Reviewer #1 (Public Review):

It is a strength of the current manuscript that it provides a near-complete picture of how the metamorphosis of a higher brain centre comes about at the cellular level. The visualization of the data and analyses is a weakness.

I do not see any point where the conclusions of the authors need to be doubted, in particular as speculations are expressly defined as such whenever they are presented.

The fact that molecular or genetic analyses of how the described metamorphic processes are organized are not presented should, I think, not compromise enthusiasm about what is provided at the cellular level.

Reviewer #1 (Public Review):

In this manuscript, the authors use time-lapse microscopy in growing intestinal organdies and computational modelling to demonstrate a paradigm for the control of a pool of proliferative cells. They find strong correlations in the proliferative behaviour of sister cells. They propose a compartmentalised model, where cells in one compartment all have a high propensity to produce two proliferating daughter cells while cells in the other department produce daughter cells who both cease to proliferate.

The work establishes a previously suggested paradigm for the control of fluctuations in a pool of proliferating cells. This paradigm might be relevant for tissues other than the intestine such that this work will be of relevance to the general field of stem cell biology. I found this work to be a nice combination of modelling and the conclusions overall convincing. The authors could improve upon the precision in their wording and the discussion of the scope of their modelling results.

Reviewer #1 (Public Review):

In this study, Apiz-Saab et al. build up prior work by the Muir lab, which examined the metabolite composition in the tumor microenvironment and found that some metabolites like arginine are present in very different levels from that in our standard culture media. In this study, the authors have formulated a custom media based on the composition of the tumor interstitial fluid (TIF media or TIFM) and found that pancreatic cancer cells cultured in this media have a metabolic state more like tumors in vivo. This is primarily driven by very low levels of arginine, which induces arginine biosynthesis is the cancer cells to cope with this nutrient limited state. Using genetic and pharmacological approaches, the authors demonstrate that arginase expression within tumor-infiltrating myeloid cells drives tumor microenvironmental arginine depletion in vivo.

Strengths:

This is a very rigorous, well-designed study and the findings are broadly interesting for the metabolism, immunometabolism, and pancreatic cancer communities. The methods are comprehensive and the experimental details in the legends are complete. The discussion is particularly well developed and does an excellent job of putting the findings in the context of the field.

Weaknesses:

The claim that arginine biosynthesis is an adaptation to myeloid arginine depletion could be further supported in vivo.

Yale researchers develop RNA-based therapy that clears SARS-CoV-2 from mice. (n.d.). EurekAlert! Retrieved January 9, 2022, from https://www.eurekalert.org/news-releases/934454

Reviewer #1 (Public Review):

Gupta et al. investigate a new molecular mechanism whereby the ETS transcription factor, ETV1, is upregulated in prostate cancer. Through a series of experiments in prostate epithelial and prostate cancer cell lines, including gene knockdown, knockout and reconstitution, they demonstrated that the concomitant loss of ERF and CIC enhance malignant phenotypes such as cell viability, invasiveness and migratory capacity. Their in vitro results were supported by in vivo subcutaneous tumour xenograft assays in immunodeficient mice. Additional analyses of publicly available data and multiple in-house assays indicated that ERF and CIC target ETV1, acting as transcriptional repressors and modulating ETV1-mediated transcriptional pathways. Finally, the authors show that ETV1 chemical and genetic inhibition moderately decrease cell viability and significantly decrease invasiveness in ERF and CIC deficient prostate cancer cells.

A major strength of this paper is the range and number of analyses performed to test their hypothesis that CIC and ERF cooperate to suppress ETS target genes in prostate cancer. The authors combine both publicly available and in-house data to answer their research questions, which are logically set out in the results section. However, there are also limitations specific to these data that slightly diminish the quality of the paper and make interpretation of their results difficult for the reader.

The premise of the molecular work is based on data from the cBioPortal but it is difficult to fully grasp the results presented due to study and assay numbers being omitted and figures being hard to interpret. The significance (or lack thereof) is also not specified in the text for a number of the subsequent cell line analyses and could be made clearer, especially when the authors are describing a trend rather than significant results. A key analysis method, single-sample Gene Set Enrichment Analysis, used to answer a question central to the paper's conclusions (whether ERF and CIC regulate ETV1 transcription), is poorly explained and presented in the methods and results sections. Furthermore, the methods section does not align with the results section, there is a missing methodology (e.g., how was the PNT2 gene expression data generated?), there are instances of figures being misnumbered and/or insufficiently described/labelled, and missing supplementary data. Finally, while the authors present what appears to be very clinically relevant data showing sensitivity to ETV1 inhibition was enhanced in cells with both ERF and CIC loss, they only present experiments in a single prostate cancer cell line. Given the potential clinical relevance of these data, further in vitro and in vivo assays in the other available cell lines would have provided further evidence for their conclusions, especially given the higher metastatic potential of one of these (PC-3 cells).

Despite the limitations described above, the interpretation and overall conclusions the authors draw from their analyses are generally sound. The study represents an advance in our understanding of how ETS family transcription factors are dysregulated in prostate cancer and suggests a new sub-class of prostate cancer patients based on somatic tumour alterations. Significantly, these patients could one day benefit from targeted ETV1 inhibitors, which are currently being assessed in clinical trials for other cancers.

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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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?

Maybe cartography can be revived: https://www.reddit.com/r/interestingasfuck/comments/urp3sd/lifesize_floor_plans_for_a_better_understanding/

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 #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.

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 #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 #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 #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 #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 #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 #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.

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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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.

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?

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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #1 (Public Review):

In this manuscript, the authors relied on their well-established directed differentiation approach to differentiate pluripotent stem cells (hESCs) towards growth plate (BMP4-treated) or articular (TGFb3-treated) chondrocytes. Integrating RNA-seq data from hESCs-derived growth plate or articular chondrocytes with data from in vivo (fetal) counterparts, the authors showed similarities (and some divergences) in the transcriptional networks of in the in vitro-differentiated cells, uncovering genes with potential novel roles in cartilage biology. Integrating ATAC-seq (to assess chromatin accessibility) and transcriptomics data, the authors both characterized the regulatory landscapes in these cells, and also uncovered lineage-specific gene-regulatory networks. Using targeted ChIP-qPCR, and leveraging available ChIP-seq datasets, the authors validated the functional interactions of two well-described DNA-binding trans-acting factors (RUNX2 and RELA) with putative genomic targets (both previously involved and with non-explored/novel roles in cartilage biology). Taken together, these analyses provide novel insight into the molecular mechanisms contributing to growth plate and articular cartilage specification.

Strengths:

This is a very well-written manuscript. The findings are of relevance to understanding cartilage development and maintenance and are of potential impact to understand (and correct) cartilage damage and pathology. The experiments are well conducted, and the conclusions and claims are supported by the data. The authors performed a superb job characterizing and defining gene regulatory networks, elegantly integrating in vitro systems with in vivo datasets, and combining transcriptomics and epigenomics tools. These approaches uncovered regulatory networks and novel genes with unexplored roles and contributions to growth plate and articular cartilage development.

Weaknesses:

The functional implication of the findings is somewhat limited: while the authors did evaluate and confirm interactions of selected transcription factors with putative target genes, the mechanistic contribution of these findings to chondrocyte specification is not fully explored.

Reviewer #1 (Public Review):

This study presents novel experimental data from a mutant mouse model lacking microglia (Pu.1-/- mouse line) which indicates that these cells have an important role in the embryonic establishment of critical neural circuits in the brainstem generating breathing motor behavior in mice. Microglia are known to have important roles in shaping neural circuit assembly during development by controlling cell death, synapse refining, neurogenesis, and axon tract formation, but such roles have not been examined in the development of functional respiratory circuits. The authors examined the anatomical and functional characteristics of two main respiratory neuronal groups-in the embryonic parafacial (epF) and the preBötzinger complex (preBötC) regions that operate together in the developing brainstem to generate the rhythmic neural signals that are necessary to establish normal breathing behavior and ensure survival at birth. They present evidence that these respiratory networks become functional at typical developmental stages in the absence of microglia, but exhibit anomalies in rhythm generation (slower respiratory rhythm) and the mutants are unable to sustain breathing behavior at birth, consistent the observed neonatal death. Their data suggest that these deficits are associated with reduced cell numbers and abnormal rhythmogenesis in epF, and reduced commissural axonal projections of the preBötC circuits responsible for generating inspiratory rhythm.

Strengths of this study include the authors' use of the Pu.1-/- mutant in combination with technically well-executed, novel anatomical reconstruction of distributions of microglia in the developing hindbrain, neuronal activity imaging in the epF of the embryonic brainstem in vitro, and electrophysiological recording approaches in slices to assess aspects of the anatomical and functional status of the epF and preBötC relative to the control wild type mice. They also examine inspiratory drive transmission to phrenic motoneurons in vitro to assess the functional status of spinal respiratory motor output critical for breathing behavior at birth. Furthermore, their behavioral measurements by plethysmography document show that late-term (E18.5) Pu.1-/- embryos are unable to sustain breathing activity ex utero, which is consistent with the observed neonatal death of the mutants.

A limitation of the study is that the microglia-related mechanisms involved in regulating cell numbers in epF and the proper bilateral connectivity of preBötC circuits have not been investigated. Therefore it remains unknown if the reduced cell numbers in epF in the Pu.1-/- mutant is a defect, for example, of neurogenesis/neuronal migration or abnormal control of cell death, and if the defect of preBötC connectivity is actually related to the aggregation of microglia along the midline (possibly affecting commissural axonal tract formation), as the authors suggest.

Reviewer #1 (Public Review):

Blake and colleagues examine programs of alternative splicing controlled during T cell activation. Using CD4+ T cells from human donors, cells were stimulated with anti-CD28, anti-CD3, and combined anti-CD3/28 antibodies. RNA was then isolated at 2 time points, sequenced, and analyzed for changes in spliced isoform ratios. T Cell Receptor stimulation alone via anti-CD3 is known to induce the anergic state resulting from suboptimal stimulation, while CD28 costimulation with CD3 induces many genes to a higher level of expression similar to stimulation by antigen-presenting cells. Analyzing the splicing responses to these stimuli, the authors find that CD28 costimulation also enhances the splicing changes that accompany T cell activation. A subset of these splicing targets encode apoptotic regulators including Caspase-9, Bax, and Bim. They show that forced expression of the isoforms that are increased by costimulation results in reduced apoptosis in Jurkat cells treated with apoptotic inducers. Using kinase inhibitor treatments they show that Jnk kinase activity is required for the splicing changes in the three apoptotic regulators.

Reviewer #1 (Public Review):

This paper examines wtf genes in relatives of S. pombe to investigate the evolutionary history of the gene family. Classic theory suggests that distorters like wtf should be fairly transient - the fitness cost due to spore killing should select for suppressors and even if a selfish allele manages to fix, its advantage disappears (under either scenario, the drive function stops, and the allele degrades over time through random mutation). Despite these predictions, the authors provide convincing synteny data to argue that wtf genes were likely present more than 100 million years ago in the common ancestor of S. pombe and its relatives. Using phylogenetic approaches, the authors also show that since this ancient origin, wtf genes have evolved dynamically by gene duplication and gene conversion within descendant lineages. Additionally, by studying the genomic regions surrounding these genes, they discover an association in S. octosporus and S. osmophilus with 5S rDNA, which, like associated LTRs in S. pombe, might facilitate this duplication history. Finally, using transformation experiments, the authors demonstrate that these newly identified wtf genes have the very same poison and antidote functions originally described in S. pombe.

This work is a significant advance in our understanding of the evolution of wtf genes, moving beyond S. pombe to several other distantly related fission yeast species. More generally, it suggests a plausible mechanism for the continued existence of wtf genes across long evolutionary time scales.

Joint Public Review:

Here the authors develop and evaluate a new hybrid-capture sequencing approach for coronavirus (CoV) surveillance in bats. The intended goal is to overcome limitations in amplicon sequencing, which is the current standard method for viral surveillance in animal species. Whereas amplicon sequencing is only suitable for targeted analysis of the highly conserved RdRp gene in bat CoVs, the new hybrid-capture approach affords a great breadth of coverage across the full genome in diverse CoV species. This promises to improve the identification and phylogenetic analysis of bat CoVs. The authors conclude by making practical recommendations about how their new method can be applied to usefully complement existing technologies in the field.

The new method appears to suffer from a lower sensitivity for CoV detection than amplicon sequencing, and also struggles to yield complete sequences across the bat CoV spike protein, which is a highly divergent region. The authors have appropriately acknowledged these weaknesses, and show how other complementary tools can alleviate them - for example by using deep metagenome sequencing to resolve the spike protein in new CoV strains discovered through hybrid capture sequencing.

This is an excellent paper in my opinion. The study addresses an important problem - improved methodologies for CoV viral surveillance in bats, a common source of zoonotic viral transmission events. The authors developed a new method that has obvious utility. They have fairly evaluated this method against existing approaches (targeted amplicon sequencing and deep metagenomic sequencing) using appropriate data. In addition to describing a useful new method, the study also produced some novel results that are likely valuable - that of complete (or near-complete) genome sequences for several novel bat coronaviruses. The authors discuss the outcomes in a fair and balanced fashion and make some simple, practical recommendations about how their new tool might best be used. Finally, the article was very well written; clear, concise, and fluent.

Reviewer #1 (Public Review):

The manuscript is clear and well-written and provides a novel and interesting explanation of different illusions in visual numerosity perception. However, the model used in the manuscript is very similar to Dehaene and Changeux (1993) and the manuscript does not clearly identify novel computational principles underlying the number sense, as the title would suggest. Thus, while we were all enthusiastic about the topic and the overall findings, the paper currently reads as a bit of a replication of the influential Dehaene & Changeux (1993)-model, and the authors need to do more to compare/contrast to bring out the main results that they think are novel.

Major concerns:<br /> 1. The model presented in the current manuscript is very similar to the Dehaene and Changeux 1993 model. The main difference is in the implementation of lateral inhibition in the DoG layer where the 1993 model used a recurrent implementation, and the current model uses divisive normalization (see minor concern #1). The lateral inhibition was also identified as a critical component of numerosity estimation in the 1993 model, so the novelty in elucidating the computational principles underlying the number sense in the current manuscript is not evident.

If the authors hypothesize that the particular implementation of lateral inhibition used here is more relevant and critical for the number sense than the forms used in previous work (e.g., the recurrent implementation of the 1993 model or the local response normalization of the more recent models), then a direct comparison of the effects of the different forms is necessary to show this. If not, then the focus of the manuscript should be shifted (e.g., changing the title) to the novel aspects of the manuscript such as the use of the model to explain various visual illusions and adaptation and context effects.

Reviewer #1 (Public Review):

In this manuscript by Woods et al, the authors study the small heat shock protein HSPB5, specifically focusing on two cataract-associated mutations. They show that the mutations, which are located in the ACD core of the protein, disrupt the interaction of the core with the unfolded N-termini and generate a much more dynamic version of the protein. A surprising feature of the mutants is that they enhance in vitro chaperone activity directed against damaged GammaD-crystallin.

Reviewer #1 (Public Review):

This is an interesting new bladder function monitoring approach in rodents that can accurately monitor bladder filling and emptying in freely moving non-anaesthetized animals that does not require implantation of a suprapubic catheter. This was accomplished by using machine learning to define the bladder wall from fluoroscopic images of mice injected with iodinated radiocontrast media taken at 30 images/second over 2-3 hours. While this approach cannot provide any information on intravesical pressures, it can provide much more accurate and detailed information on bladder filling, urethral flow rate, intra contraction intervals and residual bladder volume than assays of voiding spots on paper or metabolic cages monitoring of urine production with microbalances.

Reviewer #1 (Public Review):

Jara et al studied the interaction between the dynein intermediate chain (IC) and the three dimeric light chains (Tctex, LC7, LC8), dynactin p150, and the nuclear distribution protein (NudE). The authors are able to produce the entire intrinsically disordered N-terminal domain of IC from Chaetomium thermophilum (CT) allowing them to study the assembly and regulatory mechanism of IC with five different partners.

The authors convincingly demonstrate that IC is maintained in an auto-inhibitory conformation through NMR titrations of separate constructs of IC. Using a combination of NMR, ITC, SV-AUC, SEC, and SEC-MALS, they demonstrate that release of this auto-inhibitory conformation, through binding of LC7, is required for binding to NudE and to some extent p150. Importantly, the presence of this auto-inhibited state is validated in the context of the full-length IC protein expressed and purified from insect cells.<br /> The work provides novel insight into how dynein assembly is regulated (Fig. 10) and illustrates the unique interaction mechanisms that can be exploited by intrinsically disordered proteins.

The conclusions of the manuscript are supported, for most parts, by experimental data, however, some aspects require some clarification or should be further supported by experimental data:

1) The authors propose that the two-step binding isotherm observed for p150 is due to binding to both the SAH and H2 regions of IC(1-88), while NudE shows a single binding event by ITC due to interaction with the SAH region only. The NMR experiments of IC(1-88) do not provide sufficient support for this hypothesis (Fig. 6B, bottom panel). Additional experimental data would be needed to fully support this conclusion.

2) ITC, NMR, and AUC data are presented for the binding of NudE to IC(1-260). Some more clarification is needed in terms of the interpretation of these data, also in the context of the observations on IC(FL). The experimental observations do not seem to be explainable simply by a weak complex or a concentration-dependent effect as suggested by the authors.

3) The difference in sedimentation coefficient of the dynein subcomplex + NudE and of the dynein subcomplex + p150 is surprisingly large suggesting significantly different shapes of the two bound complexes. Some discussion of this issue is present in the manuscript, but no clear explanation is provided. It would seem necessary to confirm these observations with other complementary techniques.

4) The authors suggest that the binding of LC7 releases the auto-inhibitory interaction of IC, however, NMR does not directly support this conclusion (Fig. 7B). Some discussion of why this long-range interaction inhibits the binding of NudE, but not LC7 itself, should be included.

Reviewer #1 (Public Review):

In this paper, Jan Kubanek attempts to derive an 'effective decision strategy' that is optimal (and therefore normative) given certain constraints resulting from computational capacity limitations. The author first points out that neoclassical economics (i.e., expected utility theory, EUT) provides normative predictions for decisions to maximize utility. Next, he (correctly) points out that finding the optimal solutions to decision problems requires computational resources that are unlikely to exist in actually existing decision-makers (animals and humans). He claims that this fact is the most severe problem for concluding that EUT is an accurate description of actual human or animal decision processes. I disagree with him on this point as I will lay out in more detail below. Next, the author attempts to find an 'efficient' (i.e., computationally reasonable) decision strategy that comes close to the original normative framework. He claims that such a strategy is EDM, whereby decisions are made by allocating relative effort in proportion to the relative reward of each option.

Overall, I find this paper hard to judge. The considerations described in this paper are certainly interesting and I have no reason to presume that the mathematical derivations described are wrong (without having made an effort to follow and check it in detail). Still, I find the paper, in the end, sterile and I fear it will have only limited impact. I think the manuscript should be expanded in three different directions to make it more relevant for the neuroscientific understanding of decision making. First, the author needs to show that EDM can also explain other known violations of EUT related to the axiom of regularity (i.e., preferences between two options should not be affected by the presence of inferior options). This seems relevant because these behavioral effects robustly violate the choice allocation strategy of EDM. Second, EDM is so abstract that the actual structure and capacity of the nervous system are nearly irrelevant. The author should consider more deeply the computational requirements and capacities of different types of brains; fruit flies, frogs, and primates, and the consequences of these differences for what is (or should be) achievable in terms of optimal behavior. Third, the paper contains no test for EDM. This is in part because EDM is at no point compared to the predictions of alternative theories.

My specific concerns are as follows:

(1) The author claims that the most severe problem of EUT is that it is computationally implausible. However, I disagree. It could be claimed that EUT describes an (unattainable) optimal state that actual brains try to accomplish with limited resources. (In essence, the current paper follows this strategy). I think the situation is much direr. During the last 70 years, a small army of psychologists and behavioral economists have described a large number of violations of EUT's normative predictions: the Allais paradox, framing effects, the behavioral tendencies summarized in Prospect theory, and others. These differences between behavior and normative predictions are important because they violate basic assumptions of the normative theory.

(2) The most interesting case of such violations is a set of well-known behavioral effects that occur in the context of multi alternative-multi attribute decision making. They are known as the attraction, similarity, and compromise effects (there is a large literature; more recently: Dumbalska T, Li V, Tsetsos K, Summerfield C. A map of decoy influence in human multi alternative choice. Proc Natl Acad Sci U S A. 2020 Oct 6;117(40):25169-25178. doi: 10.1073/pnas.2005058117. Epub 2020 Sep 21.) These biases have received so much attention because they violate a very basic axiom of EUT. Choices between two options should not be affected by the presence of a third option that is inferior to both of them. However, that is exactly what happens in these choice biases. The effects have been shown in many species ranging from humans to amphibians to invertebrates. As far as I can see, EDM cannot explain how choice allocation between two options A and B that have equal value would be changed by the inclusion of a new option D so that is of lower value than A or B in such a way that D is not chosen at all, but A is chosen more often than B if D is similar in attributes to A (the 'attraction' effect). If I am mistaken, the inclusion of an explanation of how this would work would be of major importance.

(3) EDM as described in this manuscript is completely static, that is it ignores actual computational processes that underlie decision making. This is in opposition to an important modern branch of decision research that has stressed the importance of understanding processes (and their limitations) to understand how choices are made. Examples are: (1) Roe RM, Busemeyer JR, Townsend JT. Multialternative decision field theory: a dynamic connectionist model of decision making. Psychol Rev. 2001 Apr;108(2):370-92. doi: 10.1037/0033-295x.108.2.370. PMID: 11381834.; (2) Tsetsos K, Usher M, Chater N. Preference reversal in multiattribute choice. Psychol Rev. 2010 Oct;117(4):1275-93. doi: 10.1037/a0020580. PMID: 21038979. The relationship between EDM and algorithmic implementations should be explored.

(4) Most importantly, what is missing is a clear prediction for a finding (behavioral or neuronal) that would only be predicted, but not by any other theory of decision making. Without such a proposed test, the idea has no scientific merit.

Reviewer #1 (Public Review):

Aberra et al.'s work is focused on identifying genes that exhibit opposing effects on type 2 diabetes and abdominal obesity. Identification of such genes would provide stepping stones for a better understanding of why some individuals with obesity are not developing type 2 diabetes, knowledge that ultimately could shed light on the complex interplay between fat distribution and type 2 diabetes.

Aberra et al. use a number of computational tools to identify genetic variants associated with both type 2 diabetes and waist-hip ratio (both adjusted for body mass index). They identify six genetic loci that associate with both phenotypes, but exhibit discordant effects.

To better understand which tissues and genes are potentially mediating the discordant effects, the authors use GTEx data to co-localize eQTLs with genetic variants at the six discordant loci. They identified four genes, at two of the discordant loci, that are regulated by an eQTL co-localizing with one of the discordant variants. Using the Finnish METSIM cohort and correlation analysis, the authors show that expression of these genes is associated with both glycemic and obesity risk-phenotypes.

The manuscript is very concise and well-written. All computational analyses seem well thought through and executed. I have two suggestions that potentially could help the authors to improve their work.

The authors write that they "[...] predict the mechanisms of action at discordant loci" (L. 286), which seems too strong a claim given their data. Potentially the following points could help to provide more evidence on the functional context to the four prioritized genes and more guidance on how mechanistic insights could be advanced further:

1) Aberra et al. indicate that the 2p21 locus harboring the THADA gene and its antisense RNA are differentially open during preadipocyte development. Are these RNAs differentially expressed during specific stages of adipocyte development and are they differentially expressed in certain human adipocyte clusters? Relevant datasets to address these questions could be (https://www.nature.com/articles/s41467-020-16019-9, https://pubmed.ncbi.nlm.nih.gov/32066997/, https://pubmed.ncbi.nlm.nih.gov/35296864/, and https://pubmed.ncbi.nlm.nih.gov/33116305/.

2) The authors' work may serve as an example on how to shortlist relevant genetic variants for variant-to-function approaches. It could be instructive to the metabolism community if the authors' in the Discussion could dedicate a paragraph to carefully discuss how one best could further explore the function of the discordant variants they identify and the genes they implicate. For instance, how could one (i) experimentally prove that the given variants regulate the predicted effector genes, (ii) further understand the mechanisms with which they impact adipocyte biology, and (iii) further establish evidence that they have a discordant effect on glycemic and lipid traits.

Reviewer #1 (Public Review):

The manuscript titled "Notch signaling functions in non-canonical juxtacrine manner in platelets to amplify thrombogenicity" by Chaurasia et al describes that human platelets have notable expression of Notch1 and its ligand DLL-4, which function in a non-canonical manner to synergize with physiological platelet agonists, leading to prothrombotic phenotype. Targeting Notch signaling specifically DLL-4-Notch1-NICD axis can be a potential approach to develop anti-platelet/anti-thrombotic therapeutic.

Start with an essential question that matters to you.

Reviewer #1 (Public Review):

This work addresses the problem of accurately estimating dynamics parameters in single particle tracking applications. The authors give a very extensive overview of the current problems and solutions while dealing with imaging of diffusive motion of subcellular particles and challenges that one faces while trying to estimate the main parameters of interest, such as diffusion constants. The authors properly address the issues with short trajectories, which are typical in practice and propose two advanced approached, which successfully deal with the mentioned shortcomings (short trajectories from which it is difficult to estimate parameters reliably, and the measurement errors that contaminate the input data). The proposed techniques are very interesting, and the way how those pure mathematical (and long existing) concepts are applied for this specific application of single particle tracking is rather novel. The proposed methodology is supported by a thorough validation, which includes simulations of all possible conditions (numbers of trajectories, distributions of the diffusion constants within the population of particles, the levels of inaccuracies in the measurements, etc.). Additionally, the experiments with the real data are also very convincing. The authors do focus on a regular Brownian diffusion and hopefully will show the applicability of these approaches to more typical applications containing anomalous diffusion. The availability of the code, which the authors provide on github, is very important, especially to less (technically) skilled audience from the field of experimental biology, who would like to apply those techniques to their data.

Reviewer #1 (Public Review):

The present study aims to define the main immune cell subsets found in the hemolymph of the white shrimp, P. vannamei. This is significant because this species is heavily farmed around the world to meet the demand of the human consumption market. Yet, farmed shrimp suffer from infectious diseases and therefore we need to understand how their immune system works to design strategies that decrease infection losses.

Classification of crustacean (and other invertebrates) hemocytes is difficult due to the lack of antibodies to use traditional flow cytometry approaches. Furthermore, hemocyte purification is not easy, cells die and clump, again precluding flow cytometry studies. Thus, the majority of what we know about shrimp hemocytes is based on morphological classification. This study contributes significantly to advancing our knowledge of shrimp Immunobiology by defining hemocyte subsets based on their transcriptional profiles.

Another strength of the paper is that some function in vivo assays (phagocytosis) are presented in an attempt to validate the single-cell data. The authors frame their question or try to frame their question with a more evolutionary angle, such as whether the macrophage-like cell is the evolutionary precursor of human macrophages. I think that this question is not really achievable because the evolution of innate immune systems may have diverged in many branches of the metazoan tree of life. The authors, however, identify gene markers that are conserved in macrophages from shrimp and humans and that is a fair conclusion. There are some methodological caveats to the study and the manuscript needs to be heavily edited to improve language as well as to increase the depth of the interpretation.

In summary, there are interesting findings in this manuscript but the manuscript needs to be significantly improved so that its quality and impact are elevated.

Reviewer #1 (Public Review):

The manuscript by LoMastro et al. investigates whether Plk4, the master regulator of centriole biogenesis in cycling cells, has a similar role during the differentiation of multi-ciliated cells, which produce tens to hundreds of centrioles during multi-ciliogenesis. Contrasting previous work that did not find an important role for Plk4 in this process based on chemical inhibition, the authors in the current study use genetic approaches and mouse models to show that Plk4 and its kinase activity are essential for centriole amplification and multi-ciliogenesis in two different multi-ciliated cell types in vitro and in vivo. In addition, they show that centriole amplification drives cell surface area expansion.

The study addresses an important question regarding the role of Plk4 in centriole amplification during multi-ciliogenesis. It convincingly establishes that contrary to previous findings, the Plk4-dependent control of centriole biogenesis that is well-established in cycling cells is conserved also during differentiation of multi-ciliated cells. The presented data is of very high quality, phenotypes are well described and quantified, the conclusions are clear, and obtained in both in vitro and in vivo models. The authors also test chemical inhibition of Plk4 as used in previous work and show that the lack of a strong phenotypes under these conditions is likely due to incomplete Plk4 inhibition.

Reviewer #1 (Public Review):

The authors of this study adopted Cas9-mediated enrichment of target locus and Nanopore long-read sequencing to accurately count repeat numbers in the CNBP gene, which is notorious for precise calling before. They also compared their result with that of the conventional approach, validating their approach. It is an interesting read and shows a pathway that a clinic can take in the near future.

However, this paper's novel contributions need to be emphasised as there are some papers that utilized Nanopore sequencing to elucidate short repeats (https://pubmed.ncbi.nlm.nih.gov/35245110/; https://bmcmedgenomics.biomedcentral.com/articles/10.1186/s12920-020-00853-3). Another issue is the clinical utility of the approach. Although it is precise, it is not totally clear whether this accuracy is required in clinical practice, as the repeat status does not completely correlate with phenotypic severity.

Lastly, it is not clear about the familial cases (A1-A4). What are their relationships and why their copy numbers are not exactly the same? Is it because of extreme recombination and variation even in a family or just represent limited accuracy?

They lack a validation cohort, with prospective patients.