable new tool to understand individual differences in cognitive abilities. However, brain structure to behavior associations are notoriously variable across studies and have been argued to require very large sample sizes to obtain reproducible results.

The authors decided to analyse performance on a single task - the Autobiographical Memory Interview - and identified three candidate white matter tracts that connect the hippocampal region with other brain regions. While it is clear why these three tracts were chosen, it is less obvious why the authors chose to investigate associations with the Autobiographical Memory Interview and not other memory tests that were part of the battery of tests administered to the participants. It is reasonable to assume that something as general as the conduction velocity of a white matter tract would have an effect on memory ability across a range of tasks, so to single out one seems an unnecessarily narrow focus.

The results of the study are interesting and highlight a key role of the parahippocampal cingulum bundle in autobiographical memory recall. The results are corrected for multiple comparisons across the three fiber tracts of interest and the recall of "external details" provides a nice control compared to the "internal details" which are the measure of interest. The main findings are extended to show that it is likely to be an increase in axon diameter and an increase in neurite coherency that characterize those individuals with better autobiographical recall. Despite these positives, it remains unclear whether memory recall, in general, is better in people with higher g-ratios in this tract (as implied in the Abstract), or if this effect is specific to scores on the Autobiographical Memory Interview.

Reviewer #3 (Public Review):

The manuscript adds useful information about how structural properties of the brain are related to individual differences in autobiographical memory. A novel metric is used to assess features of white matter in tracts that are important for information exchange between the hippocampus and other brain regions. In one of these, the parahippocampal bundle, a relationship between the MR g-ratio and autobiographical memory recall is identified. This represents new and interesting information. The authors interpret the results in line with the theory that speed of signal transmission is important for cognitive function.

Reviewer #1 (Public Review):

Rasicci et al. have developed a FRET biosensor that is designed to light up when cardiac myosin folds. This structure is extremely important to understand, and its link to the super-relaxed (SRX) state has not been fully shown. Their study provides a comprehensive review of the literature and provides compelling data that the 15 heptad+leucine zipper+GFP construct does function well and that the DCM mutant E525K has a similar IVM velocity despite a reduced ATPase compared with HMM. They rely on the ionic strength-dependent changes in the rate of MantATP release to argue that the E525K mutation stabilizes the 'interacting heads motif' (IHM) state, which makes logical sense.

Strengths:

Well written and comprehensive.<br /> Utilizes the appropriate fluorescence-based sensor for measuring the folding of the myosin structure.<br /> Provides a detailed range of techniques to support the premise of the study

Weaknesses:

Over-interpretation of the outcomes from this study means that the IHM and SRX are the same. Similar studies, e.g. Anderson 2018 and Chu 2021 support the opposite view that IHM and SRX are not necessarily the same, Anderson (and Rohde 2018) point out that S1 has some element of a reduced ATPase, this clearly cannot be due to folding of the molecule. Also, mavacamten was used in these studies to show that even S1 is inhibited suggesting that SRX and IHM are not connected. This is not to say that with enough supporting evidence that these observations cannot be over-ridden, it is just not clear that there is enough in this study to support this conclusion.

I felt that the authors passed over the recent Chu 2021 paper too quickly, the Thomas group used a FRET sensor as well and provides a direct comparison as a technique, but with opposite conclusions. They also have supporting data in Rohde 2018 that their constructs were less ionic strength sensitive. It would be useful to understand what the authors think about this.

This section is likely to be important for end-users because initial expectations might be that these terms would inherently include such information.

Reviewer #1 (Public Review):

In their manuscript, Krug et al describe a CRISPR/Cas9 knock-out strategy for the creation of a pigment-less killifish (Nothobranchius furzeri) they term "klara". They target and inactivate three genes in parallel (mitfa, ltk, csf1ra). They employ the generated mutant offspring for studying mating preference as well as additional genome editing (knock-out of slc452) or the HDR-mediated knock-in to generate an inducible model for aging (inducible NTR mediated cell death by tagging the ORF of cdkn1a).

The authors present a valuable resource, a large bouquet of different, well-designed and described controlled experiments. The authors demonstrate the versatility of the established tool that may be of immediate use for the Noto community. Given the efficiency of the triple inactivation, the de novo inactivation may be more time and cost-efficient compared to the traditional sharing of the mutant animals.

When creating transgenic lines via HDR-mediated integration of donor sequences, the authors use the advanced protection of the donor construct by the addition of a 5'biotin. They validate integration by PCR genotyping and sequencing. However, these "proper" PCR bands can derive from in vitro recombination during the PCR (Won and Dawid, PlosOne2017), if a large number of cycles are used during PCR amplification. While PCR is often misleading, Southern Blot analysis delivers robust and unambiguous results. Here a single-copy integration is not relevant to the message of the manuscript.

Reviewer #3 (Public Review):

Krug et al. used emerging model species in biomedical research, Nothobranchius furzeri, to construct a triple mutant line that lacks all three major pigments found in fish (melanophores, iridophores, xanthophores). It demonstrates clearly that multiple genes can be inactivated simultaneously in this species, and that a new line can be a source of additional genetic manipulations. This is because their condition, vigour, and fecundity are standard compared to the wild type, which is convincingly demonstrated.

The introduction is appropriate and results generally correctly report what has been achieved, which is then adequately addressed in the discussion. Methods, as far as I can estimate, are sufficient to replicate the work.

The only substantial point I raise relates to the sexual selection (mate choice) part of the work. While it has no major effect on the overall conclusion, I think their interpretation needs to be reconsidered.

When reporting the results of mate choice experiment (L219ff), the authors state that males of wild and Klara type preferred wild-type females, because 75% of laid eggs belonged to wild-type females. However, another possibility is that Klara females had reduced fecundity, and the lower share of eggs had nothing to do with mate choice. In the same way, "90% of eggs were fertilized by wild-type males" (L223) is used to conclude that they were preferred by females (active mate choice). However, male success in N. furzeri is largely driven by male dominance (and not female mate choice) and it is more likely (and more precise to state) that wild-type males were more successful in male-male competition for access to females (and fertilize their eggs). This is especially so because wild-type males were larger (L. 322) and body size plays a major role in establishing dominance between N. furzeri males. This is then also pertaining to interpretation in discussion (L 318).

While I think this needs to be corrected to avoid misinterpretation, it has a minor impact on the overall high standard of the work or on general interpretation.

Reviewer #1 (Public Review):

This pre-registration study by Kerrén et al. examined the hypothesis that the brain resolves competition between overlapping memories based on phase separation of hippocampus theta oscillations. By applying a time-resolved decoding analysis, they demonstrate that the reactivations of target and competitor memories are locked to the varied phase of theta-band oscillation after repeated recalls. Moreover, subjects with larger phase separation show less memory interference. The study provides new evidence supporting the phase-coding neural mechanism to alleviate memory interference of multiple items.

Overall, this is a very interesting report testing an influential oscillatory-phase-based hypothesis in the memory field and would bring broad impacts to other fields, such as perception, attention, and decision making, given that lessening inference of distractor to target is a fundamental challenge.

Meanwhile, several aspects of the results need more evidence to strengthen the conclusion. The major weakness is the lack of significant decoding for the target and competitor by themselves, although I understand that the main hypothesis focuses on their different phase-locking relationship. Meanwhile, less significant decoding performance, I believe, is very crucial to verify the LDA analysis and data quality. Similarly, the original decoding performance time course did not show a clear out-of-phase pattern as revealed in the phase analysis. Overall, the authors need more results to confirm that the phase separation results are based on genuine reactivations and out-of-phase relationships.

Reviewer #3 (Public Review):

This manuscript uses MEG data acquired from human participants to examine whether representations of competing memories are associated with different phases of the theta rhythm in the human hippocampus. In brief, the authors use a proactive interference task in which subjects are asked to associate a word with two competing images and then subsequently recall the most recent image. Using pattern classifiers on the MEG data, the authors are able to decode reactivated content of the target and competitor memories and find that these patterns appear locked to different phases of the hippocampal rhythm. They also show that those subjects with worse memory performance had fewer differences in the phases to which target and competitor memories are locked. Together, the data provide support for a computational model of competing memories which suggests that oscillatory inhibition can be leveraged to strengthen or weaken inhibition of target and competitor memories (oscillating interference resolution model). One of the main strengths of the manuscript is that this is a pre-registered study, and so the specific hypotheses tested here have previously been reported. The current manuscript does not deviate too significantly from the pre-registered hypotheses and plan and reports the results of those proposed analyses. As such, this manuscript, therefore, presents a valuable addition to the literature, since it reports the results of a clearly established set of hypotheses testing a very specific question regarding memory interference.

Reviewer #1 (Public Review):

In this manuscript, the authors use C. elegans as a model system to show that calcium-dependent exocytosis of synaptic vesicles is differentially coupled to two different types of calcium channels. The authors take advantage of the fact that each major calcium channel family is represented by only a single gene in C. elegans, with CaV1 corresponding to L-type, CaV2 to P/Q-type, and CaV3 to T-type calcium channels, respectively Moreover, C. elegans contains only a single ryanodine-receptor channel that is responsible for releasing calcium from intracellular stores. While it is well established that CaV2 (as in other species) is mainly responsible for exocytotic transmitter release, the role of the other channels is not clear. Here the authors use smart genetic approaches involving tissue-specific deletion of individual channels and combinations of double mutants to document that CaV1 activity is responsible for the exocytosis of a distinct class of synaptic vesicles that is farther away from the active zone, couples to a distinct short form of Unc13, and that cooperates with RyR, with the release-relevant calcium release probably mainly being derived from intracellular stores.

With the caveat that this reviewer is not an expert in C.elegans, I consider this data-rich manuscript excellent, adding important information to the role of N-type calcium channels in transmission at the neuromuscular junction in C. elegans. As far as I can judge, the data are of high quality, and even the rather tricky spatial resolution of the adjacent release sites and the selective association of RyR with CaV1 and the short form of UNC13 using superresolution fluorescence microscopy is convincing. The manuscript is well written, and the data are succinctly discussed. As discussed by the authors it remains unclear whether such a mechanism also occurs in mammalian synapses, e.g. synapses in which exocytosis is also triggered by graded potential changes rather than by action potentials.

Reviewer #3 (Public Review):

In this manuscript, Jorgensen and colleagues elegantly used cutting-edge technologies to understand how different Ca entries lead to two different types of presynaptic release. They demonstrated that at the worm neuromuscular junctions two different classes of voltage-gated calcium channels, CaV2 and CaV1, mediate the release of distinct pools of synaptic vesicles. CaV2 channels are concentrated in densely packed clusters near the molecularly and EM-defined active zone structures. This type of release is dependent on synaptic vesicle priming protein UNC-13L. By contrast, they found that CaV1 channels are dispersed in synaptic varicosity and are coupled to internal calcium stores via the ryanodine receptor. CaV1 and ryanodine receptors mediate the fusion of vesicles docked broadly in synaptic varicosity and are colocalized with the vesicle priming protein UNC-13S.

The authors were able to direct their hypotheses because they have established powerful experimental methods such as rapid freezing EM coupled with neuronal stimulation. They used genetic null mutants for most of their experiments. They created endogenously labeled proteins to test the localization of proteins in live preparations. They used a combination and electrophysiological and behavioral assays. Since they worked with a system that has a small number of synaptic connections, they can reliably study the same set of synapses. The rigor of these experiments is extremely high.

The comprehensive approaches and the clear-cut results made this manuscript easily the top two or three papers I have read in the last couple of years of any journals.

Reviewer #1 (Public Review):

This work describes a new method, Proteinfer, which uses dilated neural networks to predict protein function, using EC terms and GO terms. The software is fast and the server-side performance is fast and reliable. The method is very clearly described. However, it is hard to judge the accuracy of this method based on the current manuscript, and some more work is needed to do so.

I would like to address the following statement by the authors: (p3, left column): "We focus on Swiss Prot to ensure that our models learn from human-curated labels, rather than labels generated by electronic annotation".

There is a subtle but important point to be made here: while SwissProt (SP) entries are human-curated, they might still have their function annotated ("labeled") electronically only. The SP entry comprises the sequence, source organism, paper(s) (if any), annotations, cross-references, etc. A validated entry does not mean that the annotation was necessarily validated manually: but rather that there is a paper backing the veracity of the sequence itself, and that it is not an automatic generation from a genome project.<br /> Example: 009L_FRG3G is a reviewed entry, and has four function annotations, all generated by BLAST, with an IEA (inferred by electronic annotation) evidence code. Most GO annotations in SwissProt are generated that way: a reviewed Swissprot entry, unlike what the authors imply, does not guarantee that the function annotation was made by non-electronic means. If the authors would like to use non-electronic annotations for functional labels, they should use those that are annotated with the GO experimental evidence codes (or, at the very least, not exclusively annotated with IEA). Therefore, most of the annotations in the authors' gold standard protein annotations are simply generated by BLAST and not reviewed by a person. Essentially the authors are comparing predictions with predictions, or at least not taking care not to do so. This is an important point that the authors need to address since there is no apparent gold standard they are using.

The above statement is relevant to GO. But since EC is mapped 1:1 to GO molecular function ontology (as a subset, there are many terms in GO MFO that are not enzymes of course), the authors can easily apply this to EC-based entries as well.

This may explain why, in Figure S8(b), BLAST retains such a high and even plateau of the precision-recall curve: BLAST hits are used throughout as gold-standard, and therefore BLAST performs so well. This is in contrast, say to CAFA assessments which use as a gold standard only those proteins which have experimental GO evidence codes, and therefore BLAST performs much poorer upon assessment.

Pooling GO DAGs together: It is unclear how the authors generate performance data over GO as a whole. GO is really 3 disjoint DAGs (molecular function ontology or MFO, Biological Process or BPO, Cellular component or CCO). Any assessment of performance should be over each DAG separately, to make biological sense. Pooling together the three GO DAGs which describe completely different aspects of the function is not informative. Interestingly enough, in the browser applications, the GO DAG results are distinctly separated into the respective DAGs.

Figure 3 and lack of baseline methods: the text refers to Figures 3A and 3B, but I could only see one figure with no panels. Is there an error here? It is not possible at this point to talk about the results in this figure as described. It looks like Figure 3A is missing, with Fmax scores. In any case, Figure 3(b?) has precision-recall curves showing the performance of predictions is the highest on Isomerases and lowest in hydrolases. It is hard to tell the Fmax values, but they seem reasonably high. However, there is no comparison with a baseline method such as BLAST or Naive, and those should be inserted. It is important to compare Proteinfer with these baseline methods to answer the following questions: (1) Does Proteinfer perform better than the go-to method of choice for most biologists? (2) does it perform better than what is expected given the frequency of these terms in the dataset? For an explanation of the Naive method which answers the latter question, see: (https://www.nature.com/articles/nmeth.2340)

Reviewer #3 (Public Review):

In this work, the authors employ a deep convolutional neural network approach to map protein sequence to function. The rationales are that (i) once trained, the neural network would offer fast predictions for new sequences, facilitating exploration and discovery without the need for extensive computational resources, (ii) that the embedding of protein sequences in a fixed-dimensional space would allow potential analyses and interpretation of sequence-function relationships across proteins, and (iii) predicting protein function in a way that is different from alignment-based approaches could lead to new insights or superior performance, at least in certain regimes, thereby complementing existing approaches. I believe the authors demonstrate i and iii convincingly, whereas ii was left open-ended.

A strength of the work is showing that the trained CNNs perform generally on par with existing alignment based-methods such as BLASTp, with a precision-recall tradeoff that differs from BLASTp. Because the method is more precise at lower recall values, whereas BLASTp has higher recall at lower precision values, it is indeed a good complement to BLASTp, as demonstrated by the top performance of the ensemble approach containing both methods.

Another strength of the work is its emphasis on usability and interpretability, as demonstrated in the graphical interface, use of class activation mapping for sub-sequence attribution, and the analysis of hierarchical functional clustering when projecting the high-dimensional embedding into UMAP projections.

However, a main weakness is the premise that this approach is new. For example, the authors claim that existing deep learning "models cannot infer functional annotation for full-length protein sequences." However, as the proposed method is a straightforward deep neural network implementation, there have been other very similar approaches published for protein function prediction. For example, Cai, Wang, and Deng, Frontiers in Bioengineering and Biotechnology (2020),<br /> the latter also being a CNN approach. As such, it is difficult to assess how this approach differs from or builds on previous work.

A second weakness is that it was not clear what new insights the UMAP projections of the sequence embedding could offer. For example, the authors mention that "a generalized mapping between sequence space and the space of protein functions...is useful for tasks other than those for which the models were trained." However, such tasks were not explicitly explained. The hierarchical clustering of enzymatic proteins shown in Fig. 5 and the clustering of non-enzymatic proteins in Fig. 6 are consistent with the expectation of separability in the high-dimensional embedding space that would be necessary for good CNN performance (although the sub-groups are sometimes not well-separated. For example, only the second level and leaf level are well-separated in the enzyme classification UMAP hierarchy). Therefore, the value-added of the UMAP representation should be something like using these plots to gain insight into a family or sub-family of enzymes.

The clear presentation, ease of use, and computationally accessible downstream analytics of this work make it of broad utility to the field.

Reviewer #1 (Public Review):

In the present study, Ibáñez-Solé1 et al evaluate transcriptional noise across aging and tissues in several publicly available mouse and human datasets.  Initially, the authors compare 4 generalized approaches to quantify transcriptional noise across cell types and later implement a new approach which uses iterative clustering to assess cellular noise.  Based on implementation of this approach (scallop), the authors survey noise across seven sc-seq datasets relevant for aging.  Here, the authors conclude that enhanced transcriptional noise is not a hallmark of aging, rather changes in cell identity and abundances, namely immune and endothelial cells.  The development of new tools to quantify transcriptional noise from sc-seq data presents appeal, as these datasets are increasing exponentially.  Further, the conclusion that increased transcriptional noise is not a defined aspect of aging is clearly an important contribution; however, given the provocative nature of this claim, more comprehensive and systematic analyses should be performed.  In particular, the robustness and appeal of scallop is still not sufficiently demonstrated and given the complexity  (multiple tissues, species and diverse relative age ranges) of datasets analyzed, a more thorough comparison should be performed.  I list a few thoughts below:

Initially, the authors develop Decibel, which centralizes noise quantification methods. The authors provide schematics shown in Fig 1, and compare noise estimates with aging in Fig 2 - Supplement 2.   Since the authors emphasize the necessary use of scallop as a "better" pipeline, more systematic comparisons to the other methods should be made side-by-side.  For example, scallop noise estimates (Fig 2) compared to other euclidean distance-based measures (Fig 2 supplement 2) looks fairly similar.  Are downstream observations (ex lung immune composition changes more than noise) supported from these methods as well?  If so, this would strengthen the overall conclusion on noise with age, but if not, it would be relevant to understand why.

Similarly, the 'validation of scallop seems mostly based on the ability to localize noisy vs stable cells in Fig 1 supplement 1 and relative robustness within dataset to input parameters (Fig 1 supplement 2).  A more systematic analysis should be performed to robustly establish this method.  For example, noise cell clustering comparisons across the 7 datasets used.  In addition, the Levy et all 2020 implemented a pathway-based approach to validate.  Specifically, surrogate genes were derived from GCL value where KEGG preservation was used as an output.  Similar additional types of analyses should be performed in scallop 

The conclusion that immune and endothelial cell transcriptional shifts associate more with age than noise are quite compelling, but seem entirely restricted to the mouse and human lung datasets.  It would be interesting to know if pan-tissues these same cell types enrich age-related effects or whether this phenomenon is localized

Related to these, there does not seem to be a specific rationale for why these datasets (the seven used in total or the lung for deep-dive), were selected.  Clearly, many mouse and human sc-RNA-seq datasets exist with large variations in age so expanding the datasets analyzed and/or providing sufficient rationale as to why these ones are appearing for noise analyses would be helpful. For example, querying "aging" across sc-seq datasets in Single cell portal yields 79 available datasets: https://singlecell.broadinstitute.org/single_cell?type=study&page=1&terms=aging&facets=organism_age%3A0%7C103%7Cyears

The analysis that noise is indistinguishable from cell fate shifts is compelling, but again relies on one specific example where alternative surfactant genes are used as markers.  The same question arises if this observation holds up to other cell types within other organs.  For example the human cell atlas contains over dozens of tissue with large variations in age (https://www.science.org/doi/10.1126/science.abl4290).

Reviewer #3 (Public Review):

In this manuscript, Ibáñez-Solé et al aim to clarify the answer to a very basic and important question that has gained a lot of attention in the past ~5 years due to fast-increasing pace of research in the aging field and development/optimization of single-cell gene expression quantification techniques: how does noise in gene expression change during the course of cellular/tissue aging? As the authors clearly describe, there have been multiple datasets available in the literature but one could not say the same for the number of available analysis pipelines, especially a pipeline that quantifies membership of single cells to their assigned cell type cluster. To address these needs, Ibáñez-Solé et al developed: 1. a toolkit (named Decibel) to implement the common methods for the quantification of age-related noise in scRNAseq data; and 2. a method (named Scallop) for obtaining membership information for single-cells regarding their assigned cell-type cluster. Their analyses showed that previously-published aging datasets had large variability between tissues and datasets, and importantly the author's results show that noise-increase in aging could not be claimed as a universal phenotype (as previously suggested by various studies).

Comments:

1. In two relevant papers (doi.org/10.1038/s41467-017-00752-9 and doi.org/10.1016/j.isci.2018.08.011), previous work had already shown what haploid/diploid genetic backgrounds could show in terms of intercellular/intracellular noise. Due to the direct nature of age/noise quantification in these papers, one cannot blame any computational pipeline-related issues for the "unconventional" results. The authors should cite and sufficiently discuss the noise-related results of these papers in their Discussion section. These two papers collectively show how the specific gene, its protein half-life and ploidy can lead to similar/different noise outcomes.

2. While the authors correctly put a lot of emphasis on studying the same cell type or tissue for a faithful interpretation of noise-related results, they ignore another important factor: tracking the same cell over time instead of calculating noise from single-cell populations at supposedly-different age points. Obviously, scRNAseq cannot analyze the same cell twice, but inability to assess noise-in-aging in the same cell over time is still an important concern. Noise could/does affect the generation durations and therefore neighboring cells in the same cluster may not have experienced the same amount of mitotic aging, for example. Also, perhaps a cell has already entered senescence at early age in the same tissue. This caveat should be properly discussed.

3. Another weakness of this study is that the authors did not show the source/cause of decreasing/stable/increasing noise during aging. Understanding the source of loss of cell type identity is also important but this manuscript was about noise in aging, so it would have been nice if there could be some attempts to explain why noise is having this/that trend in differentially aged cell types in specific tissues.

4. In the discussion section, the authors say that "Most importantly, Scallop measures transcriptional noise by membership to cell type-specific clusters which is a re-definition of the original formulation of noise by Raser and O'Shea." It is not clear what the authors refer to by "the original formulation of noise by Raser and O'Shea". Intrinsic/extrinsic noise formulations?? Please be more specific.

Reviewer #1 (Public Review):

Xian et al. systematically evaluated age and sex-dependent differences in paw skin and sciatic nerves (SCN) tissues of naïve mice, utilizing DIA-PASEF, a highly sensitive and reproducible proteomics approach. The authors demonstrated that the deep proteome profiling enabled a discovery of significant differences between male and female mice and adolescent and adult mice such as homeostasis and epidermal signaling in skin and, myelination and neuronal development in SCN that are known to be relevant to the pathophysiology. The authors claim the need for the appropriate age and sex matching in the experiment design and suggest the work as a unique systems biology proteome resource in mouse disease model. As I understand this is the first attempt to molecularly characterize the impact of mouse age and sex that would help warrant the reproducibility of the preclinical research.

Reviewer #3 (Public Review):

The paper emphasizes the importance of testing males and females in parallel when designing mice experiments as well as being consistent with age. In agreement with this, significant differences were observed between mice of different sexes and of varying ages. It also offers many insights into how DIA-PASEF workflows can improve performance in proteomics.

I would suggest to the authors they explain how experiments could be designed in a small scale in case there are time and financial constraints so that both female and male mice can be used simultaneously. It would also be beneficial to read over any challenges associated with the DIA-PASEF analysis. Enrich the discussion with performance comparison between DIA-PASEF and DDA-PASEF for mice proteomics data male versus female.<br /> Were there any unique proteins only found by DIA-PASEF?

Reviewer #1 (Public Review):

This manuscript reports the results of studies on the effects of an ActRIIB-Fc ligand trap inhibitor of myostatin on muscle contractures that develop when brachial plexus nerve roots are severed at 6 after birth. One component of this pathological response seems to be a failure to add sarcomeres as the skeleton grows resulting in short muscles. The authors use a carefully performed set of animal studies to test the effects of the ligand trap on denervation-induced limitations in range of motion in young mice. They also investigate several biochemical mechanisms that might contribute to contractures and be modified by the ligand trap. Finally, the test for gender discordance in the protective effect of a proteasome inhibitor against contractures. The major finding of these studies is that the ligand trap improved the range of motion at the elbow and shoulder in female mice but not in males. The major caveat to interpreting the data is that group sizes are relatively small such that the study may have been underpowered to detect smaller effects on a range of motion and biochemical endpoints.

Reviewer #3 (Public Review):

This timely manuscript describes the sex dimorphisms in neonatal development as it applies to muscle injury and denervation. More and more studies are identifying sex differences in skeletal muscle function and dysfunction. This is one more study to point out differences. A missing piece to the field and this study are the mechanistic links between skeletal muscle function/dysfunction and sex differences. This paper starts to point to a mechanism highlighting the non-canonical AKT pathway. This is a very well-written manuscript with a clear experimental plan and workflow. I have no major concerns.

My biggest question is the molecular mechanism linking sex differences and skeletal muscle function and dysfunction. However, this is perhaps a follow-up study to the already complete study the authors present.

Joint Public Review:

Tiedemann et al. evaluated the neural response to insulin before and after a 3-month caloric restriction diet compared to an active waiting group in 50 elderly persons overweight and with obesity. Peripheral insulin sensitivity and central insulin sensitivity of the mesolimbic reward circuitry predicted weight loss success and improvement after caloric restriction. This is the first longitudinal study showing an improvement in central insulin sensitivity in humans.

Strengths<br /> The major strength of the paper is the longitudinal study design in persons at high risk to develop type 2 diabetes with multiple fMRI visits, with a state-of-the-art fMRI design. Moreover, the study uses a placebo-controlled approach to evaluate insulin sensitivity in the brain. Another strength is the fact the control group is an active waiting group, which means that they received dietary counselling after the study and additionally benefited from the study by receiving training courses on stress management.

Weakness<br /> A weakness of the study is the lack of metabolic assessments of the participants. Only fasting blood samples were available. No oral glucose tolerance test or hyperinsulinemic-euglycemic clamps were acquired to quantify peripheral or whole-body insulin resistance. Whether peripheral insulin sensitivity and central insulin sensitivity are, in fact, independently involved in the success or treatment outcome of intervention still needs to be validated in future studies.<br /> Another weakness is the food cue picture set implemented in the study. The different categories of pictures were not carefully matched for psychological and physical variables (such as the complexity of a picture). This could potentially influence neural food cue reactivity.<br /> The study evaluated the regional BOLD response of the reward circuitry showing that insulin signaling in the VTA and nucleus accumbens play an important role in successful weight loss. The role of insulin signaling on projections (or connectivity) of the mesolimbic reward circuitry was not evaluated in the current study.

Reviewer #1 (Public Review):

This manuscript discusses evolutionary patterns of manipulation of others' allocation of investment in individual reproduction relative to group productivity. Three traits are considered: this investment, manipulation of others' investment, and resistance to this investment. The main result of the manuscript is that the joint evolution of these traits can lead to the maintenance of diversity through, as documented here, cyclic (or noisier) dynamics. Although there are some analytical results, this main conclusion is instead supported by individual-based simulations, which seem correctly performed (but for clonal populations, as emphasized below).

There could be material for a good paper here but the organization of the manuscript makes it difficult to fully evaluate. The narrative is highly condensed, with the drawbacks that this often entails in terms of accurately conveying the results of a study, as illustrated here by the following issue.

The population is apparently assumed to be clonal (more than just "haploid"), meaning that there is no recombination between the loci controlling the three traits. In the one case where this assumption is relaxed (quite artificially), the cyclic dynamics disappear (section 4.4 of the appendix). This is crucial information that cannot be appreciated in the main text.

The paragraph at line 368 offers a simple explanation for the joint dynamics of traits. However, this explanation would hold identically for a sexual population and a clonal population, whereas these two cases seem to have completely different dynamics. Thus, there is something essential to explain these differences, that is missing from the given explanation.

This is especially important because the finding that the joint evolution of several traits can lead to some form of diversity maintenance is not surprising. As the discussion acknowledges (but the introduction seems to downplay), it is also well understood that manipulation and counter-adaptations to it can occur in many contexts and lead to the maintenance of diversity. For this reason, similar results in the present case are not surprising, and the main outcome of the study should be to provide a deeper understanding of the forces leading to the different outcomes in the current models.

I do not see clearly what distinguishes "manipulative cheating" from other forms of manipulations that have been previously discussed in the literature (e.g, as cited lines 461). Couldn't this be clarified by some kind of mathematical criterion?

Reviewer #3 (Public Review):

The paper uses a mixture of game-theoretical models and individual-based simulations to study the coevolution of manipulation and resistance to manipulation in social interactions. This is a very impressive piece of theoretical research that will likely open new directions for both theoretical and empirical work.

Reviewer #1 (Public Review):

The study uses public behavioral and fMRI data to study the range adaptation properties of the orbitofrontal cortex (OFC) during risky choice that requires integrating potential gains and losses. The authors demonstrate how spill-over effects from the range of gains to the sensitivity to losses, cannot be explained by simple efficient coding accounts. The authors construct an artificial neural network (ANN) and show that Hebbian plasticity between attribute-specific and integration units can account for the context-dependent effect in behavior and fMRI data.

This is an interesting study that discusses a potential mechanism for context effects often seen in decision-making. A major concern is that the manuscript focuses on Hebbian plasticity as the key mechanism, whereas the results show that the choice of activation functions (sigmoidal vs. gaussian) has a comparable contribution to explaining behavior but is not discussed. In addition, the performance of even the best model is not very convincing for extreme ranges of expected value. There are additional moderate and minor concerns with result presentation and interpretation.

Reviewer #3 (Public Review):

The authors investigate range adaptation in the orbitofrontal cortex by taking advantage of an existing data set on willingness to gamble where two different groups experienced a wider or a narrower range of gains but the same range of losses. They find that sensitivity not only to gains but also to losses changes as a function of the gain range, such that for the part that was common to the two groups, people in the wide range group were less willing to gamble than people in the narrow range group. Moreover, a two-layer artificial network with Hebbian plasticity explains the behavioral effects of ranges and multivariate neural representations of value in the orbitofrontal cortex. The authors conclude that range adaptation occurs at the level of the integration layer rather than at the level of the attribute-specific input layer (where gains and losses are separate). The paper provides a welcome addition to the literature on how range adaptations may come about but would benefit from a couple of clarifications.

Major:<br /> 1) It appears like the Gaussian assumption may explain as much or even more of the variance as the plasticity assumption. However, the results do not really address this point. It would be good to provide some information about it for the behavioral findings, check whether the impression also holds for OFC and vmPFC activity, and discuss what the Gaussian assumption implies for the representation of value as such. After all, the monotonicity assumption pervades most previous research on value representation and seems to have been supported reasonably well so far (sometimes with the refinement that positive and negative coding monotonic signals/neurons may be intermixed). Relatedly, one may assume that the Gaussian assumption primarily holds for chosen value cells. But Figure 6 suggests that offer value units are more common in the model. Please explain.

2) The paper dismisses simplistic efficient coding scenarios that operate on neurons that transmit gain/loss information based on either finding common coding of gain and loss information but no difference between range groups or a difference between range groups but no common coding of gain and loss information. Did the authors also consider common coding of a) expected value, b) gains only, and differences between range groups in (a) and (b) signals, instead of looking at both gains and losses? Because the range manipulation primarily concerned gains rather than both gains and losses, there may be more power in looking at gains only. It may also be worth mentioning that at least for simple reward prediction error signals, a within-subject design, and regions other than the OFC, the simplistic analysis approach can find both effects (Kirschner et al., 2018, Brain). Of course, some of the mentioned or other differences may explain the difference in findings.

Reviewer #1 (Public Review):

Lymphatic drainage of CNS antigens has received lots of attention recently due to the (re)discovery of dural meningeal lymphatics. The role of dural lymphatics in CNS antigen drainage has been shown to be important in several contexts (AD, glioma, etc.). Few studies have focused on their role in CNS infections so far. In their study, Kovacs et al. dissected the role of meningeal lymphatic drainage in T cell responses during chronic Toxoplasma gondii infection in mice. They first show that T.g. infection drives DC accumulation in the dura and CSF at 6 weeks post-infection, which matches with the replication peak of T.g. in the brain, and with T cell expansion/activation in the dCLN. This effect on T cells in the dCLN was abrogated upon surgical blockade of dural drainage. However, dural lymphatic blockade did not affect T.g. burden, nor CD4+ and CD8+ T cell number and IFNg production in the brain in the chronic phase. They finally show that during chronic infection, antigen-specific T cells are generated not only in the dCLN but also in the periphery (ILN), which could account for the presence of T cells in the brain after surgical blockade of the lymphatics.

This study is interesting and some aspects are clearly convincing, but some precisions have to be made. More importantly, with similar tools, key experiments (as explained in the last paragraph: icv infection or chronic peripheral parasite clearance, etc.) have to be generated to bring more impact to this study and could reveal a new role for dCLN T cells.

Reviewer #1 (Public Review):

In this manuscript, Soto-Feliciano et al. investigate the tumor suppressive role of MLL3 in hepatocellular carcinoma (HCC). The authors used a variety of techniques including hydrodynamic tail vain injection (HTVI), CRISPR deletion, and shRNA to disrupt MLL3 expression in mouse models. They clearly show that MLL3 acts as a tumor suppressor in the context of MYC-induced HCC. They show that MLL3 acts by activating the Cdkn2a locus. Genomic analysis showed that MLL3 binds to enhancers and promoters, and specifically interacts with the Cdkn2a promoter. When MLL3 was downregulated, Cdkn2a levels fell and this corresponded to changes in relevant histone marks targeted by MLL3. The authors were also able to show that reintroduced MLL3 expression in a dox inducible system could rescue CDKN2A locus expression, which in turn reduced colony formation and induced apoptosis. Human genomic correlation showed that MLL3 and Cdkn2a mutations are generally mutually exclusive. Overall, the conclusions of the manuscript are well supported by a logical series of experiments with good controls and orthogonal approaches. While it would be useful to examine another HCC model such a CTNNB1-driven model, the current paper is convincing in its conclusions.

Reviewer #3 (Public Review):

The enhancer chromatin-modifying enzyme MLL3 functions as a tumor suppressor in multiple human cancers, however, the mechanisms underlying its tumor suppressive function remain unclear. The manuscript of Soto-Feliciano et al. focused on Myc-driven liver cancer and aimed to address and fill the gap. The authors used an elegant genetic design and approach to manipulate the overexpression of the Myc oncogene and knockout of the Mll3 tumor suppressor gene in mouse liver cancer models. Their genetic mouse models showed that loss of Mll3 constrains Myc-driven liver tumorigenesis, with tumors having a slightly later onset compared to mice with Myc overexpression in conjunction with p53 inactivation. Because MLL3 is a major histone-modifying enzyme for enhancer-associated H3K4 monomethylation and is responsible for enhancer activation and the following target gene transcription, they performed ChIP-seq analysis to study the roles of Mll3 in Myc-driven mouse liver cancer. Interestingly, their ChIP-seq studies revealed that loss of Mll3 preferentially limits Mll3 enrichments at promoters and thereby attenuates promoter-associated H3K4 trimethylation and target gene transcription, whereas the unchanged Mll3 genomic binding between the two genotypes (Myc;sgp53 and Myc;sgMll3) is largely located within enhancer (intergenic) regions. They further demonstrated that the cdkn2a locus is a genomic and transcriptional target of Mll3 in Myc-driven mouse liver cancer. Supporting their findings, genomic inactivations of MLL3 and CDKN2A displays mutual exclusivity in human liver cancer and many other cancer types. Furthermore, they described a possible mechanism for MLL3's role in MYC-driven liver cancer that MLL3 mediates MYC-induced apoptosis in a CDKN2A-dependent manner by manipulating Myc overexpression, Mll3 function, and Cdkn2a regulation in their genetic mice models. This manuscript describes a potential function of MLL3 in the control of tumor suppressor gene expression via modulating their promoter chromatin landscapes. More importantly, loss of normal function of MLL3 or the downstream effector CDKN2A may impair MYC-induced apoptosis, and in turn, lead to MYC-induced tumorigenesis.

Overall, the manuscript is well written, organized, and focused on an interesting topic, and with data presented supports the authors' claims.

Reviewer #1 (Public Review):

Mikelov et al. investigated IgH repertoires of memory B cells, plasmablasts, and plasma cells from peripheral blood collected at three time-points over the course of a year. In order to obtain deep and unbiased repertoire sequences, authors adopted uniquely developed IgH repertoire profiling technology. Based on collected peripheral blood data, authors claim that; 1) A high degree of clonal persistence in individual memory B cell subsets with inter-individual convergence in memory and ASCs. 2) ASC clonotypes are transient over time and related to memory B cells. 3) Reactivation of persisting memory B cells with new rounds of affinity maturation during proliferation and differentiation into ASCs. 4) Both positive and negative selection contribute to persisting and reactivated lineages preserving the functionality and specificity of BCRs.

The present study provides useful technical application for the analysis of longitudinal B cell repertoires, and bioinformatics and statistical data analysis are impressive. Regarding point 1), clonal persistence of memory B cells is already well known. On the other hand, inter-individual convergence between memory B cells and plasma cells might not be shown in healthy individuals even though the biological significance of circulating plasma cells is questionable.

Regarding 2), temporal stability of plasma cell clonotypes has been demonstrated already in the bone marrow with serial biopsies over time (Wu et al. DOI: 10.1038/ncomms13838). The Association of clonotypes between memory and plasma cells in the blood of healthy donors might be new, however, again its biological significance is questionable.

Regarding 3) and 4), it is hard to generalize observations from the presented data because the analysis was based on just four donor cases with different health conditions, i.e. a combination of healthy and allergic. The cell number of plasmablasts and plasma cells isolated from peripheral blood is extremely low compared to memory B cells, and in fact, the vast majority of ASCs reside in the tissues such as lymphoid organs, bone marrow, and mucosal tissues rather than in circulating blood (Mandric et al. DOI: 10.1038/s41467-020-16857-7). As the most critical problem, direct pieces of evidence to claim points, 3) and 4) are missing.

Reviewer #1 (Public Review):

This study uses the mouse calyx of Held synapse as a model to explore the presynaptic role of rac1, a regulator of actin signaling in the brain. Many of the now-classical methods and theory pioneered by Neher and colleagues are brought to bear on this problem. Additionally, the authors were able to make a cell-specific knockout of rac1 by developing a novel viral construct to express cre in the globular bushy cells of the cochlear nucleus; by doing this in a rac1 floxed mouse, they were able to KO rac1 in these neurons starting at around P14. The authors found that KO of rac1 enhanced EPSC amplitude, vesicle release probability, quantal release rates, EPSC onset time and jitter during high-frequency activity, and fast recovery rates from depression. Because the calyx synapses are the largest and most reliable of central nerve terminals, all these various effects had no effect on suprathreshold transmission during 'in vivo-like' stimulus protocols. Moreover, there was no effect morphologically on the synapse. Through some unavoidably serpentine reasoning, the authors suggest that loss of rac1 affects the so-called molecular priming of vesicles, possibly due to a restructuring of actin barriers at the active zone. The experimental analysis is at a very high level, and the work is definitely an important contribution to the field of presynaptic physiology and biophysics. It will be important to test the effects of the KO on other synapses that are not such high-performers as the calyx, and this direction might reveal significant effects on information processing by altered rac1 expression.

Major points:<br /> 1. The measurement of onset delay was used to test whether rac1-/- affects positional priming. While there is a clear effect of the KO on the latency to EPSC onset, there is no singular interpretation one can take, due to the ambiguity of the 'onset delay'. Note that in the Results authors state Lines 201-203: "The time between presynaptic AP and EPSC onset (EPSC onset delay) is determined by the distance between SVs and VGCC which defines the time it takes for Ca2+ to bind to the Ca2+ sensor and trigger SV release (Fedchyshyn and Wang, 2007)." However, in Methods "The duration between stimulus and EPSC onset was defined as EPSC onset delay." Thus the 'onset' measured is not between presynaptic spike and EPSC but from axonal stimulus and EPSC. KO of rac might also affect spike generation, spike conduction, calcium channel function, etc. Indeed some additional options are offered in the Discussion. Since the change in onset is ~100usec at most, a number of small factors all could contribute here. Moreover, the authors conclude that the KO does NOT affect positional priming since they would have expected the onset to shorten, given the other enhancements observed in earlier sections.<br /> It seems to me that all the authors can really conclude is that the onset shifted and they do not know why. If onset is driven by multiple factors, and differentially affected in the KO, then all bets are off. Thus, data in this section might be removed, or at least the authors could further qualify their interpretations given this ambiguity.

2. If the idea is that the loss of Rac1 leads to a reduced actin barrier at the active zone, is there an ultrastructural way to visualize this, labeling for actin for example? Authors conclude that new techniques are needed, but perhaps this is 'just' an EM question.

3. Authors use 1 mM kynurenic acid in the bath to avoid postsynaptic receptor saturation. But since this is a competitive antagonist and since the KO shows a large increase in release, could saturation or desensitization have been enhanced in the KO? This would affect the interpretation of recovery rates in the KO, which are quite fast.

Reviewer #1 (Public Review):

Baldazzi and coworkers propose a resource allocation model for E. coli steady-state cell growth that allows a joint description of growth rate and yield (fraction of substrate converted into biomass) and compare it with a compiled dataset based on batch growth data from different strains and two growth conditions (as well as some chemostat growth data). The model includes a description of alternative respiration and fermentation pathways with different energy efficiency. The model predicts bounds on the achievable state growth rate vs yield space that are compared with data, as well as glucose uptake and acetate secretion rates, which are compared with data.

In my view, the main merits of the model are (i) the compiled dataset of growth-yield-uptake-secretion parameters and (ii) the proposition of a resource-allocation model that includes the energy budget. Contrary to most current models in this area, the biomass includes other cellular components (DNA, RNA, metabolites, ...) in addition to proteins.

The main limitations are that the trends in the data do not emerge well and the predictions of the model are not presented in a transparent way. I believe that considerable extra work is needed in order to valorize the effort and highlight the trends in both data and model. For the data, it suffices to present more "sections" of the dataset (preferably as 2D XY plots) and more reflection on their meaning. Regarding the model, I think more effort is needed towards "breaking it open" and providing insight into why the model makes certain predictions and which ones are not trivial.

Reviewer #1 (Public Review):

The study investigated the role of oxytocin (OT) in the paraventricular hypothalamic nucleus (PVH) and oxytocin receptor (OTR) in the hypothalamus in the regulation of food intake using conditional knockout of oxytocin in adults by adreno-associated virus (AAV) approaches. Although pharmacological studies have demonstrated that oxytocin/oxytocin receptors regulate food intake behavior, constitutive knockout (KO) models are not aligned with pharmacological studies. To overcome this discrepancy between pharmacological and developmental genetic approaches, the authors used AAV to knockout oxytocin/oxytocin receptors in adults of mice, circumventing developmental compensations. They found that adult KO of OT in the PVH, but not supraoptic nucleus (SO), led to obesity due to hyperphagia. They also investigated whether OTR in the anterior or posterior side of the hypothalamus contributes to OT-mediated food intake behavior, and found that the posterior hypothalamic areas are key for that. Overall, the experiments are thoughtfully designed, and the manuscript is well written. However, there are questions that authors need to address, in particular their OT KO model.

Reviewer #3 (Public Review):

The manuscript by Inada et al. examines the role of hypothalamic oxytocin (OT) signaling in feeding behavior. They demonstrate that conditional knockout (KO) of OT in the adult paraventricular hypothalamic nucleus (PVH) increases body weight through increases in food intake, and that conditional knockout of the OT receptor in the posterior hypothalamus has a similar effect. The authors therefore conclude that OT signaling in the posterior hypothalamus, presumably through oxytocin produced in the PVH, contributes to energy balance control.

Strengths:<br /> There has been conflicting literature on the role of OT in feeding behavior. Although pharmacological and genetic approaches have suggested an anorexic effect of OT, knockout of OT or OT receptor has minimal effect on feeding. To address this apparent discrepancy, the authors use conditional knockout models to manipulate OT signaling. This allows not only temporal control of OT and OT receptor, but also allows investigation of signaling in different brain regions (versus, for example, whole body or organ). That the conditional knockout mice display hyperphagia and obesity begins to settle this conflict in the literature.

Weaknesses:

1) There is not much conceptual advance in the study. The data largely confirm what pharmacological and RNAi knockdown studies have previously demonstrated.

2) The finding that IP injection of OT partially rescues the phenotype of the KO mouse lacks rigor and proper controls. It is important to show that the dose of OT used does not influence body weight in wildtype mice in order to make the conclusion that it "rescues" the phenotype of the KP mouse.

3) There is little anatomical precision in the manipulation of OT receptors in the "posterior hypothalamus." Understanding which of these brain regions (e.g. ARH, VMH, LHA, DMH, others?) is involved in mediating these effects would be very informative.

Reviewer #1 (Public Review):

Activation of TEAD-dependent transcription by YAP/TAZ has been implicated in the development and progression of a significant number of malignancies. For example, loss of function mutations in NF2 or LATS1/2 (known upstream regulators that promote YAP phosphorylation and its retention and degradation in the cytoplasm) promote YAP nuclear entry and association with TEAD to drive oncogenic gene transcription and occurs in a significant majority of mesothelioma patients. High levels of nuclear YAP have also been reported for a number of other cancer cell types. As such, the YAP-TEAD complex represents a promising target for drug discovery and therapeutic intervention. Based on the essential functional role for TEAD palmitoylation at a conserved cysteine site, discovered by the authors of this manuscript, several groups have successfully targeted this site using both reversible binding non-covalent TEAD inhibitors (i.e., flufenamic acid (FA), MGH-CP1, compound 2 and VT101~107), as well as covalent TEAD inhibitors (i.e., TED-347, DC-TEADin02, and K-975), which have been demonstrated to inhibit YAP-TEAD function and display anti-tumor activity in cells and in vivo.

Here, Hu et al. disclose the discovery of a new class of reversible TEAD inhibitors that putatively binds across a new site within the auto-PLM pocket across the TEAD family of proteins. To date, reported inhibitors that target this site (flufenamic acid (FA), TED-347, MYF-01-037, K975, VT103, MGH-CP1) function by binding to the same hydrophobic palmitate binding pocket (PBP) which is highly conserved. The novel TEAD inhibitor is identified from a screen of 30,000 compounds using recombinant TEAD2 and TEAD4 proteins and TEAD2 and TEAD4 click-ELISA assays. A molecule termed TM2 was found to show the strongest inhibition on TEAD auto-palmitoylation (IC50= 156nM for TEAD2 and 38nM for TEAD4) and is chosen for further characterization. A co-crystal structure of TEAD2 YAP binding domain in complex with TM2 was solved and revealed that TM2 not only occupied the familiar hydrophobic PLM site but also binds within a new pocket. Notably, the charge properties of this site might be compatible with the development of more drug-like analogs with enhanced hydrophilic properties. TM2 is profiled against several malignant pleural mesothelioma (MPM) cell lines (IC50 = 26-157nM) and activity correlates well with in vitro TEAD palmitoylation inhibition. Notably, consistent with results for related inhibitor classes, TM2 is not active as a monotherapy in cells beyond NF2 deficient cell lines, although cell type selective cytotoxicity is clearly shown.

Strengths:

This newly reported chemical series is highly tractable and could provide an excellent starting point for the development of effective TEAD inhibitors. The reported ability of TM2 to bind to a new site within the conserved TEAD auto-PBP site could enable this area of drug discovery.

Weaknesses:

The authors claim that TM2 is "more specific" than existing inhibitors, with respect to its ability to regulate YAP/TAZ-TEAD target gene expression. However, this statement is based on a comparison of unrelatable RNA-Seq datasets. TM2-regulated expression is evaluated in the context of cell (mono)culture following a continuous duration of target engagement. Comparison is made to the covalent inhibitor K975 based on a published dataset involving analysis of a drug-treated NCI-H226 tumor xenograft (i.e, in vivo drug treatment). Heterogeneity in cells, time point of analysis and duration and target engagement preclude the possibility of any reasonable comparison of these datasets. The ability of TM2 to bind within a new site is a significant finding. The comparison of TM2 binding mode to other inhibitors is based on structures of non-TEAD2 co-crystal structures. It will help significantly to clarify the differences in the side chains within this site across TEAD family members (i.e., TEAD1-4). The robustness of the proposed synergistic activity of TM2 in non-responsive cell lines is unclear.

Reviewer #1 (Public Review):

This is timely and foundational work that links cellular neurophysiology with extracellular single-unit recordings used to study LC function during behavior.

The strengths of this paper include:<br /> 1. Providing an updated assessment of LC cell morphology and cell types since much of the prior work was completed in the late 1970s and early to mid-1980s.<br /> 2. Connecting LC cell morphology with membrane properties and action potential shape.<br /> 3. Showing that neurons of the same type have electrical coupling

Collectively, these findings help to link LC neuron morphology and firing properties with recent work using extracellular recordings that identify different types of LC single units by waveform shape.

Another strength of this work is that it addresses recent findings suggesting the LC neurons may release glutamate by showing that, at least within the LC, there is no local glutamatergic excitatory transmission.

Weaknesses:<br /> The authors also propose to test the role of single LC neuron activity in evoking lateral inhibition, as well as proposing that electrical coupling between LC cell pairs is organized into a train pattern. The former point is based on a weak premise and the latter point has weak support in their data given the analyses performed.

Point 1: lateral inhibition in the LC<br /> The authors write in the abstract that "chemical transmission among LC noradrenergic neurons was not detected" and this was a surprising claim given the wealth of prior evidence supporting this in vitro and in vivo (Ennis & Aston-Jones 1986. Brain Res 374, 299-305; Aghajanian, Cedarbaum & Wang 1977. Brain Res 136, 570-577; Cedarbaum & Aghajanian. 1978 Life Sci 23, 1383-1392).

Huang et al. 2007 (Huang et al. 2007. Proc National Acad Sci 104, 1401-1406) showed that local inhibition in the LC is highly dependent on the frequency of action potentials, such that local release requires multiple APs in short succession and then requires some time for the hyperpolarization to appear (even over 1 sec). This work suggests that it is not a "concentration issue" per se, rather it is just that a single AP will not cause local NE release in the LC. Although the authors did try 5APs at 50Hz this may not be enough to generate local NE release according to this prior work. A longer duration may be needed. Additionally, although the authors incubated the slices with a NET inhibitor, that will not increase volume transmission unless there is actually NE release, which may have not happened under the conditions tested. In sum, there is no reason to expect that a single AP from one neuron would cause an immediate (within the 100 msec shown in Fig 3B) hyperpolarization of a nearby neuron. Therefore, the premise of the experiment that driving one neuron to fire one AP (or even 5AP's at 50Hz in some) is not an actual test of lateral inhibition mediated by NE volume neurotransmission in the LC. Strong claims that "chemical transmission...was not detected" require substantial support and testing of a range of AP frequencies and durations. Given the wealth of evidence supporting lateral inhibition of the LC, this claim seems unwarranted.

Point 2: Train-like connection pattern<br /> Demonstrating that connected cell pairs often share a common member is an important demonstration of a connection motif in the LC. However, a "train" connection implies that you can pass from A to B to C to D (and in reverse). However, the authors do not do an analysis to test whether this occurs. Therefore, "train" is not an appropriate term to describe the interesting connection motif that they observed.

In fact, writing A↔B↔C in the paper implies a train without direct support for that form of electrical transmission. For example, in Fig. 6C, it is clear that cell 6 is coupled to cell 1 and that cell 6 is also coupled to cell 8. In both cases, the connection is bilateral. Using the author's formatting of A↔B↔C , would correspond with Cell 6 being B and cells 1 and 8 being A and C (or vice versa). However, writing A↔B↔C implies a train, whereas one can instead draw this connection pattern where B is a common source:<br /> A C<br /> . .<br /> . .<br /> B

An analysis showing that spikes in A can pass through B and later appear in C is necessary to support the use of "train". The example in Fig. 6C argues against train at least for this one example.

Although the analysis is possible to do with the authors' substantial and unique data set, it should be also noted that prior work on putative electrical coupling in extracellular recordings from rat LC showed that trains among 3 single units occurred at an almost negligible rate because out of 12 rats "Only 1 triplet out of 22,100 possible triplet patterns (0.005%) was found in one rat and 4 triplets out of 1,330 possible triplet patterns (0.301%) were found in the other rat." and moreover patterns beyond 3 units were never observed (Totah et al 2018. Neuron 99, 1055-1068.e6).

Reviewer #1 (Public Review):

The authors generated a mouse model for GEN1 and a B-cell conditional MUS81 flox allele to create B-cells devoid of both proteins using two Cre drivers. Mb1-Cre driven by the Cd79a promoter depletes MUS81 in pre-pro B cells, whereas Cd23-Cre is driven by the Fcer2a promotor to deplete MUS81 in immature, transitional and late B cells. The results show that GEN1 and MUS81 are necessary for differentiation into pro-B cells, the expansion and maintenance of pro-B cells, or both. The CD23-Cre line showed that naïve B cells can persist without MUS81 and GEN1, but they are impaired for germinal center formation. Ex vivo analysis identified a proliferation defect upon stimulation and increased cell death due to apoptosis. RNA seq analysis of such cells revealed activation of the p53 pathway and the type I interferon response. Metaphase chromosomal analysis revealed hallmarks of genomic instability including chromosomal abnormalities, including breaks, fragments, fusions, and radials. The symmetry of the breakages suggests that they arose from unresolved HR intermediates.

Strengths:<br /> This is a well-executed study, and the results support the conclusions. While the role of both nucleases has been extensively studied in cellular systems, this is the first analysis at the organismic level with a focus on B-cell development.

Weaknesses:<br /> The nature of the actual substrate(s) for MUS81 and GEN1 remains unclear and the significance of unresolved HR intermediates for the observed phenotype remains an inference from the type of chromosome aberrations observed. The DNA damage analyses presented would benefit from being clarified and extended.

Reviewer #3 (Public Review):

To investigate their role in B cell development and function, the authors conditionally delete of the structure-specific endonucleases GEN1 and MUS81 at early and late stages of B cell development. Using MB1-Cre, the authors find GEN1 and MUS81 play redundant and essential roles in B cell development, leading to an almost complete depletion of B cells in the pro-B and later stages that was rigorously shown. Conditional deletion of Mus81 in transitional B cells by CD23-cre circumvented this developmental delay, but led to a severe defect in germinal center formation in lymph nodes, Peyer's patches and the spleen specifically in double-deficient cells though total B cell numbers were similar to WT. Further characterization by in vitro stimulated cells revealed that loss of both Gen1 and Mus81 dramatically reduces cell proliferation, induces G2/M checkpoint activation, apoptosis and genome instability. The authors conclude that these defects are caused by MUS81/GEN1's shared role in processing recombination intermediates created by replication stress but do not show the cells experience replication stress. Further, there is no characterization of class switch recombination or IgH damage in the cells, which feels noticeably absent. Finally, the DNA damage analyses presented would benefit from being clarified and extended.

Overall this is an elegant and straightforward dissection of the role of GEN1 and MUS81 in B cell development, but in its current form the manuscript does not directly connect the observed phenotypes to the molecular role of GEN1/MUS81 in DSB repair.

Reviewer #1 (Public Review):

This paper provides a progress report on methods development that was initiated previously by the same authors to identify macromolecular complexes in cryo-EM images of cells. Whereas others have proposed to perform this task in 3D reconstructions from tomographic tilt series, the method discussed here (2DTM) uses template matching with known reference structures against individual 2D projection images. This concept was introduced in previous work. In the current paper, the authors show that 2DTM can be used to classify distinct molecular populations. They demonstrate this by distinguishing cytoplasmic mature 60S ribosome particles from a nuclear pre-60S population. They also define a maximum likelihood metric that assigns a probability for each particle belonging to each class. The paper reads a bit dense, and one could discuss how big the advance is over the previous work by the same authors. But the general topic (of being able to identify distinct particle populations in cells) is an important one.

However, I think one major concern needs to be addressed much more openly in a revised version of the paper: that of potential model bias of incorrect references. The single-particle field previously suffered the problems of Einstein from noise can cause with the debacle around an incorrect HIV trimer reconstruction that was the result of picking particles with a high-resolution reference. I realise that the problem here is a different one, but similar problems of model bias may exist. In fact, the observation on page 18 that the reconstruction from the picked particles was estimated by FSC to be 3.5A, yet the resulting map had to be filtered at 10A to limit the noise, is a strong indication that model bias does play a large role in the identification of particles. This bias must affect the measured SNR scores, and thus the metrics presented. It also suggests that part of the identified picks may in fact not be true 60S ribosomes, but false positives. This would then affect the conclusions drawn. If the authors disagree (and I suspect they do), they should set out clear arguments for their case. Also, they should discuss how potential overfitting or model bias would affect their new metrics for particle classification in the discussion. Currently, the only reference to the dangers of overfitting is on page 16, merely referring to their previous paper.

Perhaps points for additional discussion could include:<br /> 1) In the light of overfitting, I was wondering whether one could detect the 80S ribosomes also through 2DTM using the 40S subunit as a reference.<br /> 2) The authors have been wise in selecting the 60S ribosome as a test case. Probably, because of its size and RNA content, for many instances of this complex the SNR is sufficiently high for detection. However, if less careful authors would choose a smaller target, what would happen? What would be the pitfalls and how could they be avoided?

Reviewer #3 (Public Review):

Lucas et al. expand upon their prior work using 2D high-resolution template-matching (2DTM) to localize macromolecules directly in cells. This clearly presented work contains multiple key highlights using the Saccharomyces cerevisiae 60S maturation process as an example. It demonstrates that focused ion beam (FIB)-milling preserves sufficient high-resolution (better than 4 Å) information for the 2DTM to effectively locate macromolecules in the dense cellular environment. In addition, it demonstrates that the classification of the detected macromolecules can be effectively determined by comparison of the signal-to-noise ratios obtained with 2DTM against templates with relatively minor differences. Furthermore, the authors detail a maximum likelihood approach to specify the confidence of the class assignment for a macromolecule within a mixed population. The authors take advantage of extensive prior knowledge of the 60S biogenesis process to thoroughly evaluate and demonstrate the utility of the 2DTM methodology and accompanying classification strategy.

2DTM has great potential to motivate a broader adoption of cryo-EM for those more interested in robust localization of macromolecules of known structure rather than de novo high-resolution structure determination through conventional averaging approaches. Conventional averaging approaches for cryo-EM data notably suffer at the level of classification for which the results can vary greatly based on choice of a multitude of parameters. The classification strategy presented here for 2DTM should be reproducible and the parameter choice (i.e., priors) more straightforward.

Reviewer #3 (Public Review):

Numerous studies have demonstrated that the neural dynamics on different brain areas encode elapsed time, yet it has proven challenging to examine how these population clocks emerge over the course of learning because most temporal tasks require many training sessions. In this manuscript the authors use a simple timing task that can be learned in a single day, and accompany the changes in neural dynamics in the mPFC and STR of the first and second day on the task. The most interesting finding is a switch in which the mPFC provides a better code than the STR for elapsed time on the first day, but the STR provides a better code than the mPFC on the second day. Consistent with the increased encoding of time in the mPFC early in training, muscimol inactivation of the mPFC impaired learning of the task, but not performance in trained animals. Overall this study provides a number of novel contributions to our understanding of temporal processing, and show the first example of learning-dependent switch from the dynamics of the mPFC to that of the STR encode time.

Reviewer #1 (Public Review):

This is an interesting study supporting the notion mPFC is involved in early learning stages while the striatum becomes more engaged as animals become proficient in a temporal task. However, I have several concerns about whether the results support the main conclusions of the paper. First and foremost, it is difficult to dissociate the role of mPFC and the striatum linked with a better representation of elapsed time with learning from the operational learning aspects of the task. The latter include the increase in attention of sensory inputs associated with the nose poke, an increase in precision of movement kinematics (less body and face movements during the nose poke), and a more developed reward expectation from learning to time the 1.5 s. The authors should perform careful analysis to try to dissociate the learning of temporal and non-temporal factors and the involvement of the two areas. Second, I have comments of the decoding analysis. It is now well known that the neural activity associated with timed behaviors scales with time. Since the decoding was performed on truncated trials at 1.5s, the analysis will not capture the neural pattern of activation in longer trials. Thus, this is decoding of absolute elapsed time using activity of neurons that probably are encoding relative trial length. In addition, it seems that both areas encode the beginning and end of the trials, with high densities in the diagonal only on the initial and final bins, rather than the elapsed time across all the trials. These results could be related with learning of non-temporal factors discussed previously. Furthermore, the decoding of elapsed time both areas went down from early to late trials in the experiment of one session, supporting the notion that the striatum does not take over, although the rats learned to time the interval. This is contrary to the conclusions of the paper. Finally, animals with mPFC inactivations did not change behavior of the first session, but they partially learned on sessions two, three and five (with an increase mu2). How are these findings matching with the observation that mPFC decoding performance dramatically lowers on the second day?

Reviewer #1 (Public Review):

In this manuscript by Chen et al., the authors use live-cell single-molecule imaging to dissect the role of DNA binding domains (DBD) and activation domains (AD) in transcription factor mobility in the nucleus. They focus on the family of Hypoxia-Inducible factors isoforms, which dimerize and bind chromatin to induce a transcriptional response. The main finding is that activation domains can be involved in DNA binding as indicated by careful observations of the diffusion/reaction kinetics of transcription factors in the nucleus. For example, different bound fractions of HIF-1beta and HIF-2alpha are observed in the presence of different binding partners and chimeras. The paradigm of interchangeable parts of transcription factors has been eroded over the years (the recent work of Naama Barkai comes to mind, cited herein), so the present observations are not unexpected per se. Yet, the measurements are rigorous and well-performed and have the important benefit of being in living cells. Enthusiasm is also dampened by the exclusive use of one technique and one analysis to reach conclusions.

Reviewer #3 (Public Review):

In this work, Chen et al. measured the DNA binding dynamics of HIF transcription factors using single-particle tracking. In particular, they examined the impact of heterodimerization between the alpha and beta subunits, the integrity of the DNA binding domain and the nature of the transactivation domain in DNA binding. As expected, they found that the stoichiometry between the heterodimerization partners directly impacts the bound fraction of the beta subunit which is devoid of a DNA binding domain. More interestingly, using domain swaps between HIF-1alpha and HIF2-alpha they found that the transactivation domain of the alpha subunit plays a major role in determining the bound fraction of the beta subunit (and thus the heterodimer). This work is important because it increases our understanding of how TF search the genome, beyond the traditional conception of the "addressing tag" provided solely by the DNA binding domain. This work is thus of interest to the broad audience of scientists studying gene regulation.

Reviewer #1 (Public Review):

The authors report a public browser in which users can easily investigate associations between PRSs for a wide range of traits, and a large set of metabolites measured by the Nightingale platform in UKBB. This browser can potentially be used for identifying novel biomarkers for disease traits or, alternatively, for identifying novel causal pathways for traits of interest.

Overall I have no major technical concerns about the study, but I would encourage the authors to revisit whether they can find a more compelling example that can better showcase the work that they have done. I understand that this is partly a resource paper but I think the resource itself can have more impact if the paper provides a clearer use-case for how it can drive novel biological insight.

PRS construction: It's unclear how well the PRS work. Should the reader prefer the stringent or lenient PRS? Perhaps there could be some validation with traits that have decent sample sizes in UKBB. Was there any filtering to remove traits with few GWS hits, low sample sizes, or low SNP heritability as these are unlikely to produce useful PRSs?

Reviewer #3 (Public Review):

Fang et al. created an atlas for associations between the genetic liability of common risk factors or complex disorders and the abundance of small molecules as well as the characteristics of major apolipoproteins in blood. The whole study is well executed, and the statistical framework is sound. A clear strength of the study is the large array of common risk factors and disease analyzed by means of polygenic risk scores (PRS). Further, the development of an open access platform with appealing graphical display of study results is another strength of the work. Such a reference catalog can help to identify novel biomarkers for diseases and possible causative mechanisms. The authors further show, how such a systematic investigation can also help to distinguish cause from causation. For example, an inflammatory molecule readily measured by the NMR platform and strongly associated in observational studies, is likely to be a consequence rather than a cause for common complex diseases.

However, in its current form, the study suffers from some weakness that would need to be addressed to improve the applicability of the 'atlas'. This includes a distinction of locus-specific versus real polygenic effects, that is, to what extend are findings for a PRS driven by strong single genetic variants that have been shown to have dramatic impact on small molecule concentrations in blood. Further, it is unclear how much NMR spectroscopy adds over and above established clinical biomarkers, such as LDL-cholesterol or total triglycerides. This is in particular important, since the authors do not adequately distinguish between small molecules, such as amino acids, and characteristics of lipoprotein particles, e.g., the cholesterol content of VLDL, LDL or HDL particles, the latter presenting the vast majority of measures provided by the NMR platform. Finally, the study would benefit from more intriguing or novel examples, how such an atlas could help to identify novel biomarkers or potential causal metabolites, or lipoprotein measures other than the long-established markers named in the manuscript, such as creatinine or lipoproteins.

Reviewer #1 (Public Review):

This manuscript sets up a well-reasoned study and capitalizes on a very impressive long-term dataset. Their methods are generally sound and well explained. The argument for paternal kin recognition here could be compelling, but at this stage, it does not seem possible to rule out a simpler cognitive mechanism, i.e. gorillas direct their aggression toward unfamiliar and/or outgroup individuals. The authors note the difficulty of indexing familiarity versus kinship because there were very few out-group dyads (two individuals from different natal groups) that were related. But were there any in-group dyads that were not related? If so, then it might be plausible to add the natal group as a co-variate to parse these mechanisms. A result in either direction could be very compelling. Another concern is dealing with age- which is incredibly difficult with data like this and the authors have offered a reasonable compromise here by settling on a single term (age difference). It makes good sense that the difference in age between two partners should contribute to shaping the interactions between a dyad, however, each of the social behaviors here is more or less common depending on age in most species (including gorillas to the best of my knowledge) with social play generally decreasing with age while social grooming and aggression increase. I understand that it is not the intent of this manuscript to describe the ontogeny of social bonds, however, as the dataset includes individuals ranging from infants through adults, is it possible that such age-related changes in social behavior are affecting the results? Whether or not the authors find strong indications that gorillas can recognize paternal kin, specifically, beyond in-group/out-group familiarity, this manuscript represents an important contribution to the field.

Reviewer #3 (Public Review):

In this study the authors investigated whether mountain gorillas of varying ages behave differently toward their siblings compared to non-siblings, and, how this bias varied based on whether individuals were full or maternal half-siblings vs. paternal siblings, opposite or same-sex siblings, and close or far apart in age (with age as a continuous variable).

This study has two major strengths:<br /> One is its long-term dataset. Authors document social interactions for 157 individuals over 14 years on wild mountain gorillas. This is amazing!

A second major strength is the opportunity this dataset and study system provides to test predictions about proposed mechanisms for kin recognition in primates. The authors do a good job of making these details about their study system and their predictions clear:

Kin selection is a proposed mechanism for the evolution of cooperative behavior. For it to operate, animals must have some mechanism by which to recognize their genetic kin and affiliate and cooperate differentially with these kin than with non-kin. However primatological studies have revealed that routes to kin recognition that are immediately clear. First, there are many examples of cooperation with non-kin. Second, in certain species, individuals bias affiliation and cooperation toward maternal but not paternal kin. Because these maternal-kin-biased species are ones with low male reproductive skew (many females mate with many males and many males father infants) and where mothers are sole caregivers of offspring, both the mating system and the familiarity of growing up together under the care of the same mother (especially if close in age) are proposed to drive affiliative and cooperative biases. Mountain gorillas provide a strong model to test these predictions because there is low male reproductive skew and individuals may live in cohesive groups with both maternal siblings and paternal siblings of all ages throughout their lives.

However, this study has two major weaknesses.

First, it lacks clarity in the actual measures of kin bias: that is - how dyadic social interactions and relationships manifest in mountain gorillas and how these change throughout life as relevant to the measures used.<br /> For example, the authors provide little information on the ages of the siblings involved in the study (only that the median was 9.7 years). How do these ages match to different developmental stages and dimensions of mountain gorilla social interaction? For instance, the frequency of play, one of the three social affiliative social measures employed, might vary considerably based on age. In many other species, it occurs more often between immature individuals or between a mature and immature individual rather than between mature ones.

Relatedly, siblings who affiliate frequently do not necessarily need to have reduced aggression. Studies of dyadic affiliative bonds in baboons and chimpanzees both indicate that in certain contexts individuals who affiliate more may also have increased conflict. What might distinguish certain more cooperative bonds from others, for example, is what happens after this conflict. This is not something the authors need to measure in this study but it would be helpful to have such nuances of relationships discussed, or at least to provide the reader with more context for interpreting the behavioral results of affiliation and aggression as assays for kin-bias and potential fitness benefits associated with this bias.

Second, relatedly - there was no basis provided for the evolutionary function of sibling affiliation - that is, how might affiliation as measured by proximity, grooming, and play, contribute to cooperation and/or improved fitness in mountain gorillas? The existence of some form of dyadic social bond benefit (such as alliances, or improved survival) is necessary for kin selection to be in play. What might the functions of sibling relationships be in mountain gorillas? What are modes of dyadic cooperation like alliances described in other species (e.g. alliances between cercopithecine monkey mothers and sisters)? Providing some theoretical justification/context for the existence of benefits that might be enabled by kin selection in mountain gorillas would strengthen the study considerably.

One example of where such a nuanced explanation of both social measures and relationship function was provided well is when the authors interpreted their finding that opposite-sex non-siblings showed heightened rates of aggression compared to opposite-sex siblings and same-sex siblings and same-sex non-siblings. Here, they discussed how an opposite-sex non-sibling relationship is one that has functional importance relevant to reproduction and that increased aggression might represent sexual coercion.

Reviewer #1 (Public Review):

This is a randomized controlled trial to assess the impact on the COVID-19 progression (hospitalization, oxygen supplementation need, and/or death within 14 days) of 3 different mAbs. As only one of the assessed mAbs was shown to be active in vitro against the Omicron variant, which emerged after the study onset, the trial was interrupted for potential futility. The authors compared the activity of three mAbs in patients infected with Delta and in those with Omicron variant. Symptoms duration was shorter in patients treated with sotrovimab than in those treated with the other mAbs, mainly among Omicron infected patients. No disease progression was observed in the Delta group, while two patients infected with omicron and treated with bamlanivimab/etesevimab presented progression.

The strength of this study is that it provides clinical data about the efficacy of different mAbs on COVID-19 progression against the Omicron variant assessed by a pragmatic RCT.

The limitation is the reduced sample size.

The results are useful in confirming the usefulness of Sotrovimab in the management of patients with Omicron BA 1 and BA 1.1. They cannot be applied to other Omicron sub-lineages.

Considering the availability of antiviral agents for early use in patients with asymptomatic or mild infection but with risk factors for COVID-19 progression and the longer duration of symptoms observed for casirivimab/imdevimab compared with sotrovimab, the conclusion about the potential role of this mAb against Omicron seems inappropriate.

Reviewer #3 (Public Review):

The MANTICO trial was a 319-patient randomized comparative effectiveness trial of three monoclonal antibodies for COVID-19, during a period of time when the Delta variant was starting to become replaced by the Omicron variant. Due to this unique time period and patient-level variant typing, the trial was able to compare the three antibodies, stratified by variant. Overall, their clinical findings were consistent with in vitro data regarding these antibodies versus variants; this result is of interest as authorization and treatment decisions are being made based on in vitro data, which do not always prove consistent with clinical outcomes.

The major strength is the randomized design, which allows strong causal inference. The major weakness is the limited sample size, due to 2 of the antibodies becoming unavailable, thus forcing the authors to stop the trial early. In addition, as fortunately almost all patients did well, the primary outcome of hospitalization, need for oxygen, or death was non-informative, as were most secondary outcomes, and the authors hinged their conclusions on 1 of multiple secondary outcomes (thus raising the possibility of false discovery due to multiple comparisons).

Nonetheless, the authors largely achieved their aims, and their results generally support their conclusions.

The likely impact of the work is that it reassures the public that authorization and treatment decisions being made on in vitro data (test tube experiments) are likely reliable, as this study found clinical outcomes consistent with in vitro data. Thus, although the current variants are different from the variants treated in this trial, their overall results are compelling.

Reviewer #1 (Public Review):

While some microbes have evolved to need an animal host for completion of the life cycle or survival, termed "obligate pathogens" (i.e. Plasmodium falciparum, viruses, Neisseria gonorrhoeae), there are many bacteria and fungi that can cause disease in insect or mammalian hosts, but can survive okay in the environment and do not need a host in order to survive, persist, and replicate. These microbes are often described as "accidental pathogens," where they evolved in the absence of a host to survive durable conditions in the environment, and only caused disease when they are accidentally exposed to a host (i.e. breathing in spores or traumatic inoculation). In this work, the authors set out to determine whether selection for Saccharomyces cerevisiae's ability to bind plastic in the absence of a host resulted in enhanced virulence when the yeast was exposed to a host. In doing so, they would be experimentally showing that adaptation to environmental conditions without a host, can "accidentally" produce pathogens. In this work, the authors indeed demonstrated that S. cerevisiae which had been selected for its ability to adhere to plastics evolved multicellular phenotypes and enhanced virulence in the Galleria mellonella wax moth host. In doing so, they effectively show experimental proof of the accidental virulence hypothesis, which can serve as the basis for future studies to better understand existing non-obligate microbes, and how new changes/exposures in the environment can cause the adaptation of microbes, which may result in the emergence of new "accidental" human pathogens. The work also investigated the evolutionary relationship between the different forms of multicellularity and uncovered that there was a strong correlation between the emergence of multiple multicellular phenotypes over cycles of plastic adhesion selection, which is a new finding compared to previous reports that these phenotypes in the environment or in laboratory/mutant strains are independent.

The conclusions drawn in this paper are well supported by the data, and the experiments are well-designed and straightforward and presented in a way that is generally able to be understood. This study is of great interest to both microbiologists who study how microbes adapt to the environment and those who study microbes in the context of infectious disease. This study experimentally proves the tenet of the accidental virulence hypothesis that adaptation/evolutionary selection to environmental factors may incidentally enhance the ability to survive within hosts.

Additionally, the authors conducted the experiments in a robust, well-controlled manner, and systematically analyzed the multicellular phenotypes. They assayed the multicellular phenotypes in a clearly defined manner and were able to characterize the traits of a large number of individual clones grown encompassing different timepoints, sexual and asexual reproduction, strains, and selection. Additionally, their virulence studies in G. mellonella show an extraordinary amount of work, which allowed them to see the increase in virulence (~30%) in the hyper-multicellular phenotype after performing infections using 40 different isolates from the experiment.

The authors also sought to find a genetic basis for the increase of multicellular phenotypes and virulence following plastic adherence selection. To do so, they studied the length of the FLO11 gene in their S. cerevisiae isolates. FLO11 length had been previously implicated in fungal adhesion and fungal virulence, which could offer a reason for the enhanced virulence phenotype following selection for plastic adherence. While their findings show the ancestral and control isolates did not have increased FLO11 length while many of the plastic-selected isolates did, there was not a definitive correlation between the multicellular phenotypes and FLO11 length. The authors did not pursue an additional investigation into the genetic basis of these adaptations.

Two aspects the reader must consider are the host and the microbe used in these virulence experiments. S. cerevisiae is an interesting choice to use because it is not considered to be a "pathogenic" microbe, although there are rare cases in which it can cause disease in humans. However, previous studies have shown S. cerevisiae has been shown to cause disease in G. mellonella, which can also be seen in the survival curves presented in this paper. Therefore, using G. mellonella in this study shows that the selection process resulted in increased virulence in the host. To strongly show the accidental virulence/pathogen hypothesis, a host-microbe pair where disease does not normally/typically occur could be used. In the context of this study, it could be done using a mouse model infected with S.cerevisiae. In doing so, it could show that environmental selection in the absence of a host has the ability to turn a "non-disease-causing microbe" into a disease-causing one, rather than a disease-causing microbe into a more disease-causing microbe. This could also make a more applicable leap to human infectious diseases.

Reviewer #1 (Public Review):

This is a very thorough biochemical work that investigated the ParABS system in pSM19035 by Volante et al. Volante et al showed convincingly that a specific architecture of the centromere (parS) of pSM19035 is required to assemble a stable/functional partition complex. Minimally, four consecutive parS are required for the formation of partition complex, and to efficiently activate the ATPase activity of ParA. The work is very interesting, and the discovery will allow the community to compare and contrast to the more widespread/more investigated canonical chromosomal ParABS system (where ParB is a sliding CTPase protein clamp, and a single parS site is often sufficient to assemble a working partition complex). All the main conclusions in the abstract are justified and supported by biochemical data with appropriate controls. A proposed multistep mechanism of partition complex assembly and disassembly (summarized in Fig 6) is reasonable. Perhaps the only shortcoming of this work is that the team does not yet get to the bottom of why four consecutive parS are needed.

Reviewer #3 (Public Review):

Drs. Volante, Alonso, and Mizzuchi presented a milestone experimental finding on how the distinct architecture of centromere (ParS) on bacterial plasmid drives the ParABS-mediated genome partition process. Rather than driven by cytoskeletal filament pushing or pulling as its eukaryotic counterpart, the genome partition in prokaryotes is demonstrated to operate as a burnt-bridge Brownian Ratchet, first put forward by the Mizuuchi group. To drive directed and persistent movement without linear motor proteins, this Brownian Ratchet requires two factors: 1) enough bonds (10s' to 100s') bridging the PC-bound ParB to the nucleoid-bound ParA to largely quench the diffusive motion of the PC, and 2) the PC-bound ParB 'kicks" off the nucleoid-bound ParA that can replenish the nucleoid only after a sufficient time delay, which rectifies the initial symmetry-breaking into a directed and persistent movement. Although the time delay in ParA replenishment is established as a common feature across different bacteria, the binding properties of PC-bound ParB vary greatly, which begs the question of how Brownian Ratcheting adapts to different cellular milieu to fulfill the functional fidelity.

The finding in this work presented a new but important twist in the Brownian Ratchet paradigm. The authors showed that in the pSM19035 plasmid partition system, only four contiguous ParB-binding repeats in ParS are required for the ParA-ParB interactions that drive the PC partition. In other words, only four chemical bonds are needed for the PC partition. Crucially, the authors further demonstrated that distinct orientation of the ParB-binding repeats is required for this fidelity by their state-of-art biochemistry and reconstitution experiments. The authors then elaborated on a possible mechanism of how the smaller number of PC-bound ParB can drive directed and persistent PC movement by interacting with nucleoid ParA. If I understand correctly, in their proposed scheme, due to their specific orientations, when two of the ParS-bound ParB molecules bind to the two nucleoid-bound ParA molecules there arises a torsional/distortional stress. Consequently, the thermal fluctuations preload the forming bonds, triggering the dissociation of the two ParB molecules from the PC. And the remaining PC-bound ParBs may kick off the ParAs that have a time delay in DNA-rebinding, while ParB molecules will replenish the ParS to initiate the next round. In this proposal, the key conceptual leap is that not only the substrate but the cargo remodels to underlie the Brownian Ratcheting.

Reviewer #1 (Public Review):

Dhawan et al examine the involvement of the perirhinal cortex (PRC) and ventral hippocampus (vHPC) in different forms of approach-avoidance conflict. The paper is well written, the methods used are appropriate for the question asked and, in general, the authors have succeeded in identifying the PRC and vHPC involvement. As such, the paper will eventually have an impact on thinking about the roles of the PRC and vHPC in different forms of learning and memory. However, this impact is conditional on the authors' responses to queries regarding experimental design, group allocations and statistical analyses.

Reviewer #3 (Public Review):

The manuscript by Dawani et al. extends previous work by the same group and others to dissect brain circuits that implement decision-making in the presence of conflicting motivation using approach-avoidance behavioral tasks. The current investigation introduces multiple behavioral paradigms in which different types of signals or cues are associated with rewarding or anxiety-inducing conditions. The authors then place these cues in conflict in an attempt to identify the involvement of key brain areas in different aspects of motivational conflict. In particular, they compare processing when objects are used as cues vs when "contextual" features of the overall environment (wall color and texture) convey the motivational conflict. They then use optogenetic inactivation of brain areas that have been implicated in this type of behavior to identify their involvement in each of the different task variations. Using these approaches, they find evidence suggesting that the perirhinal cortex is important for processing conflicting motivational signals under certain conditions. While the idea that the perirhinal cortex plays such a role had been proposed in previous models it had not been tested directly making this a novel finding. In addition, the authors are able to contrast the involvement of this circuit with that of the hippocampus, which had previously been considered the major region responsible for this type of conflict processing. Consistent with previous work, their findings suggest that the Hippocampus is involved when cues are contextual but that the Perirhinal cortex, rather than the Hippocampus, plays an analogous role when conflicting signals are communicated by combinations of objects.

In general, the behavioral experiments as well as controls are well designed, and analysis of the resulting data is also consistent with current practices. Despite this overall quality as well as the strength of some of the optogenetic effects, however, the known involvement of the perirhinal cortex in encoding and recognition of object memory, particularly for complex or combined stimuli (e.g. Haskins et al. Neuron 2008, Ohnuki et al. Comm. Bio. 2020) creates a confound that the authors do not completely overcome. Specifically, they do not exclude the possibility that this area may be involved in recognizing objects with different motivational associations when they are presented together. While some of the evidence presented argues against this possibility, additional analysis and experiments are needed to more conclusively establish that the perirhinal cortex is involved in motivational conflict itself, and that the suppression effects they observe are not simply due to its memory-related functions. In particular, it would be beneficial to suppress the region at distinct time periods within the task to isolate different contributions. Beyond this major issue, there are also several minor changes to the figures and text needed for overall clarity.

Reviewer #1 (Public Review):

Ahmed et al. examine the changes in the enhancer landscape that may contribute to the transition from Barrett's oesophagus (BO) to oesophageal adenocarcinoma (OAC), building upon their past works looking at the chromatin changes within this transition. They identified a repertoire of eRNA regions that display differential expression between OAC and BO, validating their association to enhancers using H3K27ac levels, CUT&TAG, and KAS-seq. The authors look further into the target genes and regulatory TFs that may define eRNA levels, finding several TFs - AP1, KLF5, CTCF, and HNF1 - that have previously been implicated in OAC and confirming that sets of eRNA target genes were downregulated upon depletion of these TFs. Ahmed et al. also showed that eRNA target genes were relevant to OAC phenotypes, akin to that of DEGs in whole RNA-seq datasets. The authors lastly validate the activity of certain eRNAs targeting JUP, MYBL2, and CCNE1 using functional methods to confirm enhancer activity and effects on cell viability, as well as clinical features such as the age of diagnosis and survival time.

The landscape of eRNA activity seems to be well validated. However, deeper analyses to support the relevance of the function of key eRNAs, their specificity in regulating target genes, and the interaction with other OAC features would further support these findings.

Reviewer #3 (Public Review):

This study uses RNA-seq data sets from pre-cancerous Barrett's Oesophagus (BO) and Oesophageal adenocarcinoma (OAC) patients to identify enhancer-associated (e)RNAs that are specifically associated with the transformed OAC state. Integrative genomics and functional analyses using patient data and data from an OAC-derived cell line provide evidence that eRNA-producing regions are bone fide enhancers driving the expression of genes relevant for AOC tumour biology. These analyses defined a 6-gene signature that shows a strong association with the overall survival of AOC patients but did not compare the clinical value of this signature with signatures based on genes differentially expressed in BO and OAC.

The strength of this study lies in using patient RNA-seq data to identify eRNAs and enhancers unbiased pertinent to AOC tumour biology. General application of this approach to other tumours should be possible but may be limited by the availability of high-quality RNA-seq data sets and tumour purity. Nevertheless, this novel approach provided novel insights into AOC biology.

Reviewer #1 (Public Review):

In this paper, Blair et al., analyze hippocampal place cell remapping in rats performing a shock avoidance task using miniscopes to image large-field-of-views. They use behavioral (barriers) and pharmacological (scopolamine) manipulations to interfere with place cell representations during the formation and extinction of aversive memories. By exploiting multi-cellular imaging they examine cell remapping dynamics during learning and extinction induced by the different manipulations and evaluate how they relate to behavioral readouts. The work is carefully planned and analyses successfully control for many potential confounds (but see below).

Major strengths of the paper include a) using large-field-of-view miniscope imaging to provide place cell data from rats thus favoring inter-species comparisons (most miniscope data is emerging from mouse and rats, especially better in memory tasks); b) an appropriate set of control analysis and experiments to exclude for potential confounds especially when it comes to comparison between groups (speed, number of trials, performance, within- and between-trials differences). Major weaknesses are a) the systemic effect of pharmacological manipulations and specificity regarding memory function; b) the lack of appropriate shuffled contrasted effects. Other inevitable methodological aspects (such as the effect of large GRIN lenses on the integrity of the dorsal, and ubiquitous expression of GCaMP) also require further consideration.

The paper may be of interest to the neuroscience community by setting new methodological standards and providing new data for across-species comparisons.

Reviewer #3 (Public Review):

Understanding how neural representations throughout the brain, including the hippocampus, interact with neuromodulators such as acetylcholine to support flexible and lasting episodic memories is a fundamental question of interest to a broad neuroscientific community. Here, Blair et al. build on existing literature to concurrently characterize the relationships among these elements. Using large-population widefield miniscope recordings combined with systemic scopolamine administration in rats, the authors first demonstrate that localized aversive experiences result in lasting avoidance behavior as well as changes to (a.k.a. 'partial remapping of') the hippocampal neural code, with lasting changes occurring predominantly near the aversive experience, all replicating prior work with high precision. Next, the authors show that systemic administration of the acetylcholine antagonist scopolamine during the aversive experience gives rise to a different but reliable hippocampal code during that experience. Moreover, rats on scopolamine did not exhibit lasting avoidance behavior or changes to their hippocampal codes from before or after the experience, suggesting that the instantiation of a different hippocampal code during the aversive experience shielded the existing representation and its associated behavior from experience-induced changes. Together, these results demonstrate novel, provocative links between episodic memory, the plasticity of hippocampal neural codes, and the neuromodulator acetylcholine, with a number of important implications for how this memory system functions.

In my eyes, this work has a number of strengths. One major strength is the power and precision afforded by the use of the large-field miniscope recordings. While this may leave questions of fine temporal structure unaddressable, many of the questions of interest here are best addressed with large populations of simultaneously-recorded neurons that can be confidently tracked across at least a week, all of which are strengths of this technique. Another strength of this work is the replicate and extend approach to addressing the relationships among this work's components. The links to prior work in all of these cases are well noted, the replications of prior results are often with significantly more statistical power than the original result had, and these replications raise confidence in the quality of the data and the novel results reported here.

As with all work, this too has its limitations. One fundamental limitation is the inability to speak to functional localization. That is, although this work points to provocative correlational links among acetylcholine, the plasticity of hippocampal codes, and behavioral memory expression (all of which are well-motivated by existing literature) because the administration of scopolamine is systemic and only one region can be monitored it is impossible to draw causal conclusions from this work. While it is tempting to infer that manipulating acetylcholine modulation of hippocampal plasticity is necessary and sufficient to produce these results, it is also possible that the behavioral impact of the acetylcholine manipulation is driven by regions outside of the hippocampus and that changes to the hippocampal plasticity are not behaviorally relevant, or that these changes are necessary but not sufficient to drive memory expression. A specific version of this limitation is referenced by the authors in the discussion when considering the possible impact of the manipulation on amygdala responses.

Despite its limitations, this work meaningfully complements and extends existing literature probing the links between episodic memory, the plasticity and stability of hippocampal codes, and neuromodulators such as acetylcholine.

Reviewer #1 (Public Review):

ARL3 is a small GTPase that localizes to the primary cilium and plays a role in regulating the localization of some specific ciliary membrane proteins, including PDEδ and NPHP3. Mutations in this gene cause Joubert syndrome, a type of ciliopathy characterized by cerebellar malformation, and retinal degeneration. While the majority of the diseases occur in an autosomal recessive manner, two mutations in ARL3 (D67V and Y90C) have been reported to cause autosomal dominant retinal diseases. In the current paper, Travis et al. sought to understand the pathogenesis of the diseases caused by the two autosomal dominant mutations. They found that D67V acts as a constitutive active mutation, whereas Y90C is a fast-cycling mutant, which can be activated in a guanine nucleotide exchange factor (GEF) independent manner. Since the fast-cycle mutant did not bind to the effector proteins in vitro (likely because the guanine nucleotide falls off from the mutant ARL3, which has a lower affinity to GDP/GTP), they developed a method to snapshot the interaction between ARL3 and its effector. Using this method, they showed that the Y90C mutant indeed has increased interaction with the effectors, suggesting that Y90C is an overactive form of ARL3. They then addressed how photoreceptor cells are affected by these two mutations using a mouse model and found that the mutations disrupt the proper migration of the photoreceptor cells.

Strengths:<br /> • The paper is well written, and it was easy to understand what the authors did from the figure legends and the methods section.<br /> • It was easy to find out what is known or unknown, as the paper has accurate references.<br /> • The authors developed a method to analyze a snapshot of the interaction between ARL3 and its interactors.<br /> • The paper has an in vivo model and connects the biochemical characteristics of ARL3 to in vivo cellular phenotypes.

Weaknesses:<br /> (1) I understand that authors focused on nuclear migration defect as the phenotype was first described in ARL3-Q71L transgenic mice. The similar phenotype observed in RP2 knockout mice further supports the idea that the defect is caused by the hyperactivation of ARL3. Indeed, the defect is not reported in the ARL3 knockout mice, however, I feel that it does not necessarily mean that the defect is not caused by loss of function. Although it has not been assessed, ARL3 knockout mice might have the same defect. Therefore, I think analyzing both the migration defect and trafficking defect would be more informative, rather than focusing on the migration defect. The fact that the relationship between nuclear migration defect and the retinal degeneration phenotype is not entirely clear further enhances the importance of analyzing the trafficking defect.<br /> Does the expression of ARL3-Y90C also cause the trafficking defect? If it is the case, you can separate the nuclear migration phenotype from the one caused by the trafficking defect. Would the expression of lipidated cargo(s) rescue the trafficking defect as well?<br /> I think many questions can be addressed by analyzing the localization of the lipidated cargos, such as PDE and GRK1.

(2) I am not quite sure if the nuclear migration was assessed properly. Based on the pictures in Fig.1, some of the FLAG-negative cells also seem to be migrating to INL (please see Fig.1C and Fig.1D). Is this biologically normal during development? Could this analysis be affected by the thickness of OPL, the layer between ONL and INL? Also, the picture is cut out in the middle of INL. Could authors include more layers, such as IPL, of the retina in the picture, so that we can evaluate INL and OPL better? Taking this into account, I think it is worth measuring the nuclear position of FLAG-negative cells as a negative control in all the experiments.

(3) The way that the authors showed the Y90C mutant of ARL3 is a fast-cycling mutant is not very compelling. In Figure 2C, the authors showed that ARL3 Y90C can bind to PDEδ, its effector, once it is pre-loaded with GTP. The authors also showed that the mutant can bind to its effector even without EDTA as long as an excess amount of GTP is added. The authors used endogenous ARL3 as a control to compare the effects between wild-type and mutants. I see that this experiment has multiple pitfalls. First, ideally, this type of experiment needs to be done with a purified protein using fluorescent guanine nucleotide/radioactive guanine nucleotide (e.g. nucleotide loading assay or nucleotide exchange assay) to directly access the kinetics of nucleotide exchange. However, I do understand that this is out of the authors' expertise. In the authors' experimental setting, I am not sure loading the protein with GTP in the presence of the EDTA means anything more than confirming that the protein is intact. Theoretically, wild-type and a fast-cycling mutant can load GTP with similar efficiency in the presence of EDTA. Then during immuno-precipitation, GTP falls off from the Y90C mutant faster than wild-type (because a fast-cycling mutant theoretically has a lower affinity to guanine nucleotides), assuming that GTP was not added during immuno-precipitation (GTP addition was not mentioned in the method, but could authors confirm this?). But in this case, the kinetic of GTP dissociation can be affected by many factors, including the presence of GAP in the reaction, the dissociation constant of Y90C, the volume of the buffer used, and the number of washing steps. Thus, it is not very easy to estimate the difference between wild-type and Y90C. Besides, using endogenous ARL3 rather than ARL3-wild type FLAG as a control can be dangerous. I have experienced that a tagged protein is cleaved to a protein that has a similar size to endogenous protein. (I expressed GFP-protein X in knockout cells lacking protein X, and saw the band at the position where the endogenous protein is observed in wild-type cells). So, the endogenous band that the authors showed could come from the cleaved FLAG-Arl3. (Authors can easily confirm this by having wild-type not expressing FLAG-tagged ARL3, though).

An alternative experiment that I would suggest is doing immuno-precipitation in the buffer containing: 1) no guanine nucleotide, 2) 10mM GDP, or 3) 10mM GTP in the cells expressing the following protein: 1) ARL3 wild-type FLAG, 2) ARL3 Y90C FLAG, or 3) ARL3 D129N FLAG. 10mM guanine nucleotide should be added throughout the process including washing. This experiment might also be affected by many factors, but variability should be lower than the experiment presented in Fig 2C. ARL3-wild type FLAG is also a better control here than endogenous protein.

(4) In Fig.3, the authors attempted to take a snapshot of the interaction between ARL3 and multiple effector proteins. The three bands that were enriched in the Q71L cells were found as RP2, UNC119, and BART by mass spec (Fig.3B). These bands were used as a readout for the subsequent experiments. I am not quite sure why the authors used this approach rather than using the cell line that expresses both FLAG-ARL3 and GFP tagged protein of interest, just like what the authors did in Fig3G. The reasons why I prefer the latter approach are the following: FLAG bands that correspond to the three proteins (RP2, UNC119, and BART) in wild-type cells are very close to the detection limit, 2) authors failed to confirm that the lowest band actually comes from BART, 3) authors cannot access some important effector proteins, such as PDE because 293 cells might not express them. All of the problems can be solved by using the approach that was taken in Figur 3G.<br /> If the authors chose the former approach because of some specific reason, I would appreciate it if the authors could explain that in the main text of the paper.

(5) ALR3 Y90C causes nuclear migration defect. Given that Y90C is a fast-cycling mutant (hyperactive) and has a high affinity to ARL13B, the nuclear migration defect might come from either the increased activity of ARL3 or sequestration of ARL13B, which can act as a GEF for ARL3 but potentially have other functions. If my understanding is correct, the authors concluded that the defect caused by ARL3-Y90C is likely due to hyper-activation of the protein, as Y90C/T31N mutant, which cannot bind to effectors but still retains the ability to capture ARL13B, did not cause migration defect. But I am a little confused by the fact that Y90C/R149H, which is unable to bind to ARL13B (Fig.2C) but still retains the ability to interact with the effectors (Fig.3F), did not have migration defect (Fig.7B). Wouldn't this mean that the sequestration of ARL13B could contribute to the phenotype?<br /> If my understanding is correct, the authors are trying to say that both hyper-activation of cytosolic ARL3 and the defect in endogenous ARL3 activation in cilium is necessary to cause migration defect. I am not very convinced by this hypothesis, and still think that the defect could be caused by sequestration of ARL13B to the cytoplasm.<br /> Then why Y90C/T31N did not cause the defect even though they can sequester ARL13B? This might be explained by the localization of the ARL13B mutants. If Y90C can localize to the cilium while the double mutant, Y90C/T31N, does not, then only Y90C might be able to inhibit the ARL13B function in the cilium. This could explain the lack of the defect in the cells expressing Y90C/T31N.<br /> It would be helpful to understand how exactly the fast-cycling mutant causes the defect if the authors can provide more information, including localization of ARL3 (wild-type and mutants) as well as key proteins, such as ARL13B and the effector proteins. Assessing ARL13B defect seems to be particularly important to me because ARL13B deficiency has been connected to neuronal migration defect (Higginbotham et al., 2012)<br /> What I am trying to say here is that how the defect is caused is likely very complex. So, providing more information without sticking to one specific hypothesis might be important for readers/authors to accurately interpret the data.

(6) The rescue experiments that the authors presented in Fig.5-6 are striking and would build a base for future therapy of the diseases caused by ARL3 defects. However, I believe more examinations are needed to accurately interpret the data. The authors did this rescue experiment by co-injecting ARL3-FLAG and chaperons/cargos if I understand the method section correctly. But I feel we can interpret this data correctly only when ARL3-FLAG and chaperons/cargos are co-expressed in the same cells. I think a better way to analyze the data might be by comparing the nuclear migration phenotype between ARL3-FLAG only and ARL3-FLAG;chaperons/cargos double-positive cells.

Reviewer #3 (Public Review):

This work provides mechanistic insights into two recently described dominant variants of Arl3, a small GTPase, namely mutations D67V and Y90C. The authors identified a phenotype of these dominant variants during the development of rod photoreceptors by in vivo experiments in mice. They specifically observed a defect in rod nuclear migration to their final outer nuclear layer. This phenotype has been previously observed in another constitutively active variant of Arl3, Q71L. The authors performed a series of extensive and thorough biochemical assays to clarify the mode of action of these variants, mostly the Y90C variant, comparing the behavior of these variants to previously described mutants and combining multiple variants by mutagenesis. They also developed a new in vivo crosslinking strategy to be able to identify transient states of protein-protein interactions. They finally performed phenotypic rescue experiments by co-expression of various relevant proteins interacting/involved with Arl3. They finally propose a model based on differential subcellular compartmentalization of Arl3 activation which when disrupted leads to rod nuclei misplacement. These data add to the current understanding of contribution of different Arl3 variants causing human retinal degeneration, which has strong potential translational implications.

Strengths:<br /> Relevance of Arl3 dominant variants to human retinal degeneration.<br /> Identification of Y90C variant as a "fast cycling" GTPase, and not as a predicted destabilizer of the protein structure.<br /> New method of crosslinking to enable snapshots of endogenous protein-protein interactions.

Weaknesses:<br /> - The relevance of this study is justified by the fact that newly identified dominant variants of Arl3 have been associated to retinal degeneration. However, the authors never assess a degeneration phenotype.<br /> - The authors show new dominant variants of Arl3, namely Y90C and D67V, cause rod nuclear mislocalization. This phenotype is interesting but this was previously observed with other constitutively active mutation of Arl3, Q71L, and therefore is not novel.<br /> - The main claim of this paper is that subcellular compartmentalization of Alr3 activation to the cilium (the so called gradient by the authors) is required for proper rod nuclear migration to their final outer nuclear layer destination. The authors provide multiple experiments to support this model, but this is never directly demonstrated.

Reviewer #1 (Public Review):

Weiss et al have developed a novel model of Huntington's disease (HD) by injecting a mixture of recombinant adeno-associated viral vectors (AAVs) into the caudate and putamen of rhesus macaque monkeys. There is a significant need for relevant models of HD. While many mouse models exist, current models lack genetic relevance (with repeat lengths much longer than those found in humans being used) and mice lack the anatomical relevance to humans since they have small brains with important brain regions (in particular the neostriatum) being dissimilar to those seen in humans. The authors used non-human primates because they have large brains with anatomy similar to humans. They used a mixture of recombinant adeno-associated viral vectors (AAVs) in an attempt to overcome the shortcomings of previous models using AAVs. They studied their animals over 20 months using both behavioural tasks and MRI assessment. The animal served as their own controls for the imaging, which improves the power of the study. The methods of analysis, particularly the imaging, are modern and directly relevant to assessments that can be conducted on human patients.

Strengths<br /> The major strength of the paper is that the authors used Rhesus macaque, a species that is highly relevant to studies aimed at assessing therapies and drug delivery. As a stepping-stone to humans, the macaque has many advantages, including brain size, relevant anatomy and in particular, longevity compared to mice. The approach of mixing a number of recombinant AAVs is also interesting since it overcomes some of the limitations of individual AAVs as detailed in the Introduction. The tasks used for behavioural assessment to investigate the effect of the AAV on brain and behaviour also highlight the advantages of a monkey model, since human-relevant assessments were used. The study was very well controlled, with both vehicle and AAV containing non-pathogenic length CAG repeat (10Q) used.

The behavioural assessment was comprehensive, and the motor control measures are relevant to HD. MR imaging is also very relevant to what can be measured in humans. The imaging was comprehensive and of excellent quality. Overall, the study presents some important and interesting data, because an acute monkey model has not been studied in such depth previously. The range of approaches taken to assessing the animals is comprehensive and impressive.

This is a study that will be of interest to researchers who are developing methods for studying the role of the caudate/putamen in behaviour. I agree with the statement that the model will be useful for studying the effects/causes of disruption to the cortico-basal ganglia. The effect of the lesions on cortical regions of the brain are interesting, and well presented.

Weaknesses<br /> The major weakness of the study is that with the interpretation of the results. The changes in tractography, behavior and TBM are what would be expected following lesions of the neostriatum. Indeed, all the data point to this being an acute lesion model, and in my opinion, the authors have made an interesting novel neurotoxic model (using a very relevant neurotoxin). Unfortunately, there is no detailed pathology showing what is happening at the level of the striatum or associated cortical regions (see also below). The results have been interpreted as showing a progressive model, although evidence that there is progression is limited. The whole manuscript is written as though this is a genetically-relevant progressive model of HD. But the animals are normal, and so there is no genetic context relevant to HD. While the authors present this as a new model of HD with progressive motor and cognitive changes (as seen in the title of the paper) there is little evidence presented that there are major 'progressive' changes seen. Furthermore, the idea that the changes mirror those seen in HD patients (as stated in the discussion) is somewhat misleading. While their data may be similar to some findings in patients who have early degenerative changes, there are many differences that are either not seen or not explored in the new model. In particular, evidence that the changes seen in the monkeys are relevant to progression of disease in HD patients is missing. While this appears to be a sophisticated acute lesion model that has been assessed more thoroughly than previous models, nevertheless all evidence points to it being an acute rather than a progressive model. While I agree that the data show clearly that there are changes in motor function and to a lesser extent in cognitive function caused by the injection of the AAVs, it does not follow that the changes seen are 'progressive'. Even in rats following striatal lesions, changes in behaviour have been measured for months afterwards. For example, in acute lesions of monkeys, Deglon et al showed years ago that in NHPs the behavioral sequelae of acute neurotoxic lesions change with time. This model also does not show 'progression' in the sense of a progressive disorder in HD.

The authors state in the Abstract that the injection resulted in "robust expression of mutant huntingtin in the caudate and putamen". These data are not in the manuscript. This seems to be deduced, rather than measured experimentally.

A disadvantage of the method used, that has been a bugbear of the field, is that the authors chose to use a fragment of the HD gene, with a very long repeat that is seen only in juvenile patients. While using the fragment rather than the whole gene is a sensible approach, since it is known to be toxic, I am not clear why they chose to use a juvenile length repeat rather than a repeat in the adult-onset pathological range. There are mouse models with a CAG repeat of 40-50, but short lifespan of mice has limited their usefulness. Longevity is one of the major advantages of using a monkey. Had they used a repeat of 45 or 50, this would have been a much more interesting paper, because there is little known about the toxicity of proteins with that length repeat in vivo. As it stands, the model is a non-human primate acute fragment model using a long repeat. The disadvantages of fragment models and long CAG repeats has been well discussed in the literature and is a major criticism of many of the mouse models. Consistent with the disadvantages of fragment models with long repeats, the onset of the symptoms of the monkeys is much more rapid that would be expected in either juvenile or adult-onset HD and is likely to be due to both the fragment nature of the vector and long CAG repeat. The rapid onset of phenotype is not discussed in the context of other models.

The use of a DA receptor agonist was an interesting idea, because DA agonists have been shown to exacerbate abnormal involuntary movements in HD patients. The mechanism for this is complicated, however, given that the balance of D1 and D2 receptors changes as HD pathology progresses. The authors chose to use a non-selective agonist, which caused transient changes in behaviour. However, the usefulness/relevance of the apomorphine data is unclear, particularly since the effect was only seen at the early timepoints and not at the later timepoints. If the AAV causes acute toxicity, then such changes in response to apomorphine would be expected, and this would be expected to resolve with time - as was seen.

For their cognitive testing, the authors used a task (delayed non-match to sample) that measures object recognition and familiarity. Before surgery, only 11/17 of the animals were successfully trained to complete this task. It is not clear how useful the data are when only 64% of the animals can be included. It would have been better to have choosen a task that all monkeys could perform at baseline.

The results of the motor task showed a clear deficit. This would be expected with an acute lesion of the striatum. It was interesting that there was a significant improvement from the 9-month point to the 20-month point in the 85Q lesioned monkeys, whereas the performance of the vehicle-treated monkeys plateaued. There appeared to be a deleterious effect on the 10Q monkey performance that was maintained. (This was not discussed in any detail but should be.) This begs the question as to whether or not the 85Q-lesioned monkeys would recover to a level similar to the 10Q animals if left for another 12 months.

The tractography and tensor-based morphology data are clear, and consistent with the idea that a Q85 fragment would be neurotoxic. The authors consistently suggest that this is relevant to early stages of HD, but there is little evidence presented to support this statement. The sensitivity of the MRI used shows that multiple regions were affected beyond the lesion sites. This would be expected and is new and interesting data in the non-human primate field, although it does not bring anything particularly new to the table with respect to HD (since similar findings have been shown in lesion studies using mice, rats and sheep).

For HD researchers investigating aspects of the disease, such as transition from prodromal to early symptomatic stages, or for developing treatments, the usefulness of this model will be limited. It is not clear how this monkey model will be useful for developing either disease biomarkers or therapeutic strategies for HD (as stated in the abstract). For studying biomarkers of the disease, this model lacks a number of critical parameters. First, the genetic context of the disease is missing. Second, it is known that HD has multiple sites of pathology, and that symptoms are not simply due to degeneration of the caudate/putamen and that multiple regions of the brain where mutant Htt is expressed become dysfunctional and eventually degenerate. Understanding how the caudate/putamen degenerate is important, but since last century HD researchers have been very aware that dysfunction in the HD brain occurs at many sites other than the caudate/putamen. Indeed, it is probably not only a brain disease since there is evidence of peripheral pathology in humans and other models.

The authors state that they hope the model will become a widely used resource. This seems an unlikely scenario, given the limitations of the current study and the challenges associated with using monkeys. They say that a major advantage of their technique is being able to generate large numbers of monkeys. But this is not a relevant argument if the usefulness of the model to investigate HD is not proven. Studying the role of the caudate/putamen in motor behaviour is interesting for a small field but limited in scope.

The authors suggested a number of experiments that could be done, for example, using a shorter HD-relevant CAG repeat length. But as stated above, this is a weakness of the current study, and it would be much more useful had the authors done this experiment themselves. It seems unlikely that until the authors prove its usefulness, this model will not become a widely used resource, since the disadvantages of the model outweigh the advantages. Using monkeys requires a specialist laboratory and facilities and a careful consideration of the ethics involved in animal experimentation. Unless the model offers clear advantages over other models, it is unlikely to become mainstream. It is also not clear what therapies could be tested in this model that could not be tested in other existing models. For example, given that there is no control over which cells are infected by the AAVs, or if any of the cortical pathology is due to spread of AAV from the initial sites of injections, it is not clear how antisense oligonucleotides efficacy could be tested.

Reviewer #1 (Public Review):

Zhang et al. use a mosaic vaccine design, where three SARS-CoV-2 spike receptor domains (RBD) are linked together, to make a multivalent vaccine: one of the RBDs has the Omicron sub-lineage BA.1 sequence, while another has a combination of 8 mutations from different variants. They show that rats immunized with this construct have improved neutralization of Omicron and other variants relative to a mosaic vaccine where all RBDs are ancestral, as well as relative to the Sinopharm BBIBPCorV inactivated virus vaccine either as the primary vaccination or as a booster of BBIBPCorV.

Public Review:

Using a Discrete Choice Experiment (DCE) the study asks respondents in six EU countries to choose between two persons A and B and select the one that they believe s/he should receive the COVID-19 vaccine first. Across eight different scenarios, each person is given different attributes in terms of age, COVID-19 mortality risk, employment status, and country of residence (own vs. other with low healthcare system capacity). The study found the risk of mortality and also working for essential services to be perceived as particularly important across all the countries. Moreover, living in a low-income country with poor healthcare system capacity was found to be favored when it came to allocating the vaccine first. This is particularly interesting given that the respondents were selected from those who were not vaccinated at the time of the survey but were willing to receive one.

Strengths:

• The study evidence is based on large samples from 6 EU countries.<br /> • It captures the opinion of those who had not been vaccinated at the time of the survey, hence, allocation to those in low-income countries indicated further altruism.<br /> • The method, a conditional logit estimate, and also the robustness checks are appropriate and suitable.<br /> • The study distinguishes between two key attributes of mortality risk and country of residence allowing for evaluating the importance of each factor separately. The implication of these factors can be helpful in making decisions in the future. This is in particular critical given that the initial aim of COVAX was to prioritize those who are vulnerable and the healthcare workforce across the world before launching national programs did not materialize and we are still facing large global disparities between the global north and south.

Weaknesses:

• The sample from Germany is noticeably different from the rest of the countries (particularly in terms of having a higher ratio of those who are in the high-risk category). This might have impacted the results and needs to be reflected in the study discussion. Also, there is heterogeneity between studies in terms of the time of the fieldwork and each country's conditions in regards to the vaccination roll-out and the number of infections at that time.<br /> • The manuscript narrative needs to be updated to reflect the present conditions in terms of inoculation campaigns, their success rate, and their disparities across the world.<br /> • There is space for more discussions on an interesting finding of the study that is prioritizing the vaccines according to employment status and in particular income loss.<br /> • The temporal nature of the public views at various stages of the pandemic and vaccination campaigns should also be noted.

Reviewer #1 (Public Review):

This article creates a formal definition of the 'informativeness' of a randomized clinical trial. This definition rests upon four characteristics: feasibility, reporting, importance, and risk of bias. The authors have conducted a retrospective review of trials from three disease areas and reported the application of their definition to these trials. Their primary finding is that about one quarter of the trials deemed to be eligible for assessment satisfied all four criteria, or, equivalently, about three quarters failed one or more of their criteria. Notably, industry-sponsored studies were much more likely to be informative than non-industry-sponsored studies. It would be interesting to see a version of Figure 3 that categorizes by industry/non-industry to see the differences in fall-off between the four criterion.

As the authors point out, the key limitations to this work are its inherent retrospective nature and subjectiveness of application, making any sort of prospective application of this idea all but impossible. Rather, this approach is useful as a 'thermometer' for the overall health of the type of trials satisfying the eligibility criteria of this metric. A secondary and inherent limitation of this measure is the sequential nature of the four criteria: only among the trials that have been determined to be feasible (the first criterion measured) can one measure reporting, importance, and lack of bias. And only among those trials that are both feasible and reported properly can one measure their importance and lack of bias, and so forth. Thus, except for feasibility, one cannot determine the proportion of all trials that were properly reported, were importance, or evinced lack of bias.

Reviewer #3 (Public Review):

The idea of individual ageing trajectories of single cells is important and the authors provide sufficient evidence that there is some stochasticity that directs individual cells towards certain routes of ageing - at least in budding yeast. Additionally, understanding the connection and dependence of various different processes that occur during ageing is critical and timely. However, despite the fact that the hypothesis laid out in the manuscript is tempting and the approaches taken might be the right way to tackle it, the results presented still fall short of connecting chromatin instability and protein aggregation. I have provided more detailed comments below, but in essence, I miss a clear experiment linking rRNA instability and the role of RBPs with protein aggregation and loss of proteostasis. All experiments that try to achieve this are either too unspecific (e.g. NAM as an inhibitor for Sir2, while it inhibits a wide variety of deacetylases) or do not show protein aggregation (e.g. Nop15-mNeon, which might simply stain a fragmented nucleolus).

Reviewer #1 (Public Review):

In this study, Trolle et al set out to investigate the impact of reintroduction of essential amino acid biosynthetic pathways into mammalian cells. To this end, they employed an elegant synthetic genomic approach to enable Chinese Hamster Ovary cells to endogenously produce methionine, threonine, isoleucine, and valine. Notwithstanding that attempts to functionalize biosynthesis of methionine, threonine and isoleucine were not successful, reintroduction of valine biosynthetic pathway rescued survival of Chinese Hamster Ovary cells deprived of valine. Moreover, the authors provide evidence that global mRNA abundance profiles in valine biosynthesis-proficient Chinese Hamster Ovary cells mirror those observed upon recovery from valine starvation. Collectively, these pioneering studies suggest potential for the functionalization of biosynthesis of essential amino acid in mammalian cells. Based on this, it was found that this study is of broad interest to a variety of research fields including synthetic biology, biotechnology, and biochemistry.

Strengths: This study incorporates a very elegant synthetic genomic approach to address a long-standing gap in knowledge related to exploring the ability of mammalian cells to tolerate restoration of essential amino acid biosynthesis. It was highly appreciated that this is one of the pioneering attempts to address this question. For the most part, the data were robust and supportive of the author's tenets. Finally, demonstration that Chinese Hamster Ovary cells can be rendered prototrophic for valine may open many interesting avenues in the domains of synthetic biology and biotechnology, with potential long-term applications in medicine.

Weaknesses: Relatively modest rescue of proliferation of valine-producing Chinese Hamster Ovary cells in valine-free media, apparent reduction in de novo valine synthesis during propagation of the cells and some technical issues pertinent to potential utilization of valine from breakdown of serum proteins were considered as the weaknesses of the study. Furthermore, it was thought that further molecular characterization of valine-prototrophic Chinese Hamster Ovary cells may be warranted.

Reviewer #3 (Public Review):

During evolution, eukaryotes lost the biosynthetic pathways that are responsible for the production of 9 amino acids. In this study, Wang et al successfully reintroduce the fully functional biosynthesis of these 9 amino acids back into mammalian cells. To accomplish this task, Wang et al had to introduce, into mammalian cells, >40 genes and reconstruct pathways that are naturally functional only in fungi plants, and bacteria. The entire pathway was synthesized de novo by commercial gene synthesis in 3 kilobase fragments and assembled in yeast. The work is a major bioengineering accomplishment that will serve for fundamental research into evolution and metabolism.

Reviewer #3 (Public Review):

The report is a major leap in understanding the Ca2+-central pathways underlying egress and invasion of Apicomplexa, using T. gondii as a model organism. Temporal phosphoproteomics is novel, yet even more innovative is to apply temperature stability profiling using various Ca2+ concentrations and temperatures. This provides a really unprecedented depth in the Ca2+ protein network, revealing several dynamic trends in the responses, reveals many new proteins with stability shifts in absence of apparent Ca2+-binding, and ties together many previous observations on putative channels and transporters and signaling pathways. The dynamics of PP1 are intriguing, first accumulating apical of the nucleus (secretory pathway compartment?) and then transitioning apically and to the cortex. Although this is characterized as 'pleiotrophic' I am not sure that is a correct term if this is a PKG-dependent trajectory (but can be bypassed by Ca ionophore) - all of which are somewhat artificial stimulations and therefore could present pleiomorphic under these conditions: some more caution in the results/discussion would be warranted.

Reviewer #1 (Public Review):

This manuscript uses a wide range of experimental and computational techniques to address how mice use active vision to gauge distance in a gap-jumping task. It is found that for this task, the animals can compensate for the lack of binocular vision via an increase in active head movements, to perform the overall task with similar effectiveness. When the primary visual cortex (V1) is temporarily inactivated using optogenetic techniques, the animals perform much worse, suggesting a critical role for V1 in distance estimation.

The paper makes substantial technical advances in the understanding of gap jumping in mice. The data are convincing that the animals can rely on monocular information to nearly equal effect as binocular vision. However, the reasons for this were somewhat murky: the authors concluded that the animals performed more active sensing movement, but the use of HMMs as the only means to assess this was a weakness of the manuscript. Roughly, they showed that the "recurrent" connections in an HMM were stronger with monocular vision than binocular, i.e. the mice tended to repeat certain motifs that the authors suggest were related to sensing the distance. There are several major weaknesses with this as the only approach. First, it is unclear in plain statistical terms what differs in pre-jump behavior. Second, it is unclear how these dynamical systems motifs are related to any kind of active sensing behavior. Third, and as a consequence, it is unclear any potential mechanistic benefit of the change in pre-jump behavior.

There are also a few weaknesses to the V1 analysis. First the only analysis of the effect of inhibiting V1 was basically that more of the animals chose not to jump. But, was their accuracy worse when they did jump? If not it is entirely unclear that V1 is involved in the distance estimation and in fact one could argue that they can judge the distance fine without V1 (which of course would not mean V1 were not used).

Reviewer #3 (Public Review):

In this manuscript, the authors demonstrate mice can use monocular cues to estimate distance in a new task they developed. They developed an ethologically relevant task in freely moving mice where the animals must estimate the distance of a platform to complete a jump to be rewarded. The task can be coupled to eye tracking and optogenetics. The authors provide evidence that the eye movement compensates the head movement in maintaining gaze and the initiation of the jump depends on V1. The task is in freely moving mice and offers the possibility of genetics and/or electrophysiological interrogation of the brain circuitry in the future.<br /> Strengths:

The authors achieved their aims of demonstrating mice can use monocular cues to estimate distance, and the results are simple and convincing. Regarding the specific claims in the accuracy of mice estimating the distance and whether the monocular condition caused more head movement I have a few specific comments below.

Most of mice behavior is systems neuroscience has been in head-fixed behavior. The electrophysiology and/or imaging equipment do not move with the animals. There has been recent advances in electrophysiological and imaging techniques that allows them to be tethered to the animals. This calls for ethologically relevant behavior in rodents. The authors demonstrated that they can combine eye tracking and optogenetic with the task. As freely moving electrophysiological recording techniques improve in the future. Researchers will be able to combine this with their task to further elucidate the circuitry underlying behavior.

Weaknesses:

Although the paper has a simple message, most of systems neuroscience is interested in how sensory evidence, in this case, monocular cues, are encoded in the brain, and the process in which it is transformed into action. Falling short of the goal to address the circuitry underlying the behavior, we can only judge the merit of how likely the task will be adapted by the community to elucidate insights into the neural circuitry. The behavior in its current form is impossible to speculate which monocular cue the mice used to solve the task, e.g. relative size, occlusion, motion parallax etc., therefore it will be difficult to pinpoint the relevant area of interest to start the interrogation. If the interest is in motor control, the jump has many degrees of freedom and muscles involved than the classical eye movement or arm reaching tasks. It is unclear the advantages this task has. Furthermore the timing of choice and reward is poorly controlled in the trial structure of the task, so it is unclear the additional insights it can offer regarding decision making and motivation.

An important use of mice in system neuroscience is for invasive monitoring of brain activity with electrophysiology and/or imaging. The equipment for electrophysiology and imaging often require the animals to be head fixed. This study does not attempt to expand on the behavior observed, and this will be a limitation for adaptation of the task that the authors presented.

The authors also provide an insufficient amount of details on the task. For example, how were the platform and distance manually changed by the experimenter for each trial? This is an important manual step that limits the number trials and potentially the animals' engagement in the task. In its current form, the task will unlikely be adapted by the community. Head-free behavior and the low trial number might limit the utility of the task to systems neuroscience.

Reviewer #1 (Public Review):

The congenital condition posterior urethral valves (PUV) is a major cause of end stage renal disease in young males. While prior work has partially characterized the genetic landscape of this condition, Its pathogenesis remains poorly understood so any new insights will be of broad interest to pediatric nephrologists, urologists, geneticists and developmental biologists. The study by Chan et al makes a significant contribution to this story. Here, the authors have used for the first time a diverse ancestry whole-genome sequencing approach to tackle the problem and have identified variants within/near two genes, TBX5 and PTK7, as being significantly associated with this condition in both their original cohort as well in a replication study. The data are compelling and are a good example of the power of applying a diverse ancestry approach to disease locus discovery in rare disease. They also were able to use this approach to fine map variants inferred to be causal. This study's use of WGS provided other advantages: they could identify rare exonic variants and small structural variants missed by conventional microarrays. This strategy resulted in two additional observations: 1) none of the genes previously associated with congenital bladder outflow obstruction were associated with PUVs, showing that monogenic causes of PUVs are rare; 2) an enrichment in PUV cases of rare inversions affecting candidate cis-regulatory elements, with the strongest signal for inversions affecting CTCF-only regions.

While the genetic associations appear robust, there are a number of weaknesses to this study. The most obvious and important one is that all of the findings are associative, and none are experimentally validated. The authors nicely use bioinformatic methods to show that the variant near TBX5 may map into the same topologically associated domain, but they provide no direct evidence that this variant directly affects TBX5 expression. The closest they come to providing any link is by showing possibly permissive expression of TBX5 in relevant tissues. Likewise, they suggest that the intronic variant in PTK7 may disrupt the binding domain for at least two transcription factors, though neither is experimentally evaluated, and they provide no direct evidence showing that this variant affects the expression of PTK7. It should also be noted that their immunohistochemical studies of human fetal tissue for TBX5 and PTK7 are not convincing. There appears to be widespread staining of multiple cell types, suggesting either very broad expression of both genes or poor specificity of the primary antibodies. There is, of course, no reason that a broadly expressed gene cannot have organ or tissue-specific effects when its activity is altered, but these data do not provide compelling evidence that either TBX5 or PTK7 is functionally important in this condition. Further highlighting the importance of this issue, PUVs have not been described as a clinical manifestation of disease associated with mutations of either gene in humans. Finally, it would be useful for the authors to discuss how variants in either gene or in the patterns of structural variants that they found associated with PUV intersect with sex to result in this exclusively male condition.

Reviewer #3 (Public Review):

In this manuscript, the authors attempt to identify risk factors for PUV, a rare disease with unclear pathophysiology. The study design is a well-designed GWAS, although performed on sequence data rather than SNP array data with imputation; the sequence data also allows for study of structural variants. Strengths of the study include an exemplary design and analytical approach, as well as the novelty of applying a GWAS to a rare disease. Weaknesses include a somewhat thin exposition as to what is known and unknown about the genetic architecture of PUV, some omitted analyses that could further elucidate the genetic basis of PUV, and some results in the latter half of the manuscript that have unclear impact.

I believe that the primary objective of the study was achieved -- the reported genes have reasonable evidence as candidate genes and the association signals nearby them seem to be robust. I am not familiar with PUV but if these are some of the first genes identified for the disease, they may have a significant impact on the PUV research field. They do face the same limitations of any gene identified from a GWAS, however, in that the evidence implicating them in PUV is still circumstantial, and there is a long way to go to demonstrate the mechanism linking them to disease or whether they or other genes in the same pathway could be targeted by therapeutics.

More generally, while the GWAS methodology applied is not particularly novel, the scenario of applying it to a rare disease is innovative and of value -- as we become increasingly aware that the dividing line between rare and common diseases may be blurry, GWAS for rare disease (and, conversely, sequencing studies for common disease) are important data points for advancing the field. Rare diseases are traditionally studied through very different approaches than are common diseases, so bringing rigorous statistics and analytical approaches to a rare disease is of value to the field.

Reviewer #1 (Public Review):

In this paper Bonnard et al. describe the development of a new method to quantify C. elegans feeding behaviour. The circuit mechanisms underlying feeding behaviour of worms are not fully understood and studying them potentially can lead to fundamental new insights into the control of rhythmic behaviours in general. In classical work, the up to 5Hz peristaltic movements of the worms' pharynx were assessed manually, a painful procedure which is challenging and error prone. Alternative methods have been developed, either by video recording and subsequent slo-mo manual analysis, or recording of electropharyngeogram in microfluidic devices, however, these prevent observations in unrestrained behaving animals. The approach reported here is based on a sophisticated image processing pipeline analysing freely moving worm populations of animals with fluorescently labelled pharynges. The authors convincingly show that peristaltic pumping rates can reliably be measured across development, under various feeding conditions and capturing known and previously undescribed phenotypes of feeding mutants. The method represents a significant improvement and possibly seems applicable to researchers in the community; however, it cannot measure detailed aspects of pharyngeal dynamics like it is possible with electropharyngeogram recordings. I see great potential also to perform long-term recordings, this unfortunately is not addressed in the current manuscript.

Reviewer #3 (Public Review):

In this manuscript, the authors present a method for simultaneous assessment of pharyngeal pumping (feeding) and locomotion in many C. elegans simultaneously. In this technique, imaging of the fluorescent labeled pharynx provides a measure of velocity and pumping rate, through analysis of the spatial variations in fluorescence.

The technique is clearly described, well-validated, and yields some novel results. It has the advantage that it can be performed using microscopes found in many C. elegans laboratories.

Some limitations of the method include its reliance on fluorescence imaging, which is a hindrance to genetic analysis, computational intensiveness, and phototoxic effects of fluorescence excitation that are not fully explored in the manuscript.

The authors show the utility of their method by assessing pharyngeal pumping and motor behavior (1) during development, (2) in the presence or absence of food, and (3) in the presence of two mutations affecting feeding.<br /> Although I understand these are proof-of-principle demonstrations, I still came away feeling underwhelmed by these examples. I did not see any results here that could not have been obtained fairly easily with conventional techniques.

Given these limitations, I feel the method's eventual impact in the field will be relatively small.

Reviewer #1 (Public Review):

This manuscript investigates the cellular and developmental defects underlying Wolbachia-induced cytoplasmic incompatibility (CI), which occurs when male insects harboring the endosymbiont bacteria Wolbachia fertilize eggs of uninfected females, triggering embryonic lethality at the first nuclear division. Characterization of the mechanisms of CI has implications for pest control in insects beyond Drosophila, and thus this topic will have broad interest.

Previous work, including by the Sullivan lab, has shown that CI is caused by a paternal effect in which the sperm from a Wolbachia+ male triggers a dramatic early failure in the first nuclear division within the newly fertilized Drosophila egg. In this work, the authors provide compelling evidence that there is an additional, later defect that is present in ~30% of the affected embryos. These defects occur at the mid-blastula transition and beyond. They go on to show that these later embryonic defects can be due to loss of the paternal genomic DNA (creating haploids) which could be due to the early fertilization defect, but also a chromosome segregation defect independent of haploidy or the initial fertilization defect. They use elegant single embryo PCR, pooled blastoderm genomic sequencing, and FISH methods to track the origins of the blastula defects; this is a compelling set of experiments! Taking all their results together, they conclude that the latter phenotype is due to a distinct molecular mechanism than that inducing first division defects. The paper is well written and easy to follow.

Reviewer #3 (Public Review):

This study aims to determine whether the chromosome defects induced by a bacterial endosymbiont in insects in developing embryos are a direct result of paternal chromosome defects from early embryogenesis or due to a second, independent set of defects that arise later: "we addressed whether defects observed in late CI embryos such as chromosome segregation errors and nuclear fallout are the result of first division errors or a second, distinct CI-induced defect."

Using crosses, genetics, and fluorescent microscopy, the study claims that the defects at different embryonic stages are due to independent processes, and this work thus has mechanistic relevance to how bacteria inflict developmental harm to insect embryogenesis. The claim is not well supported by the weight of the evidence in this paper and the literature.

The work is technically sound and proficiently completed to an expert level with appropriate statistics, but it does not provide straight-line evidence to substantiate the primary claim of the paper that later-stage embryos die for different reasons than early-stage embryos. That is no fault of the experimental rigor but rather to the difficulty of directly answering this question. It appears the field has insufficient information on the reductionist, bacterial mechanism that induces embryonic death, namely what acutely is modified by the bacteria to cause embryonic death? As such, the authors hedge that by studying different developmental stages of the embryonic defects, the answer can be surmised. However, a simple explanation for how late and early-stage embryos could die to similar mechanisms is that host cellular conditions are more or less susceptible to the same bacterial-induced change of the insect chromosomes (e.g., new chemical marks on the DNA). It's just not possible to rule this out until the acute mechanism of killing is known. For instance, some embryos may vary in their transcriptomes, proteomes, physiology, etc within a single family of fly offspring, and as such these varying embryos may be more or less susceptible to the same proximal cause of the bacteria-mediated defects. The difference is just when do they take place in development. Without knowing the bacterial mechanism of death (e.g. changes in chemical marks of the fly DNA), the study here can characterize broad strokes of chromatin biology while speculating on the weight of the evidence for whether or not different mechanisms are at play.

To evaluate the primary question of whether or not there are completely separate defects across development, the study shows several pieces of data that offer a finer resolution of the broad defects of embryos that were previously characterized by the literature. The new follow-up details are robustly supported and include percentages of embryos experiencing a defect, nuclear fallout, determination of haploidy/diploid, sequencing depths, Y chromosome tracking, and developmental-staged characterizations of the chromatin defects. However, according to the text, there is effectively a single type of data that speaks to the main question of the paper - whether or not viable embryos that escaped the first mitosis had increased mitotic errors during later developmental stages.

"Therefore, the significant increase in mitotic errors observed in diploid CI-derived embryos relative to wild-type derived embryos demonstrates the existence of a second, CI-induced defect, completely separate from the first division defect." This was already known; later-stage, chromatin defects do occur in a variety of insect species cited in the paper. In effect, the question answers itself because, in order to traverse an early lethal state that does not occur, there must be defects that ensue later in development, several of which have already been characterized, though to a lesser resolution than this study.

Moreover, the study does not link the staged chromatin errors to the CI genes using transgenic tools that are now customary in this field. That work is quite relevant to the conclusion of the paper because the authors speculate in the discussion that additional CI genes may be necessary to explain the later defects in embryogenesis versus the initial defects. This work has been completed to a degree by the papers reporting the initial discovery of the CI genes. CI transgene expression in males causes both 1st mitosis and later chromatin defects, suggesting additional genes are not necessary to explain lethality after the first mitosis. This to me is perhaps the most significant counterpoint of the narrative of the paper's claim because the acute genetic cause of CI can lead to differently timed chromatin errors.

This is solid work and a strong effort to refine the stages and types of embryonic lethality induced by bacteria, however, the claim that there are different acute mechanisms of death during embryogenesis is not well supported.

Reviewer #1 (Public Review):

This is a very interesting paper showing that postsynaptic bursts in the presence of dopamine produce input-specific LTP in hippocampal synapses 10 minutes after they were primed with negatively coincident pre- and postsynaptic activity. LTP requires NMDAR activation during priming and involves a cAMP-PKA cascade and protein synthesis. When this synaptic rule is incorporated into a computational model, reinforced learning is possible through selective reactivation of neurons. Experiments in behaving mice confirmed that neurons reactivated after an exploratory period display more activity than non-reactivated neurons.

Reviewer #3 (Public Review):

Fuchsberger et al. demonstrate that an otherwise LTD-inducing STDP protocol can produce LTP if followed by burst reactivation of post-synaptic neurons in the presence of dopamine. Using computational modeling and single-photon imaging in the CA1 in mice, they propose these findings are relevant to spatial over-representation at a reward location.

This is a follow-up of the two previous studies from the same group (Brzosko et al., 2015 and Andrade-Talavera et al., 2016) where they showed a post-before-pre STDP protocol, which by default induces a (pre-synaptic) LTD, will induce synaptic potentiation in the presence of dopamine and continuous synaptic activity. The main conceptual difference between this manuscript and these previous studies is that continuous synaptic activity can be replaced by post-synaptic burst. This means that reactivation of post-synaptic neurons without any further pre-synaptic instruction is sufficient for successful LTP induction.

Mechanistically, the two protocols (continuous vs burst activation) appear to be similar (but not identical). For example, both require the activation of post-synaptic NMDAr during STDP pairing, and both depend on the AC/PKA pathways. Additionally, there are two new observations here: The activity of voltage-gated calcium channels during bursting is required for potentiation; the burst-induced potentiation also requires protein synthesis.

The evidence provided at this stage is strong.

Major point:

It is not clear to me how the STDP studied here relates to the next part of the study, the reward-based navigation task. My interpretation is that the authors consider the activity before reaching the reward location (approaching time) as resembling the STDP priming protocol, the activity at the reward location as equivalent to the bursting protocol, and consumption of the reward as similar to dopamine application. If so, what is the circumvential evidence that the activity during the approach induces any form of plasticity? The link between the two is not obvious and I see the manuscript as two interesting but not naturally linked stories.

Reviewer #1 (Public Review):

This manuscript describes a novel role of the centrosomal protein CEP83 in mesoderm patterning, specifically the balancing between the intermediate mesoderm (IM) and the lateral plate mesoderm (LPM). The authors nicely demonstrate that CEP83 is required for the accurate formation of the IM and differentiation into the kidney lineage. This is achieved by generating CEP83 knockout iPSC lines and differentiating these into mesoderm cultures and kidney organoids. The most obvious defects in the knockout situation are ciliary abnormalities (in the mesoderm monolayer as well as 3D organoid cultures), indicative of the critical role of the cilia in normal kidney formation. Strikingly, the CEP83-deficient cultures completely fail to undergo further differentiation into kidney epithelia as shown by immunohistochemistry for nephron and podocyte markers. Thorough transcriptomic and bioinformatic analyses provide insights into these defects on a molecular basis, i.e. the CEP83 knockout appears to favor the formation of the LPM at the expense of the IM, with LPM-specific marker genes strongly upregulated in the knockout. A possible key role for the transcription factor HAND2 is discussed.

Strengths and appraisal<br /> A major strength of this work is the use of cutting-edge technologies, including the CEP83 knockout in iPSCs, mesoderm and kidney organoid cultures, and bulk and single cell transcriptomics. These techniques provide strong data, which support the conclusions of this study.

Weaknesses<br /> There are no major weaknesses.

The impact of this work is strengthened by its relevance to human kidney disease, i.e. CEP83 mutations that manifest, amongst others, in the kidney. It is also interesting for the field of researchers that use kidney organoids from human pluripotent stem cells and their ongoing attempts of improving the organoid model to more accurately recapitulate human kidney pathologies.

Reviewer #1 (Public Review):

In this study, Ravindran et al. describe heterozygous de novo variants in the CRMP1 gene in three unrelated kindreds with muscular hypotonia, autism spectrum disorder, and/or intellectual disability. Based on in silico analysis these variants are predicted to affect the CRMP1 structure. The effect of these variants on the protein structure/levels and cellular processes was analyzed. The authors show that the identified CRMP1 variants are dominant-negative and impact the oligomerization of CRMP1 proteins. Moreover, overexpression of mutant-CRMP1 variants affects neurite outgrowth of murine cortical neurons. It has been known that maternal autoantibody reactivity to CRMP1 significantly increases the odds of a child having a higher Autism Diagnostic Observation Schedule (ADOS) severity score (PMID: 33483694), and increased CRMP1 mRNA levels were identified in individuals with schizophrenia and autism spectrum disorder (PMID: 22798627). Mice lacking Crmp1 expression manifest hyperactivity, impaired learning, memory, and prepulse inhibition (PMID: 24409129). Previous findings strongly support the involvement of CRMPs in neurodevelopmental disorders. It is known that the mammalian CRMP family consists of five cytosolic family members (CRMP1-5) and are highly expressed in the developing and adult nervous system. Monoallelic CRMP5 variants can cause Ritscher-Schinzel syndrome 4 (MIM#619435).

Regarding studies:<br /> In family 1, whole-exome sequencing (WES) was performed on a HiSeq XTen Deep Sequencer (Illumina, CA, USA), with an average coverage of ~36X, which is lower than expected. CRMP1 variant segregation was confirmed by Sanger sequencing.<br /> In family 2, the variant was detected by routine trio-based WES diagnostics. Sanger confirmation was not performed. IGV images can be added as supplementary material. Furthermore, median coverage was 75× which might not be sufficient for the identification of all heterozygous variants.<br /> In family 3, trio-based whole-genome sequencing was performed. Variants >4kb were called using CNVnator (v0.4.1) and annotated with AnnotSV (v2.5.1). An average depth-of-coverage of >50x was obtained. Sanger sequencing was performed to confirm the identified mutation in the CRMP1 gene.

Regarding Results:<br /> Proband 1 (P1) was born as the second child of non-consanguineous healthy parents of Caucasian descent after an uneventful pregnancy. At delivery, a singular umbilical artery was noted. P1 has a moderate intellectual disability and behavioral abnormalities. Chromosome analysis and array-CGH were normal in the index patient (P1). The identified NM_001014809.2(CRMP1_v001):c.1766C>T variant has not been reported in publicly available databases.<br /> Proband 2 (P2) was born as the second child of non-consanguineous parents of Caucasian descent after an uneventful pregnancy and delivery. The boy was macrosomic at birth. Since there was macrosomia, how would the pregnancy be uneventful? At the last assessment at 10 years of age, obesity associated with hyperphagia was of concern; the weight of the patient should be clarified. P2 was diagnosed with autism spectrum disorder but a normal cognitive profile. The identified NM_001014809.2(CRMP1_v001):c.1280C>T variant is very rare and reported in GnomAD exomes with allele frequency 0.0000041.<br /> Proband 3 (P3) is the first of three children of a non-consanguineous family of European descent. There is a familial history of obesity on both parental sides, and the father is macrocephalic (head circumference: 60.5 cm). Macrocephaly can be isolated and benign, such as in benign familial macrocephaly. However, P3 presented with moderate intellectual disability and an autism spectrum disorder. Since P3 has a macrocephaly also, the PTEN gene should be further interrogated by detailed WGS data analysis as well as an additional orthogonal method(s) since it has pseudogenes.<br /> Array analysis revealed two maternally inherited deletions: a 668 kb deletion at 3q26.31 and a 371kb at 5q23.1, confirmed by genome sequencing and considered a variant of unknown significance. The identified NM_001014809.2(CRMP1_v001):c.1052T>C variant has never been reported in the publicly available databases.<br /> Regarding the protein purification, the transient expression, the Western analysis (denaturing and native), and neurite length (4 independent experiments) all seem clean experimental data. The Western blots are clean, and band strength supports the authors' claims. Fluorescence images of the neurons (Fig 3) were not provided but the plots of the four experiments support again the authors' conclusions.<br /> To analyze the effect of CRMP1 variants on its protein levels and cellular function, two isoform variants (CRMP1B-P475L (P1) or -T313M (P2)) were chosen for further functional analysis. CRMP1B T313M and/or -P475L expression might perturb the oligomerization of CRMP1B-wildtype in a dominant-negative manner. Based on the data P475L may exhibit a stronger dominant-negative effect than T313M.

Reviewer #3 (Public Review):

This manuscript identifies specific dominant-negative mutations in the CRMP1 gene encoding Collapsing response mediator protein 1 involved in cytoskeletal remodeling. The authors identified 3 independent probands, each with a de novo CRMP1 mutation-based upon unbiased exome or genome sequencing. Family 1 showed p.P589L/p.P475L, family 2 showed p.T427M/p.T313M and family 3 showed p.A351S/p.A237S. CRIMP1 is known to homo-oligomerize, and the paper attempts to show defects in this ability with the incorporation of patient mutations. Finally, forced expression of patient mutations into neuronal cells show defects in the length of the longest neurite.

Major weakness:

The major weakness is Figure 2, as it is not performed up to high standards like the rest of the paper. Panel A does not show any loading control and does not confirm. Panel B at 720 kDa band is not convincing. Results should be repeated with size exclusion chromatography and/or another method to determine molecular weight and should be quantified from triplicate experiments. Panel C is also not convincing and should be repeated to more carefully show results, and quantified.

Reviewer #1 (Public Review):

Codjoe and colleagues report a combined proteomic and genetic analysis of MSL protein function in the context of mechanosensing in Arabidopsis leaf epidermis. The study identifies MSL10 as being associated with proteins residing in ER-PM contact sites (EPCSs). This is a novel and interesting observation and offers a new context in which to evaluate MSL activity in mechanosensing. It is striking that genetic suppressor analysis of a gain of function msl10 allele also identifies two components of EPCSs as suppressors.

This firmly associates MSL10 with EPCS. However, beyond this association, the study does not identify a clear mechanism of action or even relevance of EPCS localization or relevance of the MSL10/VAP27/SYT1 interaction. There is some indication based on synthetic lethality between msl10 loss of function and VAP27 or SYT1 overexpression that the interaction is relevant, but most direct assays for localization are negative. As a consequence, there is much interesting speculation in the discussion, but I find this somewhat unsatisfying.

Reviewer #1 (Public Review):

In this manuscript, the authors build off prior work identifying LamA as a mycobacterial protein required for asymmetrical cell division. The authors identify PgfA as a LamA protein interaction partner. A PgfA homolog has been studied in corynebacteria where it has channel activity and is involved in lipoglycan synthesis but had not been assigned a function in mycobacteria. The authors show that PgfA is essential in mycobacteria, and interacts with MmpL3, as well as a TMM analog. The data presented are interesting, important for the field, and convincing. However, the authors also make a number of conclusions in the text for which there is no data shown.

Reviewer #3 (Public Review):

Gupta and colleagues investigate the function of the PgfA (MSMEG_0317) protein in Mycobacterium smegmatis (Msmeg). This protein was of interest due to previous work showing that it interacts with the LamA protein involved in the asymmetric polar elongation of mycobacteria. Evidence is presented that PgfA is essential for the growth of Msmeg and that it localizes primarily to the old cell pole. This asymmetric localization as well as the asymmetric localization of the trehalose monomycolate (TMM) flippase MmpL3 was shown to be dependent on LamA. Co-immunoprecipitation was used to show the MmpL3 and PgfA interact. Moreover, cells depleted of PgfA and MmpL3 were shown to have similar terminal phenotypes - the depleted cells lost cell envelope material from their surface and lysed. PgfA depleted cells were also shown to have defective outer membrane by cryo-electron tomography. Crosslinking studies were also used to show that PgfA interacts directly with TMM. Together, these data make a strong case for the involvement of PgfA in the process of mycolic acid transport to the mycomembrane, which is a significant advance in the field of mycobacterial envelope assembly.

Less convincing were results showing the depletion of PgfA affects the levels of TMM and its derivative TDM (trehalose dimycolate) in cells and that overexpression of PgfA can restore asymmetric polar growth to cells lacking LamA. I was also not convinced by the argument that PgfA and its homolog from related corynebacteria (NCgl2760) have different functions. There are many explanations for the failure of NCgl2760 to complement PgfA inactivation in Msmeg that do not require invoking different functions for the two proteins. Specific protein-protein interactions required for PgfA function could have diverged in the two organisms such that NCgl2760 is unable to interact with its required mycobacterial counterparts. Additionally, the lengths of mycolic acids differ between corynebacteria and mycobacteria, which may make the transporters incompatible across organisms.

Reviewer #1 (Public Review):

The authors provide compelling evidence that the protein kinase GCN2, which is an arm of the integrated stress response (ISR) in cells deprived of amino acids and nutrients, displays a pro-tumorigenic role in prostate cancers.

The strength of the manuscript is its novelty, the well-performed experiments, and the very good quality of data and their analysis. The weak points are focused on a better explanation of the mechanisms of GCN2 function based on the presented data. Minor issues relate to the inclusion of a few control experiments to further improve the quality of the data.

Reviewer #3 (Public Review):

The authors perform a wide range of molecular, cellular tissue, and animal model studies that demonstrate clearly that GCN2 activity impacts amino acid transporter activity and essential amino acid uptake, which is needed for PCa tumor growth in a variety of model systems. As a whole the data are convincing, and the authors have achieved their aims. One potentially translatable finding is that a small molecule inhibitor of GCN2 may be a useful candidate therapeutic tool for certain PCa patients.

Reviewer #1 (Public Review):

The authors report experiments and a mathematical model to understand how a flow network of Physarum polycephalum rearranges the channel radii in time. The topic is interesting since fluid flows in networks are ubiquitous and in many living systems the networks are not static but instead can rearrange over time. The variables that control the rearrangements, including growing and shrinking different flow channels, are still not understood though apparently often it is assumed that the local shear rate dictates time-dependent network dynamics. In this paper, the authors demonstrate using experiments that there is a time delay between the change in the flow and the change in the network geometry, and that network architecture-dependent parameters, such as the local shear rate in a channel, and the resistance of flow in a part of the network, relative to the resistance in the rest of the network, can be used to predict vein dynamics. For example, the authors observe vein dynamics by tracking vein radius and shear rate over time and identify regular behavior, e.g., usually stable veins perform looping trajectories in the shear rate-radius space shown in Figure 1, which appears to correlate with an in/decrease in shear followed by an in/decrease in vein radius yielding shear feedback on local vein adaptation. In contrast, usually in shrinking veins, the relation between shear and vein adaption is ambiguous, to use the authors' words. Their data makes clear these main features and the authors construct a mathematical model that helps understand the observed instabilities (channels shrink and disappear) or stability (channels can periodically grow and shrink). It is the features of the time dependence of the network, and identifying variables and a macroscopic model for the dynamics, that I think are the novelties of the paper and so most likely to be impactful in the field, e.g., vein fate being determined by network architecture dependent parameters, such as relative pressure and relative resistance. That said, I find some of the writing unclear and some of the figures challenging to read and understand. Also, it was unclear what might have been reported in several of the referenced papers that highlight dynamical features relevant to this paper.

Reviewer #1 (Public Review):

The manuscript by Rial et al. describes an interesting interaction between the dFOXO transcription factor and the transposable element (TE) activity in aging using Drosophila. The authors find that dFOXO deletion mutants lead to elevated TE expression. They go on to use Drosophila molecular genetics to over-express the gypsy retrotransposon coding sequences and show deleterious effects on lifespan. They show that a wild-type reverse transcriptase (RT) enzyme is required for the reduced lifespan. Interestingly, the effects are only observed in "middle-age" flies. The authors also go on to show that there are defects in circadian rhythms in the flies over-expressing gypsy.

Reviewer #1 (Public Review):

Yang et al. provide a scientifically sound and compelling manuscript.

Strengths:<br /> -Very thorough study characterizing transcriptional and secretory responses of organoids derived from mid-to-late gestation at baseline and after viral infection.<br /> -Establishes organoids as an important model to study vertically transmitted microbial infections.

Weaknesses:<br /> -Discussion and characterization of what each organoid type is specifically modeling (orientation/cell types) is important for context to fully grasp inherent strengths and weakness of model.<br /> -Despite commonly indicating TO and DO are matched, the use of this matched specificity is not utilized either experimentally or in the interpretation of data.<br /> -Given gestational age can putatively impact outcome, a better understanding of the result of each organoid line in terms of specific gestational age derived is warranted.<br /> -There is only a single readout for viral infection with quantification being % infected organoid (which could include organoids with only a single cell or hundreds of cells infected). A more fine-tuned quantification seems necessary given the conclusions of the manuscript.

Reviewer #1 (Public Review):

The authors asked to what extent early visual and visuomotor experience is essential for developing the ability to recalibrate the visuo-motor system flexibly. This kind of recalibration crucially underpins everyday actions, allowing the brain to issue effective feed-forward motor control commands that correctly account for temporary changes in sensory-motor mappings (e.g. when using tools, carrying objects, wearing new glasses). To address the role of experience in developing these recalibration abilities, they used the unusual clinical population of late-operated cataract patients: children and adolescents who initially had many years of sensory experience that is atypical in that it lacked effective pattern vision. They used a standard sensory-motor task in which participants point to targets with and without displacement of the visual image via a prism lens: after the prism displacement, the visuo-motor mapping needs to be recalibrated to enable effective pointing. They compared late-operated cataract patients with controls matched in age, controls matched in both age and visual acuity (via added visual blur), as well as an extensive broader comparison group of typically developing 6- to 17-year-olds. Their key findings were that recalibration was less effective - both in the initial effect and in the subsequent after-effect - in the patient group than in control groups; this was not related to chronological age but was related to time post-operation, such that performance came to match controls after around 2 years of improved visual experience. The authors conclude that flexible sensory recalibration abilities normally rely on extensive sensory-motor experience in childhood, and suggest that the underlying computational problem is establishing the correct correspondences between sensory and motor coordinate frames. This may be achieved through extended exposure to the sensory consequences of self-generated movements.

Strengths of the approach include use of the established (although rare and difficult to access) model population of late-operated cataract patients and a well-established experimental task (pointing after displacement of the visual image by viewing through prism lenses). The task has a known typical time-course of behaviour - supplemented here by an extensive additional study on typical development using the exact same main task, which even alone would be a meaningful contribution to literature on sensory-motor development. The procedure, measures, analysis, and the approach to control groups are careful and rigorous. The findings are rich in showing not only an initial deficit in patient vs control groups but also an approximate time course for further learning and development after which point (by ~2 years) the patients come to match controls. A challenge is the heterogenous group, in terms of age at operation and ages at testing and follow-up. However, this is very usual and almost inevitable in the literature with this kind of population, and is dealt with well in the analyses. The approach is also well supplemented by repeated follow-up of a portion (actually more than half) of the group.

One potential issue is the role of baseline pointing precision differences across the groups. It would be useful to better understand the potential role of the reduced pointing precision that was found in the cataract group (Supplemental Figure 1B). It is not surprising that, following visual deprivation, this group's predictive feedforward visuo-motor control was less precise than that of controls, even in the baseline measures before any prism manipulation, and even when the controls' vision is comparably blurred. It seems likely (although is not shown) that during the adaptation phase and the post-adaptation phase, the variability of individuals around their (gradually shifting) mean pointing location would also be higher than in controls. I wonder how large an explanatory role there could be simply for this noisier initial visuo-motor mapping in the patient group. It might be said that, on each trial, they intend to carry out a feedforward plan with a certain endpoint, but because of noise, they are on average substantially further from that endpoint than comparable controls are. So, during recalibration, while controls are dealing mainly with cancelling out one kind of error - the constant error due to the prism adaptation - the cataract patients are also dealing with more variable errors due to their own noisier visuo-motor system. In theory, could this alone - higher initial noise in the system - explain the difference? This seems like a simpler explanation than that the system has developed differently in substantial ways to do with its abilities to learn and adapt. One starting point for checking in to this would be asking if initial pointing variability predicts recalibration (perhaps controlling for visual acuity), both at first test and in the repeated participants. Another would be looking into ways to perturb controls' baseline pointing performance further (perhaps with something like an unexpected added weight rather than more visual blurring) so that their variable pointing errors were matched to the cataract group.

Another question is how well the contrast sensitivity function (CSF) as a whole (not just the maximum acuity point) was matched - this is dealt with only briefly. I am not sure to what extent the blurring manipulation would be expected to change the shape of the CSF as a whole to be in line with that of patients, and to what extent other aspects of the CSF besides the maximum acuity point determine the precision and accuracy of ballistic pointing movements under the experimental and lighting conditions used in the study. Depending on the answers to these questions, the concern could be that visual differences relevant to control of pointing remained across the patient and blurred control groups.

Another more minor or technical issue is some lack of detail in how the calibration index, which feeds into most of the key analyses, is calculated. It is likely that many different ways of doing this would lead to similar conclusions, but it should be clear, including for the sake of replicability.

Reviewer #1 (Public Review):

Ribonucleotide reductases (RNR) share low sequence similarity, which makes it challenging to infer their phylogeny with traditional methods. To accurately decipher their evolutionary history and evolutionary relationships between different clades the authors combined a structure-based workflow developed by Spence et al. (Reference 1) and a state-of-the-art evo-velocity analysis. Thus, they present a convincing phylogenetic map of RNR, among which they found a clade Ø unknown before and determined its cryo-EM model. One strength of this study is that the analysis pattern utilized in this paper can give a good example of the analysis of protein families which are highly diverse in sequence but share an overall conserved structure core, and thus this analysis pipeline may be implemented in other protein families. The weakness of this study is that the catalytic function of RNRs from the novel clade Ø is not well characterized, such as the ferritin-like domain. It would be interesting to design comprehensive biochemical experiments on this novel clade RNR and maybe the authors will do that in the near future. On the basis of the large-scale phylogeny of RNR, the authors studied three extension/insertion regions of RNR, including N-terminal ATP-cone, C-termini of class II RNR and finger-loop-motif of Class III RNR. These discoveries systematically reveal the plasticity and evolvability of RNRs, which may lead to a model to depict the complete evolutionary history of RNRs. Another weakness is that the descriptions on these extensions/insertions are somewhat scattered and lack a summary model/illustration/table to unify all discoveries. Overall, this manuscript can promote the understanding of RNRs and protein evolution, and the methodology utilized in this study may offer a reference of other diverse protein families.

Reviewer #3 (Public Review):

In this manuscript it has been found that there is a deeply diverged ribonucleotide reductase class that can potentially be the ancestor of both class I and class II ribonucleotide reductases. Furthermore, the structure of a representative member of the new class was characterized with cryo-EM and SAXS. I found the manuscript very interesting and of high relevance. A weakness though was that I did not see anything written about enzyme activity and if the small subunit contains any free radical in the manuscript, which means that we cannot be sure that it really is a ribonucleotide reductase although the homologies and the ability of dTTP to induce dimerization is a strong indicator of that.

Another conclusion in the manuscript was that the last common ancestor of the ribonucleotride reductase classes had the ATP cone-mediated allosteric regulation that we see in approximately half to the ribonucleotide reductase today. However, although the analysis presented is interesting, I think that it is still an open question whether the last common ancestor had an ATP cone or not. Many species contain more than one class of ribonucleotide reductase and because it is a mobile element, it can easily jump from one class to another.

Reviewer #1 (Public Review):

The authors use a novel Satellite Glial Cell (SGC)-enriched promoter Blbp to target diphtheria toxin (DTX) killing, then analyze changes in sympathetic ganglia and autonomic function. These changes are compared to those resulting from similarly targeted deletion of Kir4.1 channels. To summarize, tamoxifen induction of DTX in adult mice led to >50% reduction in cervical sympathetic SGCs, a substantial decrease in adrenergic enzymes (~90-99% loss of TH and DBH), smaller neurons, and decreased pS6 (from which impaired mTOR is inferred), loss of ~25% neurons but 8-fold cFOS activation, and maintained axons and 60% increased circulating NE. Expression of certain adrenergic receptor subtypes was also found to be decreased. Conditional knockdown of Kir4.1 (by ~75% in RT-PCR) led to no apparent decrease in SGC numbers (judged by Sox2 and Blbp staining), ~60% decrease in TH and DHB, increased numbers of smaller neurons and impaired mTOR signaling, loss of about 20% neurons and increased cFOS.

Although cellular effects of DTX ablation and Kir4.1 deletion in SGCs overlap considerably, the overlap does not include changes in autonomic function, where the DTX and Kir4.1-targeted deletion mice were quite different. DTX led to increased sympathetic activity (increased pupil size without apparent parasympathetic change in constriction and increased rate but reduced variability in heart rate). However, none of these changes were observed in the Kir4.1-targeted mice. The authors conclude that satellite glia is important for sympathetic neurons, partly through the provision of Kir4.1 channels and spatial buffering of potassium.

Strengths of the paper include the use of the novel promoter (which is stated to have ~50-fold higher abundance in SGCs than astrocytes) and the dataset itself, which is for the most part thorough and convincing Issues include specificity of the targeting, opposite effects on sympathetic function reported from studies using DREADD activation of SGCs, and conclusions regarding Kir4.1 effects and mechanism.

Concerning specificity, CNS involvement through effects on other cell types is not totally ruled out in these studies, and effects on the same cell type but in other ganglia (parasympathetic and sensory) might be expected to impact sympathetic function. For example, as Vit (2008) reported that following shRNA knockdown of Kir4.1 in trigeminal ganglia hypersensitivity to mechanical stimulation could affect autonomic activity. The authors tested for the influence of parasympathetic using pupillary constriction, and it is somewhat surprising that there is no deficit if neuronal death and dysfunction are as profound in parasympathetic ganglia as shown here for the superior cervical ganglia.

Physiological effects of DTX but not Kir4.1 deletion increased sympathetic activity, whereas increased heart rate was also observed following chemical activation of SGCs using DREADD ligands (Xie et al., 2017). This opposite action is not discussed at length but is attributed to "context-dependence." Inconsistent results with stimuli believed to target the same substrate are worthy of additional consideration by the authors. An alternative conclusion from the finding that the similar cellular level changes in sympathetic neurons induced by DTX and Kir4.1 cKO led to distinct changes in autonomic tone is that the neuronal phenotype does not dictate whole animal physiology.

Spatial buffering is given as the proposed benefit of Kir4.1 channels to the sympathetic neurons. However, this concept arose from studies in which clearance of local extracellular space was limited, and astrocytes were appreciated to be connected to a vast syncytium allowing siphoning away from the high levels near active neurons. The organization in peripheral ganglia differs in three major respects: Despite narrow extracellular space, there is no true barrier to diffusion of K ions from the neurons (one factor that makes drug targeting peripheral neurons appealing), SGCs are very thin (and thus without spatial consequence to uptake), and the coupling among the SGCs is local to those surrounding individual neurons, with very little coupling under normal conditions to other distal SGC-neuron units.

Reviewer #3 (Public Review):

In this manuscript, Mapps et al. report on the very interesting finding that satellite glia deletion significantly impacts sympathetic neuron function and survival. Specifically, loss of the glia results in reduced mTOR signaling, norepinephrine production, and a loss of neurons. Surprisingly, there was an increase in neuronal activity, leading to increased physiological effects such as increased heart rate and pupil dilation. The authors also demonstrate that many of these effects can be mimicked by glial K+ channel, Kir4.1, deletion, indicating that loss of the glia disrupts K+ buffering around the neurons. This is a very novel finding that reveals an important role for satellite glia in sympathetic physiology. It is comprehensive and well controlled. There are just a few issues that the authors should consider.

In Fig. 1C-D, how many dpi was the TUNEL assay performed? It would be helpful to know how quickly the neurons die after glial depletion and if the cell death continues or plateaus. The authors should also co-label using neuronal and glial markers to evaluate whether the apoptotic cells are primarily neurons or glia. They report a loss of neurons, but how much of that is reflected in the TUNEL labeling is not clear.

In Figs. 1C and 5C TUNEK analysis, there are quite a few TUNEL+ puncta outside of the ganglia, suggesting that there may be apoptosis in other adjacent tissues when the glia removed or Kir4.1 is deleted. The authors should comment on that if it were something consistently observed.

The loss of neurons upon glial cell loss or Kir4.1 deletion is interesting. The authors discuss how neuron death could occur, but did they observe TUNEL+ cells in regions where the glia had been deleted? Given that the diphtheria toxin did not ablate all glia, were the neurons left with little or no surrounding glia more likely to die? This may be difficult to tell, but from the images in 1E, it looks like some neurons lack nearby glia. This would be a potential explanation for why only a fraction of the neurons died; those neurons with associated glia may be more protected.

It would be helpful to clarify a bit more what the control mice used for comparison were. From the text, it seems as if they were the same mice but not treated with tamoxifen. Were they given diphtheria toxin? In addition, did the authors check for any effects of tamoxifen alone? Given that estrogen can affect many physiological parameters, including cardiac function, tamoxifen alone could have some effect, e.g., Kuo et al., PMID: 20392827.

Interestingly, TH levels in BLBP:iDTA mutant axons appeared to be similar to that in controls, despite the marked reduction in TH mRNA and protein levels in neuronal cell bodies (Figure S2A). The Kaplan lab (PMC7164330) showed that TH mRNA trafficking and local synthesis play an important role in synthesizing catecholamines in the axon and presynaptic terminal. Although a bit beyond the scope of this study, it would be interesting to determine whether TH mRNA transport is altered by deletion of the glia. The authors might check to see if TH transcripts are reduced in axons by something like RNAscope.

Reviewer #1 (Public Review):

The authors ask an interesting question as to whether working memory contains more than one conjunctive representation of multiple task features required for a future response with one of these representations being more likely to become relevant at the time of the response. With RSA the authors use a multivariate approach that seems to become the standard in modern EEG research.

I have three major concerns that are currently limiting the meaningfulness of the manuscript: For one, the paradigm uses stimuli with properties that could potentially influence involuntary attention and interfere in a Stroop-like manner with the required responses (i.e., 2 out of 3 cues involve the terms "horizontal" or "vertical" while the stimuli contain horizontal and vertical bars). It is not clear to me whether these potential interactions might bring about what is identified as conjunctive representations or whether they cause these representations to be quite weak. Second, the relatively weak conjunctive representations are making it difficult to interpret null effects such as the absence of certain correlations. Third, if the conjunctive representations truly are reflections of working memory activity, then it would help to include a control condition where memory load is reduced so as to demonstrate that representational strength varies as a function of load.

Depending on whether these concerns or some of them can be addressed or ruled out this manuscript has the potential of becoming influential in the field.

Reviewer #3 (Public Review):

This study aims to address the important question of whether working memory can hold multiple conjunctive task representations. The authors combined a retro-cue working memory paradigm with their previous task design that cleverly constructed multiple conjunctive tasks with the same set of stimuli, rules, and responses. They used advanced EEG analytical skills to provide the temporal dynamics of concurrent working memory representation of multiple task representations and task features (e.g., stimulus and responses) and how their representation strength changes as a function of priority and task relevance. The results generally support the authors' conclusion that multiple task representations can be simultaneously manipulated in working memory.

My only concern is that in Figure 4, the strongest priority by task-relevance interaction occurred earlier in the response than the conjunction representation, which seems to be opposite to the assumption that the conjunction representation produces the response and thus requires more discussion on why this is the case. This study expands the working memory research by showing that working memory can simultaneously hold and manipulate multiple task representations. It also provides solid foundation for future work to investigate the control mechanisms on working memory representations of task conjunctions.

Reviewer #1 (Public Review):

The manuscript is well written, clearly describes the scientific background and hypotheses, and provides a sound illustration of the results, which can advance our current understanding of the neural basis of decision-making processes. The main conclusion is that pallidal stimulation in patients with dystonia leads to an increased number of exploratory choices, i.e. choosing the option with a lower expected value instead of exploiting the option with the highest expected value. There are, however, some shortcomings that limit the interpretability of the data in its current form regarding the lack of a healthy control group, inconsistency between frequentist and Bayesian statistics applied, and the limited specificity of the connectome correlation analysis. These shortcomings should be addressed by the authors in order to improve the paper.

Detailed description of comments:

(1) Generalizability:<br /> Studying dystonia patients gives the unique opportunity to study the effects of electrical pallidal stimulation on decision-making in humans and given that dystonia primarily affects movements rather than cognition/decision-making this might also well be representative of healthy people. This (i.e. the similarity between task performance of patients and healthy people) is, however, not demonstrated in this study. In the introduction, the authors state that reward prediction error is intact in dystonic patients, but the paper that they cite for this (ref 34) is titled '... abnormal reward learning in cervical dystonia'. Furthermore, albeit clearly less pronounced than movement symptoms cognitive problems are present in dystonia patients (see Jahanshahi 2017 Movement Disorders). I would therefore recommend enrolling a healthy control group allowing to compare DBS ON and DBS OFF to healthy people.

(2) Statistics:<br /> I understand that Bayesian statistics cannot always directly be compared to non-Bayesian frequentist statistics. However, to me, the frequentist and Bayesian statistics are not consistent in this study. ANOVAs, etc are applied on subject-averages data using a p-value of 0.05 to distinguish between significant vs. non-significant results. In the Bayesian modelling analysis, the 95% HDI is computed. While this number is arbitrary (just as a p-value of 0.05) it still has a rationale to it given that in the scientific community 95% is also used for frequentist confidence intervals. Therefore, I think that 95% would be the most consistent choice here. However, none of the model parameters differ between ON vs. OFF regarding the 95% HDIs, since they overlap with 0 (see 'Contrast' in table 1). Especially the decision threshold and drift rate scaling parameter HDIs have a large overlap with 0, but they are still interpreted as significant based on the Bayes factor. The Bayes factor, however, is not used for the behavioral analyses. For example, there are no effects of DBS on decision times, but at the computational level, several parameters (which predict the decision time) are affected. I think for the sake of consistency of analyses within the paper the statistics of the Bayesian analyses should rely on the 95% HDI.

(3) Connectome correlation analysis:<br /> If I understand it correctly, the connectome analysis relates behavioral effects of stimulation to whole-brain networks rather than just local effects in the pallidum by testing whether patients who showed stronger effects of stimulation have electrodes that are closer to connections with different brain areas. In the abstract, the results of this analysis are reported as "... was predicted by the degree of functional connectivity between the stimulating electrode and prefrontal and sensorimotor cortices". In the discussion, it is stated that "...DBS-induced enhanced exploration correlated with the functional connectivity of the stimulation volume in the GPI to frontal cortical regions identified previously in functional imaging studies of explore-exploit decision making ... The exploration-enhancing effects of GPI-DBS in our study were predicted by functional connectivity to brain regions whose neurons encode uncertainty [27] and predict behavioural switching[430 29, 30]". However, figure 4 essentially shows that almost the whole brain correlates with inter-individual differences in behavior reaching correlation coefficients as strong as -0.7 e.g. lower brain stem, cerebellum, and occipital cortex, none of which are mentioned in the paper. To me, it seems that there are correlations with very large and very distributed cortical areas rather than with specific areas in the prefrontal and sensorimotor cortex as stated in the paper.<br /> Related to this point: The variable used for the connectomic correlation analysis is not the same variable that was affected by DBS in the statistical analysis. The statistical analysis found that P(explore) differed between DBS ON vs OFF irrespective of the session. Instead the "maximum within-session increase in P(Explore) DBS-ON - P(Explore ) DBS-OFF" was used.

In general, could you please explain this analysis in more detail? If I understand it correctly each voxel had a value for 'connectivity' to the stimulation field and a value for 'behavioral effect' and across patients, this then gave an R-map. How was figure 4 thresholded (only the maximum positive and negative Rs are given in the color bar)? Then p-values are listed. One is 0.04 and another one is 0.009. What is the difference between the two? These values seem to reflect the correlation of similarity between the individual map with the group map and the behavioral variable, but was the correlation with the behavioral variable not already used for creating the R-map? Describing the analysis in more detail might help make it more understandable to the audience not familiar with the analysis (including me).

4) It is my understanding that high exploration (e.g. P(Explore) of 0.2) should be related to poorer task performance since the optimal strategy would always use the high-value option and only switch rarely to identify the reversal(s). Why is it then that DBS can affect exploration but not the sum of rewards if the two are related? Should DBS not affect the sum of rewards if it for example was more pronounced in its effect on P(explore)?

5) Would the authors have predicted different effects for subthalamic deep brain stimulation? The DBS effects on the GPi are mainly interpreted in terms of reduced firing rate/activity. Since the STN exerts glutamatergic innervation of the GPi, should STN suppression lead to similar results? Conversely, GPe exerts GABAergic innervation of the STN. Should GPe suppression lead to the opposite behavioral effect? Were some of the electrodes localized within or close to the GPe rather than GPi and if so, did these patients show different behavioral effects?

6) Was the OFF vs ON DBS order counterbalanced? 3 patients did not complete the task OFF, and the ON dataset was not available in another patient. Did the authors check if the DBS order was relevant for the DBS effect on P(explore)?

Reviewer #3 (Public Review):

The manuscript examines the neural bases of the exploration/exploitation tradeoff - a crucial component of decision-making, that determines whether we choose the best option or explore less beneficial, but perhaps more informative alternatives. The authors specifically focus on the role of a substructure of the basal ganglia (the globus pallidus internus, or GPi) in modulating the amount of exploration in a simple learning task. This is a straightforward, well-designed study - albeit with a small patient sample, as is often the case in clinical data involving deep brain stimulation - and the computational modelling is rigorous. The presented work convincingly argues for the role of the GPi in suppressing exploration and enhancing exploitative choices.

Strengths of the present work<br /> 1) Testing DBS patients is a somewhat rare opportunity to directly observe the impact of stimulating or inactivating specific neural areas on human behavior. The present task's pallidal-DPS cohort and the ON/OFF stimulation manipulation make for a strong argument that the observed differences in behavior and model parameters are indeed due to the GPi, and the author's proposed neural framework for how the GPi modulates exploration is well-supported and convincing.

2) The computational modelling is rigorous; the authors have shown how their selected model complements the data and model-free analyses, as well as conducted posterior predictive checks to test the extent to which recovered model parameters are actually informative.

3) This line of investigation is always relevant and timely, as most daily decisions from small-scale human decisions to large-scale AI machines involve calibrating exploration and exploitation in some form. Further insight into the neural mechanisms of this tradeoff, therefore, holds significance and countless potential applications.

Other Comments<br /> 1) While historically, 'exploration' was simply defined - as in the present work - as simply choosing the non-greedy/non-maximizing option, in the past decade or so more recent work has crucially distinguished between types of exploration that are explicitly aimed at seeking new information (i.e. directed exploration - specifically choosing the options that are less well-known, in order to build a more accurate world representation) and those that are independent of the informativeness or other properties of the other choice options (i.e. decision noise). Existing literature provides evidence for separate neural substrates for the two, and any model that will enrich our understanding of how the brain calibrates the explore/exploit tradeoff should at least touch on how these separate types of exploration fit into the proposed framework. It would therefore help contextualize and strengthen the presented work to include more discussion on precisely which type of exploration the GPi is modulating.

2) While the proposed model is well-presented and checked, some further clarification for readers who are not familiar with RLDDM might improve clarity. Furthermore, the model-free performance analyses as well as the brain connectivity analyses, while they clearly show a link between GPi stimulation and the overall amount of exploration, do not delve too deeply into the specific patterns of the exploratory behavior (e.g. by showing within-task fluctuations through a moving window of average exploration, or by describing further the differences in decision time between explore and exploit trials, etc.). The basic performance analyses are consistent with the authors' hypotheses and support the conclusions, but a more in-depth check of specific exploration patterns might help clarify the mechanism better.

Reviewer #1 (Public Review):

The development and patterning of the pharyngeal arches of the vertebrate embryo have not been as well studied as many other more classical areas of embryonic development, such as the developing limb. However congenital malformations of pharyngeal arch derivatives are common, and elucidation of the mechanisms of pharyngeal arch development would be informative for human and animal health, as well as adding to our knowledge of biological mechanisms of patterning during organogenesis.

Using an unusual model - the development of the skate gill arch, and underpinned by a complementary analysis of pharyngeal arch patterning in the chicken embryo, this paper builds on previous work by the authors as well as more established paradigms of embryonic development and patterning, to understand the organisers and molecular pathways they express which contribute to gill arch/pharyngeal arch patterning.

Based on previous work that showed that the primary organiser of skate gill arch patterning - the GAER, expressed SHH, the authors used fate mapping techniques to establish the origin and subsequent morphogenesis of the GAER. They found that it has an endodermal origin. They repeated this experiment on chicken and found it to be the same.

They subsequently followed the expression of SHH and FGF8 through gill arch development, to show both the morphogenesis of the gill arch and that these genes go from a complementary gene expression to having an overlapping gene expression which is most highly in the posterior arch environment. The posterior expression and activation of the SHH and FGF pathways are also shown to be highest in the posterior gill arch- thus this is proposed as the primary mechanism by which the gill arch is 'polarised'.

Further work identified that the anterior gill arch expresses components of the Wnt signalling pathway, in a complementary way to FGF/SHH. Pharmalogical inhibition of Wnt signalling produces extra, non-polarised gill arches, suggestive of not only a loss of polarity but also a change in the distribution of gill arches- perhaps due to a modification of a Turing-type mechanism that would space the cartilages appropriately. At a molecular level, SHH expression did not change, but the activation of the SHH signalling pathway expanded. This perhaps suggests that Wnt signalling acts to restrain/inhibit SHH pathway activation, increasing and underpinning the mechanism of polarisation of the gill arch.

Fundamentally I think the work is strong overall. Each section of the paper is based on a clear platform of data and a hypothesis, which link together to really tell us about how this tissue is patterned - the organisers and the signalling pathways and the interactions between them. The fate mapping, sequencing, pharmacological inhibition, and HCR ISH are conclusive, although I presume due to using the usual skate a model the replicate numbers are quite low.

I do find the paper overly complex in interpretation and the figure quality of summary figures lacking in detail so that a non-gill arch expert can struggle to understand the findings. While the additional work in chicken pharyngeal arch is also strong, it is not overtly covered in the main body of the paper - and I think this is a mistake. I think interest here is uncovering mechanisms of vertebrate development - in which case a stronger comparison between chicks would demonstrate the similarities. I would suggest including make figures in which these species are shown together.

I also feel that there is much that could be discussed - not only about the formation of a polarised tissue but about how the gill arches are spaced - is this a Turing-type mechanism? Are there similarities that can be drawn with the limb or other systems which generate repeating structures? An interpretation of this could interest a wider group than only those that work on pharyngeal arch development.

In summary, I think this is an exciting paper using an unusual model and an understudied but important area of embryonic development which gives us an insight into how some of our commonly held dogmas may apply across different systems.

Reviewer #3 (Public Review):

In this study, the authors aimed to provide evidence of a novel developmental mechanism regulating brachial arch formation in the little skate. More specifically, the authors leveraged previous studies establishing the role of Hedgehog signaling in early little skate brachial arch development and built upon these studies by discovering the embryonic identity of Shh-expressing cells and the role of canonical Wnt signaling in regulating proper anterior brachial arch formation. The authors nicely combined the use of the spatiotemporal expression of various Hedgehog and Fgf signaling members with transcriptomic analysis and pharmacologic experiments to assess genetic relationships. In general, this manuscript is of high quality and will appeal to a diverse array of scientific disciplines. Moreover, the relationship between Shh-Fgf8 and the importance of Wnt signaling in the context of brachial arch formation in the little skate may be more broadly applied to other cartilaginous fishes or other aquatic vertebrate species in general. As the little skate is largely an unexplored model organism, this study exemplifies the utility of the little skate and emphasizes the wide array of methods that can be implored to further identify this species' development on a molecular basis. Future studies should consider the generation of genetically modified skate species, as current functional interrogation is limited to pharmacological approaches. Although this study has been eloquently conducted, there is some extraneous information that takes away from the major conclusions of the story in addition to some gaps in experimental data that are required to clarify their findings.

Reviewer #1 (Public Review):

Bakoyiannis et al. investigated the distinct contribution of ventral hippocampal outputs to the nucleus accumbens and medial prefrontal cortex on memory in mice exposed to a high-fat diet (HFD) beginning in adolescence. The authors first characterize the hippocampal to accumbens or mPFC circuits using intersectional viral approaches. They then replicate their previous finding that adolescent HFD contributes to the overactivation of the ventral hippocampus during contextual learning via quantification of c-fos+ cells. In this manuscript, the authors further explore the distinct contribution of these two outputs from the ventral hippocampus using chemogenetics to specifically inhibit one circuit or the other. Interestingly, the authors find that inhibition of either circuit returns c-fos+ cell number to control levels, but the effects on memory are dissociable. They demonstrate that inhibition of output to the NAc rescues HFD-induced deficits on object recognition, while inhibition of mPFC outputs rescues HFD-induced deficits on object location recall. The authors further confirmed that chemogenetic manipulations resulted in alterations in c-fos+ cells that were specific to CA1, and not CA3 or DG. Behaviorally, they excluded any contribution of anxiety on recall, finding no effect on the elevated plus maze.

The strengths of this manuscript include robust behavioral findings that can be attributed to specific circuits. The conclusions of this paper are largely well supported by the data, although some of the methods could provide more detail and the statistical approaches used for analysis need improvement.

Reliance on only one measure of anxiety to exclude this as a confound on recall performance is a weakness of the manuscript. To be more convincing that anxiety is not a confound, more than one behavioral assay should be performed.

Reviewer #3 (Public Review):

"Obesogenic diet induces circuit-specific memory deficits in mice" by Bakoyiannis et al., investigates the role of specific ventral hippocampal circuits (specifically to nucleus accumbens and mPFC) in high-fat diet-induced memory deficits. The authors had previously shown that increases in activity in the ventral hippocampus accompany high-fat diet-induced memory deficits, and that inhibition of activity thereby normalizes those memory deficits. In this manuscript, the authors extend these findings to specific projections, showing that they normalize different types of memories by inhibiting the two different pathways.

The strengths of the paper include the pathway-specific manipulations that reveal a difference between the two types of memory. The results are a modest step forward for the field of feeding and learning and memory and would be of interest to that subgroup of neuroscientists. However, the paper also has a number of weaknesses which I detail below.

1. First, the authors show an effect of cfos from both pathways in Figure 2 on object learning. However, the inactivation studies show a pathway-specific effect on object recognition and object location, with no experiments to delineate how this divergence occurs. The authors do not specify whether they compared cfos in the control group between NAcc and mPFC projections (presumably they did some controls with each injection), which might reveal differences.

2. Related to this, it is unclear how the pathways end up diverging for memory if they do not show any differences in cfos during training. Perhaps there are pathway-specific differences in cfos following the ORM and OLM tests? It is difficult to support the claim that there are pathway differences in memory following inactivation if we do not see any pathway-specific change in activity.

3. Figure 2 and Figure 3 are also hard to interpret because of the usage of a 1-way ANOVA which is not the appropriate statistical test when there are two independent variables (HFD and DREADD manipulation). Indeed, noticing the statistical test also reveals that a critical control missing: HFD -, hM4di+CNO +. It is possible that inactivation simply brings down cfos levels regardless of diet. While this might benefit memory in the case of HFD, it is critical to know whether the manipulation is specific to the overactivation caused by HFD or just provides a general decrease in activity.

Reviewer #1 (Public Review):

My impression is of a careful and thorough study, that potentially could provide a paradigm for future studies in this direction. A potential causative pathway for hepatic-related sarcopenia is identified. Parallel studies are made in both experimental and human clinical systems.

Reviewer #3 (Public Review):

In this study, Dr Tamai et al. investigated the association between bile acid level and skeletal muscle mass using a rat model and patients with HCCs. The authors found that LCA level was closely associated with skeletal muscle mass in both CLD rats and human patients with HCCs.

Reviewer #1 (Public Review):

Many animal studies have shown that the first and second heart fields give rise to the heart in normal embryonic development. For obvious reasons, this has not been well-studied in humans. Thus these investigators applied hiPSC technology to recapitulate human heart development using small molecules to modulate WNT signaling and thus induce mesodermal lineage differentiation. They set up a triple reporter genetic system (TBX5-Cre/MYL2-tdTomato/CCR5-CM-Lox-STOP-Lox-TurboGFP reporter) in two hiPSC lines and demonstrated that > 90% of ventricular cardiomyocytes were derived from the TBX5/MYL2 lineage. They used RT-qPCR to verify over 12 different time points during the course of the differentiation protocol that cells begin to express markers of the FHF lineage and eventually markers of ventricular cardiomyocytes.

Reviewer #3 (Public Review):

Galdos, et al., have developed a novel lineage tracing technique using genetically encoded fluorophores in human-induced pluripotent stem cells to identify first heart field cells and ventricular cardiomyocytes during differentiation. To label the FHF lineage, the authors use a CRISPR/Cas9 strategy to express a floxed TurboGfp and add a P2A-Cre recombinase sequence at the stop codon of Tbx5 in two well-characterized hiPSC lines. In these same lines, they then added a P2A-tdTomato construct at the stop codon of the ventricular cardiomyocyte-specific sarcomeric protein Myl2. They expected this strategy to allow them to identify cells as they commit to the first heart field lineage and ultimately FHF cells that differentiate into ventricular CMs, which should therefore represent LV CMs by virtue of their lineage. RT-qPCR confirms that over the course of the differentiation protocol cells begin to express well-studied markers of the FHF lineage and eventually markers of ventricular CMs. This matches the flow analysis of their lineage-tracing technique which is suggestive though not conclusive that their technique is identifying the cells it claims to identify.

The authors found, however, that their flow data showed that the differentiation protocol they used gave rise to >90 % FHF lineage cells, most of which were also Tnnt2+ or tdTomato+ by day 30 of differentiation. None of the cells were positive for markers of the second heart field lineage. To confirm this, the authors used scRNAseq data from multiple differentiation time points to identify the paths cells follow through their Wnt-signaling-based small molecule 2D differentiation protocol. What they find suggests there are two distinct path bifurcations using this protocol. The first is between a mesodermal lineage and an endodermal lineage, and the second is, within the mesodermal cells, a bifurcation between myocardial and epicardial lineages. They compare these results to previously published datasets from murine heart field development and see that the mesodermal pathway matches murine FHF lineage development and that there is no good match for SHF lineages. They hypothesize that a 3D differentiation protocol might lead to a subset of cells developing SHF hallmarks and test this by combining the CMs from their own scRNAseq results with those from a group that developed a novel 3D differentiation protocol to form heart organoids. They identify a cluster in the 3D differentiated cells that does not appear in their own dataset and which is enriched for cells expressing SHF markers and markers of outflow tract CMs.

Strengths:<br /> 1. The use of a Cre/lox system to permanently label putative FHF lineage cells with TurboGFP even after reduction of Tbx5 expression will make it possible to both follow the same cells over time to better understand early human heart development and to evaluate novel differentiation protocols for which cell lineages are likely to predominate. This can then be paired with fluorophores tagged to markers of later progenitors or terminally differentiated cell types (as the authors do here with Myl2) allowing isolation of distinct cell types with known lineages at distinct stages of models of human heart development. This is a potentially quite powerful tool given the limited availability of human fetal tissue and the ethical concerns inherent to using it to study development.<br /> 2. The authors have identified a clear weakness of using 2D differentiation protocols based on Wnt-signaling as models of human heart development. They show convincingly for two separate hiPSC lines that while the cells progress through the primitive streak and the emergence of the first heart field cells, the second heart field does not arise in this protocol. This homogeneity of the terminally differentiated cells may be beneficial in regenerative medicine contexts, but it is clear that for studying development and for pushing cells to OFT or RV CM fates, new techniques are required. They then demonstrate the promise of 3D organoid differentiation techniques in overcoming this hurdle.<br /> 3. This manuscript also sets up a powerful workflow for evaluating cell fate decisions over pseudotime in early heart development. The authors used well-published packages to set up their datasets to meaningfully compare scRNAseq results from their own 2D differentiation experiments with those from previously published scRNAseq results of murine heart development and 3D differentiation. For the latter, they were able to combine the datasets to identify a new cluster of cells from the 3D protocol. This workflow will prove extremely beneficial in comparing cell fate outcomes arising from disparate cardiac differentiation protocols.

Weaknesses:<br /> 1. While demonstrating that 2D differentiation of hiPSCs is an imperfect model of development is a valuable outcome of this work, this also makes it an imperfect model in which to test the robustness of their lineage tracing technique. Nearly all of the cells are shown to progress through the FHF lineages using their fluorescent techniques. This is confirmed using scRNAseq, but this means that they are unable to give a proof of principle that their method will distinguish FHF cells from SHF cells since none of the latter arises.<br /> 2. The authors validate their lineage tracing technique with bulk gene expression by RT-qPCR at different time points during differentiation. However, they never directly confirm that isolated TurboGFP+ cells show higher expression or protein levels of their target FHF markers nor that the TurboGFP+tdTomato+ cells are enriched for LV CMs. While their validation as it stands is highly suggestive that their lineage tracing technique works as advertised, the evidence is still only circumstantial.<br /> 3. The section of the paper devoted to the development and validation of their lineage tracing technique is connected to the section analyzing their scRNAseq results only loosely. Having shown by their new technique and its validation that no populations positive for SHF markers are arising during their differentiation, they turn to scRNAseq to confirm this observation. The issue here is that it requires a bit of circular reasoning. Having established that better techniques are required to study human heart development to move away from relying so heavily on our understanding of murine heart development, the authors then draw their conclusion that no SHF lineages arise during the differentiation of their hiPSC lines in part by comparing them to murine heart development. This is in no way a fatal flaw to the work but it limits the ability to use the authors' techniques to draw novel distinctions between human and murine heart development.

Reviewer #1 (Public Review):

Detecting and quantifying balancing selection is a notoriously difficult challenge. Because the distribution of times to fixation or removal of strictly neutral variants has a long tail, it can be hard to exclude the null hypothesis of neutrality when testing for balancing selection that was not established so long ago that trans-specific variants can be observed. As Aqil et al. point out, most efforts to detect balancing selection in the human genome have been focused on single nucleotide variants. The authors seek to characterize the amount of balancing selection specific for polymorphic deletions. The authors justify their focus based on the fact that deletions are more likely to have functional consequences than single nucleotide variants, making it more likely that if they have remained for many generations, this could be a signature of balancing selection. That said multiple aspects of the analysis deserve more attention.

I have two broad concerns about the manuscript that the authors need to address. First, the authors use neutral simulations to exclude that neutrality alone can explain the amount of allele sharing observed between African modern humans and the archaic genomes. My concern is that human demography models, including the one from Gravel et al. (2011) used by the author are always simplifications of the complex demographic events that shaped human populations during evolution. In the case of the specific model used by the authors, African populations were inferred by the Gravel et al. model to have a constant population size for the past ~150,000 years (parameters Taf and Naf in the original model). This is an unrealistic assumption of this model. In brief, I am wondering how much the claim of the authors that neutrality alone cannot explain patterns of allele sharing is potentially based on mis-specifications of the neutral demography model. For example, the more fine scale fluctuations of effective population sizes in Africa inferred by author L. Speidel in 2019 Nature (Figure 3) paint a different picture than the Gravel et al. model. The authors need to run extensive testing of the robustness of their conclusions to changes in the neutral demographic model used. What if the average ancestral population size was closer to 20,000? What if it was closer to 50,000 and frequency fluctuations every generation were smaller? Given how uncertain past population sizes really were and the current uncertainties about demographic reconstruction in particular relative to linked selection, the authors need to explore a range of past populations size beyond the idiosyncrasies of a specific model.

My second broad concern is that it is difficult to evaluate how novel the findings really are. It is true that the authors focus on deletions while pasts scans for balancing selection in the human genome focused on SNVs. But it could be the case that a substantial number of the deletions identified here as under balancing selection could have previously identified as such loci through linked SNVs by the scans cited by the authors. The authors need to provide quantification of how many of their deletions are truly novel balancing selection loci as opposed to balancing selection loci already identified through linked SNVs.<br /> The novelty of the balanced deletions will also be better established by providing a more quantitative and less anecdotal functional analysis. It is true that the deletions include immune loci, but are they statistically enriched for immune loci as annotated for example by Gene Ontology, in a way that shows that their distribution across the genome is not random but indeed driven by selection enriching them at loci with specific functions? In addition, do the pie charts in Figure 5E, represent a statistically significant deviation from left to right or not?

Reviewer #1 (Public Review):

The authors generated valuable snRNAseq data sets from the hippocampus region in APO E4 and E3 mouse models. Through bioinformatics analysis, they identified a list of differentially expressed genes between E4 and E3at 5, 10, 15, and 20 months. In addition, changes in cell type distribution were observed across different time points, and the number of differentially gene expressions varied across multiple cell types. Through pathway enrichment analysis, the authors identified shared pathways such as calcium signaling and MAPK/Raps1/Pld pathways. To determine the relevance of these observations with respect to human Alzheimer's disease, they verified that genes/pathways identified in their mouse models are largely conserved in APOE4-Knockin and human APE4 iPSC-derived Neurons.

A major strength of this study comes from the combination of mouse and human models using snRNAseq analysis. In addition, the authors also used comprehensive bioinformatics tools to dissect the shared genes/pathways during disease progression. While a major weakness of the study is the lack of experimental validation of the specific pathways and their impact on disease. The observational gene expression analysis cannot provide any casual information. It is unclear whether the genes and pathways identified are primary events of disease etiology or secondary events due to disease progression.

Finally, I want to congratulate the authors on creating and sharing such a comprehensive set of snRNAseq data of the APO e4 allele. This set of omics data will become a reference point for the Alzheimer's research community. Their initial analysis of this rich dataset has yielded many interesting findings that may be validated by other groups.

Reviewer #3 (Public Review):

The study by Grone and colleagues proposes to understand how APOE4 contributes to Alzheimer's disease risk by understanding how different cell types within the brain are affected at the level of the transcriptome across the lifespan. There are several strengths of the study, including the concept of profiling different cell types across the lifespan using advanced sequencing methods and the use of a model incorporating neuron-specific deletion of APOE to understand how distinct pools of APOE affect the networks identified according to the form of APOE allele being expressed. There are a number of pathways identified that may inform the field in terms of the elusive role of neuronal APOE in shaping brain function. There are a number of issues in this work that limit many of the conclusions made. For example, the ages chosen to study how APOE alleles affect gene expression in different cell types are limiting and do not unfortunately include earlier ages representing developmental or young adult ages or very advanced age, two ends of lifespan where many functional changes occur in the brain that may be regulated by APOE. Additionally, sex is not studied as a biological variable in the study, leaving the results in question as to whether the findings are limited to one sex. There are a number of other methodological issues, including a lack of clarity on how variance from different sequencing datasets generated at different times for ages within the same comparisons has been handled. In terms of the impact of the study, there is a missing functional validation of key networks that have been identified. We do not know if any of the gene expression differences identified here translate to changes in brain function, limiting our ability to know whether neuronal APOE regulates the brain and may play a role in AD as claimed. Finally, constitutive deletion of APOE within neurons may result in changes in gene expression that are shaped by developmental changes mediated by APOE. Overall, this is an interesting resource that may be useful for scientists seeking to understand the non-canonical roles of APOE in shaping gene expression in the hippocampus.

Reviewer #1 (Public Review):

The authors have performed a deep mathematical analysis of unitary data recorded from the stellate ganglion to understand how the neural code is altered in heart failure.

The study is advantaged by being performed in vivo with afferent and efferent pathways intact. The use of modern microelectrode arrays has allowed mass activity to be recorded from multiple sites simultaneously within the ganglion. The authors have a number of powerful analytical tools that have revealed quantitative changes of interest.

The data are from animals under anesthesia with an open chest and open pericardial sac and one wonders what effect this has on the neural activity given the changes in pulmonary physiology this will cause.

Some of the data are from pigs where resiniferatoxin (a chemical agent to kill sensory afferents) was applied to the epicardial surface. Given the elevation in sensitivity of cardiac afferent reflexes in heart failure (Schultz, Zucker, and others), it is surprising that this had no effect on the neural activity recorded in the heart failure animals. Either the afferents were not destroyed (no data given to demonstrate this) or these sensory fibres play no role in the changes in neural activity reported from heart failure pigs. This would go against current data and remains unclear.

Most of the stellate neurons project to non-cardiac tissues. One does not get a sense of the proportion of activity that was related to the heart (left ventricular pressure) and whether in heart failure there is an elevated activity within a confined network or recruitment of additional networks. In this regard, the manuscript is jargon-heavy and for those that are physiologists, the subtleties of the study may be lost.

Finally, the authors could provide a clearer take-home message and break out of the shackles of math talk and interpret the possible physiological relevance of the work as well as why it is important to understand the changes in stellate neural network dynamics in heart failure.

Reviewer #1 (Public Review):

The authors analyze the roles of BRC-1 and SMC-5 in C. elegans meiosis taking advantage of specific assays to distinguish DSB repair pathways: an inter-sister assay (ICR) (Mos1 induced DSB), an inter-homolog assay (IH)(Mos1 induced DSB), a SCE assay based on Edu labelling of sister chromatids, and other assays such as radiation sensitivity. In addition, due to the controlled timing of DSB induction, by recovering progeny at specific time points, the authors evaluate the properties of cells at leptotene-mid pachytene or at late pachytene-diplotene. The authors also take advantage of SNP in the ICR assay to measure conversion tract length.

The main findings are:<br /> - Intersister crossovers are increased in brc-1 and smc-5.<br /> - Intersister non-crossovers are increased in smc-5.<br /> - Interhomolog recombination is increased in both brc-1 and smc-5 for late prophase cells.<br /> - Increased mutation rate in brc-1.<br /> - Shorter non crossover conversion tracts (ICR assay) in brc-1.<br /> - TMEJ involved in DSB repair in brc-1 smc-5 double mutant.<br /> - Independent localization of Brc-1 and smc-5.

Having assays for specific events allows gaining more direct information on the DSB repair phenotypes of such mutants. The conversion tract assay is the most convincing and clear data which fits well with the role of Brc-1 in end resection. However the results of the ICR and IH assays are interesting but do not fit with previous observations on the role of Brc-1 and Smc-5 based on analysis of meiotic phenotypes, Rad-51 foci and diakinesis, these discrepancies should be addressed.

The experimental approach has some issues that should be addressed: i) the two main windows (inter-homolog and non-inter homolog) are defined based on meiotic progression in wild type. The timing in the mutants and upon Mos1 induction (which could also affect the timing of meiotic progression) should be determined. In particular, the increase of interhomolog events in brc-1 is left without a validated interpretation. ii) Potentially the phenotypes observed in the ICR and IH assays (but not EdU) may be specific to Mos1-induced DSB and may not apply to Spo11-induced breaks. iii) The use of the Edu assay could be clarified, it seems that the interpretation of configurations is challenging, thus potentially leading to selection bias among diakinesis.

Reviewer #3 (Public Review):

The manuscript by Toraason et al investigates the role of BRC-1/BRCA1 and the SMC-5/6 complex in repair pathway choice during C. elegans meiosis. The authors use a recently developed system to detect crossover and non-crossover repair events that use the sister chromatid or the same chromosome for repair of a site-specific induced DSB, a related system to look at repair outcomes using the homolog as a repair template, and a cytological approach to detect inter-sister exchanges. The authors show that BRC-1 and SMC-5 both function during meiosis to limit the formation of inter-sister crossovers but are not essential for interhomolog recombination. BRC-1 also suppresses error-prone DNA repair processes during mid-pachytene and promotes the formation of long non-crossover conversion tracts, functions that may not be reliant on SMC-5/6. Finally, the authors show genetic interactions consistent with a role of BRC-1 regulating theta-mediated end joining in smc-5 mutants; however, BRC-1 and SMC-5 do not appear to regulate one-another's localization.

The manuscript is focused on examining the consequences of brc-1 and smc-5 mutations on repair pathway choice in C. elegans meiosis. It achieves that goal. The experiments are generally well done, and the results will be of interest to investigators studying DNA repair and meiotic recombination in C. elegans.