254 Matching Annotations
  1. Last 7 days
    1. Reviewer #1 (Public Review):

      This manuscript reports a novel and original approach to examine the possible mutational paths underlying directed protein evolution.

      The authors conclude from their experiments that "Necessity was almost entirely absent" (line 209). Indeed, the vast majority of states evolved in just one replicate from one starting point. But this is the problem of a half-full glass: is it half full or half empty? If I understand Fig.4F correctly, one can still detect amino acid changes that recapitulate historical substitutions, and others that revert to the historical state, so it does not seem that necessity is "almost entirely absent". Furthermore, several of the amino acid changes that were detected may not have any effect on NOXA or BID biding, maybe they occurred because of mutational bias, drift or hitchhiking. If this is the case, then one cannot compare all acquired states in each trajectory and conclude about the importance of chance, as in this sentence for example: "Pairs of trajectories launched from the same starting point differed, on average, at 78% of their acquired states, indicating a strong role for chance" (line 219). There are causal mutations that arose repeatedly during PACE replicates from each starting genotype and these mutations do indeed confer the selected-for specificity in their "native" background (as is nicely shown in Figure 6A-B). So this, to me, is evidence for necessity.

      Loosing a binding property can probably occur via multiple ways, which are likely to be more numerous than gaining binding for a given protein. It would be nice to discuss this point in more detail.

      The experiments presented are limited to one protein family and to the binding properties to two different proteins. In living organisms, each protein is likely to exhibit particular properties such that it can bind or not bind to hundreds of different proteins, and not just two as tested here. So the constraints present in living organisms may be much larger than the ones present within this experimental evolution set up. Furthermore, the tested proteins probably encounter other constraints in their native environment besides affinity for other proteins, and it is yet unclear whether the variant forms obtained here via experimental evolution would be fine to replace the endogenous proteins in living organisms. It is therefore difficult to generalize from the obtained results to all types of evolutionary changes. In general, the conclusions should be toned down and focused on this particular example.

    1. Reviewer #1 (Public Review):

      The manuscript by MeHeust reports identification of flavinylation proteins that can potentially function as cellular redox mediators related to electron transfer systems in prokaryotes.

      The work is useful and informative. The authors used bioinformatic approach to illustrate wide distribution of these proteins in a variety of prokaryotes. Although exact functions of these proteins are not known, this work should inspire further investigation by researchers in the fields of redox enzymology and bioenergetics.

    1. Reviewer #1 (Public Review):

      The authors sought to understand the relationship between sequence conservation and biological function for a protein complex that undergoes conformational changes during its functional cycle. This included understanding the extent to which phylogenetic comparisons can guide identification of functionally important residues for a specific family member. The particular focus was on the bacteriophage clamp-clamp loader complex, with the long-term goal of understanding structure-function principles that might facilitate the design of novel AAA+ ATPase proteins.

      A systematic mutagenesis screen was used to determine the relative fitness for single site mutations throughout the bacteriophage T4 clamp-loader and clamp proteins. A feature of the screen is selection for fitness in an (almost) authentic biological context. The vast majority of residues are highly permissive to substitution, which is notable because the considerable conformational changes required during the loader-clamp reaction cycle might have been expected to place more constraints throughout the structure. It is demonstrated that tolerance to mutation within the T4 proteins does not correlated well with conservation across 1,000 other bacteriophage sequences, thereby illustrating the importance of specific context and limits of inference from phylogenetic comparisons. The only critical residue distant from a catalytic active site or binding surface is a glutamine, whose importance was not previously noted, but imparts rigidity to the structure by coupling functionally-important clusters through a hydrogen bonded network. Inspection of distantly related AAA ATPases indicates that this residue is important for many, although not all members of this large and diverse family of molecular machines.

      One major concern is that the levels of protein expression and folding are not verified. This is concerning for the Gln118 mutation because lack of fitness could result trivially from misfolding or accelerated degradation that might result from increased flexibility and conformational stability. Moreover, the authors' finding that it was not possible to purify Gln118 mutant proteins for biochemical studies is consistent with this sort of trivial explanation for apparent lack of biological function.

    1. Reviewer #1 (Public Review):

      Using a third odor for memory test, the authors found that single-trial conditioning produces a protein synthesis-dependent LTM leading the avoidance to both CS+ and CS- for more than 7 days. By acutely blocking neurotransmissions from target neurons, they showed that the merged LTM requires outputs from TH-positive dopaminergic neurons during training, and from αβ Kenyon cells and α2sc mushroom body output neurons during testing. Several lines of evidence support their claim that the long-term avoidances of CS+ and CS- after single-trial conditioning are based on the same memory component: 1, Five independent disruptions of the system produced the same level changes of avoidance from CS+ and CS-; 2, Re-exposure to either CS+ or CS- alone abolished both CS+ avoidance and CS- avoidance; 3, The same PPL1 DANs, αβ KCs, and α2sc MBONs involved in both avoidances; and 4, Similar responses of functional depression to CS+ and CS- occurred in the same α2sc MBONs. Based on these results, the authors suggest that animals can develop distinct memory strategies for occasional and repeated threatening experiences.

      While the entire manuscript is well-written and the data are well presented, the conclusion has several weaknesses. First, memories for CS+ and CS- are clearly distinct from each other at least during the first 24 hours after training (Figure 1B). During this period, CS+ memory shows a gradual decline similar to conventional LTM. Oppositely, CS- memory shows a gradual increase. When these flies were tested immediately after training, they seemed to approach CS-, a result similar to the previous study using a similar third-odor test showed that multiple-trial conditioning induces "two independent LTMs of opposite valence for avoiding CS+ and approaching CS-". How does "the approaching CS- memory" turn into "the avoiding CS- memory"? Or, "the avoiding CS- memory" is derived independently, similar to the anesthesia-resistant memory?

      If initial behavior responses to the CS+ and CS- memories are opposite and thus separated, where does each of them occur? Where and how are they merged after one day? The authors claimed that both CS+ and CS- memories require the same PPL1 DANs, αβ KCs, and α2sc MBONs. But, they manipulate PPL1 DANs with TH-Gal4 that expressed in many other dopaminergic neurons. Similarly, the αβ KCs include at least three major cell populations, each has hundreds of neuron with distinct functions. Thus, while both CS+ and CS- memories require the same family of neurons, it remains uncertain whether these events actually occur in the same neurons.

      In general, the identified merged LTM is original and brings a new concept to the field of learning and memory. The finding suggests that two forms of protein-synthesis-dependent LTM induced respectively after occasional or repetitive experiences are encoded in the brain by different neuronal mechanisms. It appears that merged LTM after single-trial training remembers the event with limited details and repetitive spaced trials of training then adds more details to the classical LTM.

    1. Reviewer #1 (Public Review):

      The study is focused on neural deficits in Smith-Lemli-Opitz syndrome (SLOS) that is caused by loss of function of 3b-hydroxysterol-D7 -reductase (DHCR7) and results in lower cholesterol. Individuals with SLOS have cognitive impairment and the authors use mouse models and human iPSCs to investigate the effects of the SLOS mutation on neural progenitor proliferation and neurogenesis. Data show that the loss of DHCR7 leads to premature differentiation of cortical progenitors and altered cortical development. However, the work offers little mechanistic insight.

    1. Reviewer #1 (Public Review):

      The regulation of highly dynamic interactions is for many biological processes of great importance. The authors study the regulatory interaction of the single transmembrane helix protein Phospholamban with the P-type ATPase SERCA which is responsible for removing calcium ions from the sarcoplasm and restoring its concentrations in the sarcoplasmic reticulum. The inhibitory interaction between both proteins is relieved by phosphorylation of a single residue in the cytoplasmic domain of Phospholamban. The authors show by a combination of solid state NMR as well as MD simulations that phosphorylation results in a order to disorder transition in the cytoplasmic part which leads to an re-arrangement of electrostatic networks which is propagated into weakened hydrophobic interactions between the transmembrane parts, thus activating SERCA. Phospholamban has been studied extensively by solid state NMR, liquid state NMR or hybrid methods. For example the phosphorylated form was studied previously, showing that it interacts differently with lipids (doi: 10.1021/bi0614028) and that Ser-16 phosphorylation alters the structural properties of the cytoplasmic domain with respect to the lipid bilayers (doi:10.1016/j.bbamem.2009.12.020). There have also been EPR and other studies, in principle showing the same effect. The current paper adds to this a new solid state method that shows additional details that could not be investigated previously. The work confirms less well determined previous models. The major new aspect is an MD simulation that provides a more detailed view than what was previously possible.

    1. Reviewer #1 (Public Review):

      The authors address the broad question of what is responsible for the large diversity of presynaptic function at synapses arising from a single type of neuron. They use a variety of sophisticated and complementary approaches to address the functional and molecular heterogeneity of hippocampal pyramidal cell to fast-spiking interneuron synapses. The rigorous functional and molecular analysis is clearly described and compelling. The conclusions are consistent with the current view that each presynaptic active zone contains a variable number of release sites, and this variability makes a substantial contribution to the heterogeneity in postsynaptic response amplitude at unitary synaptic connection. Using state-of-the-art imaging approaches, the authors report variability in the content of Munc13-1, a core component of release sites, between release sites. Although these results and conclusions are well-supported, the functional significance of Munc13-1 variability at release sites is unclear.

    1. Reviewer #1 (Public Review):

      The authors have done a great job in carefully labeling the β-catenin with fluorescent protein SGFP2 and quantitatively measuring the β-catenin behavior during Wnt pathway activation with advanced biophysical methods. This is an excellent effort on quantitative biological studies. The knock-in constructs, the cell lines the authors made are great resources for the Wnt field. And the quantification like the β-catenin concentration, β-catenin diffusion coefficient are great knowledge for future studies. The finding that S45F mutation lead to higher fraction of the slow-moving complexes is interesting. Other areas could borrow the research ideas and methods used in this manuscript. My primary concern is the difficulty of interpreting some of the quantitative results in the biological context.

      The authors have concluded that β-catenin has two major populations: free population and slow-diffusing complexed population. The authors have concluded with FCS that the diffusion coefficient of free β-catenin to be 14.9 um2/s (line 259) and the complexed β-catenin to be 0.17 um2/s (line 327). Similar to the authors' argument in the manuscript, this difference means about a 100-fold change of the complex length scale. If the complex is linear, this means a 100-fold change in molecule size, but if the complex is spherical, this means a one-million-fold increase of the molecule size. Furthermore, in the next section, with the N&B method, the authors have suggested that "few, if any, of these complexes contain multiple SGFP2-CTNNB1 molecules" (line 366). When combining the two parts of information, it is hard to imagine a complex that contains one thousand to one million molecules only have one or a few β-catenin subunits. From the biology point of view, APC is the backbone of the destruction complex, which has several β-catenin binding sites by itself. Additionally, APC also contains several Axin1 binding sites where each Axin1 can also recruit one β-catenin. It is unlikely that one APC complex contains only one β-catenin, not mentioning the potential oligomerization of APC. The conclusion that most of the β-catenin containing complexes has only one β-catenin could either be real or due to the misinterpretation of experimental data.

    1. Reviewer #1 (Public Review):

      This manuscript by Kurashina et al. describes a novel post-mitotic role in synaptic patterning for a cell fate determining gene, unc-4, and its co-repressor, unc-37. The DA neurons in C. elegans are cholinergic motor-neurons that exhibit unique synaptic tiling of their dorsal axonal segments. Mizumoto et al has previously shown that Semaphorin-Plexin signaling is required to establish the tiling between DA8 and DA9, by functioning in cis in the DA9 neuron. Using temperature sensitive mutant of unc-4, as well as a combination of CRISPR/Cas9 genome editing with the AID system for specific temporal degradation, the authors nicely examine the spatiotemporal requirement of unc-4, and show that unc-4 is required only post-mitotically for synapse tiling, but not during the development of the DA neurons. Interestingly, activity of the corepressor unc-37 is required both during development and postmitotically for correct tiling. unc-4 and unc-37 are suggested to function by inhibiting the canonical wnt signaling. Overall this is an interesting study which sheds light on our understanding of the post-developmental role of cell fate genes in synapse patterning. I only have one major issue that requires some clarification. The authors present in their introduction the results from Kerk et al, regarding the role of unc-4 as a cell fate determining gene for the VAs and DAs. Kerk et al have shown that UNC-4 is specifically required for the expression of DA genes, without affecting ACh pathway genes. Table 1, however, doesn't fully recapitulate the same results and actually shows that unc-4 and unc-37 mutants do not exhibit significant cell fate defects. The authors use these results to argue in the discussion that the synaptic patterning defects can occur independent of the cell fate transformation. The issue of unc-4 as a cell fate determining gene of A type motor neurons needs to be more clearly addressed. The authors should test whether acr-5 expression is elevated in DAs in unc-4 and unc-37 mutants (Winnier 1999, Kerk 2017). In addition, they should also analyze these DB markers in the temp shift experiments (do the VB-DB markers show up in the post embryonic knockout? And if so, will silencing them specifically in DA neurons rescue the tiling defects?). The discussion, accordingly, should also address these issues.

    1. Reviewer #1 (Public Review):

      Higashi et al. present a molecular mechanism of how the cohesin complex, a supramolecular assembly of several proteins, can topologically embrace DNA and actively extrude DNA into loops. The loop-extruding activity of cohesin and of related condensin complexes have been proposed to represent a cornerstone of genome organization. While recent in vitro studies demonstrate that cohesin and condensin complexes are capable of extruding loops, the molecular mechanisms driving loop extrusion, i.e. how ATP energy is utilized, and what underlies the processivity of the loop extrusion, remains enigmatic. The cohesin complex consist of two long flexible protein "arms" connected at the 'hinge' ends. The other, 'head' ends are linked by the kleisin protein and also can dimerize to form an ATP-binding chamber. Defining how transitions in the cohesin complex structure and its ATPase activity underlies known cohesin functions has been the object of numerous studies for over two decades. Here, the authors build upon these studies.

      The authors start by analyzing available structural data for cohesin domains and associated loading factors. First, by combining the structure of the cohesin-head-domain complex engaged with DNA in ATP-bound state and the corresponding free crystal structures, they show that the 'head module' in the ATP-bound state can tightly wrap around DNA, and upon ATP hydrolysis the DNA-embracing cavity will dilate. In other words, the complex transitions from a 'DNA-gripping state' into a 'DNA-slipping' state after ATP is hydrolyzed. Next, they show that the other DNA-binding module, the 'hinge module', does not change its interaction with the DNA after ATP hydrolysis. The authors also conclude that ATP hydrolysis weakens the interaction between the head and hinge modules, suggesting that the cohesin ring alternates between folded (with head and hinge closed) and unfolded ('free' hinge) states. The authors next carried out FRET experiments to provide experimental evidence for the predicted change in spatial arrangement between the head and hinge modules. Based on this structural analysis, they propose that whether DNA is passed (or not) through the 'kleisin gate' before binding to the head module (into the gripping state) determines if the DNA will be released inside the cohesin ring (i.e. 'topological entry') or if the DNA will remain loosely associated with the head module (i.e. 'loop extrusion') upon ATP hydrolysis. In the latter case, repetitive simultaneous binding of DNA to the head and hinge modules in a folded state followed by relaxation of the cohesin ring while DNA remains bound to the hinge module, may result in a overall 'inward' directed motion of the DNA thread relative to the head domain, i.e. loop extrusion. Stochastic simulations of a coarse-grain model further support that such a model can give rise to loop extrusion.

      The real strength of the paper is in its combination of several pieces of structural and biophysical data that results in a compelling mechanism for cohesin function. The outcome is a united model for cohesin's two characteristic activities - topological engagement of the DNA and DNA loop extrusion. Importantly, the authors explore the role of ATP hydrolysis in driving conformational changes, and, thus, the translocation of DNA, as well as the role of the DNA binding kinetics. The authors go on to relate these findings to the consequences for cohesin function inside cells, where it must content with chromatized substrates. For example, they suggest that while a single nucleosome probably can be bypassed by the cohesin complex, an array of the nucleosome may present a significant hindrance.

      Given its interdisciplinary nature and important conclusions, I believe that this paper will be of broad interest to scientists across disciplines and will influence and stimulate future consideration of how cohesin contributions to the spatiotemporal organization of chromatin.

    1. Reviewer #1 (Public Review):

      Redmond et al. use single-cell and single-nucleus RNA-sequencing to reveal the molecular heterogeneity that underlies regional differences in neural stem cells in the adult mouse V-SVZ. The authors generated two datasets: one which was whole cell RNA-seq of whole V-SVZ and one which consisted of nuclear RNA-seq of V-SVZ microdissected into anterior-posterior and dorsal-ventral quadrants. The authors first identified distinct subtypes of B cells and showed that these B cell subtypes correspond to dorsal and ventral identities. Then, they identified distinct subtypes of A cells and classified them into dorsal and ventral identities. Finally, the authors identified a handful of genes that they conclude constitute a conserved molecular signature for dorsal or ventral lineages. The text of the manuscript is well written and clear, and the figures are organized and polished. The datasets generated in this manuscript will be a great resource for the field of adult neurogenesis. However, the arguments and supporting data used to assign dorsal/ventral identities to B cells and A cells could be strengthened, and more rigorous data analysis could result in new biological insights into stem and progenitor cell heterogeneity in the V-SVZ.

    1. Reviewer #1 (Public Review):

      This study focuses on the consequences of deleting the GluA4 subunit of AMPA receptors for cerebellar synaptic transmission and cerebellar-dependent behaviors. The manuscript is well organized and the information is clearly presented. The first aim of the study is to investigate the effect of the deletion at the level of synaptic function. This is well achieved by a combination of patch-clamp recordings from cerebellar slices and modeling. It is found that deletion of the GluA4 subunit results in a strong decrease in synaptic currents from mossy fibers (MF) to granule cells (GC) as well as in two «compensatory» changes pertaining to NMDA Rs and tonic inhibition. As a consequence, MF-GC transfer is strongly reduced at high frequencies but less affected al low frequencies. The second part of the work investigates the effect of the GluA4 deletion on cerebellar-dependent behaviors. GluA4 knock-out mice are found to have no deficits in locomotion but exhibit a total absence of associative learning in an eye-blink conditioning paradigm. Both, at the slice level and at the behavioral level the strength of this work resides on the quality of the data and the rigorous analysis. A shortcoming of the work stems from the «compensatory» changes which complicate interpretation. However modeling strategies are implemented incorporating those changes and they are able to well predict the observed alterations in GC firing pattern, thus limiting the negative impact.

    1. Joint Public Review:

      The way homologous chromosomes identify one another and become paired is an intriguing phenomenon that has a long history of study, yet the molecular mechanism remains unclear. Recent studies have led to a phenomenological button model for homolog pairing, which hypothesizes that pairing is initiated at discrete sites along the length of each chromosome. The authors aimed to rigorously investigate this idea using biophysical modeling and live imaging. They first constructed a simple polymer model with buttons distributed along the chain that possess locus-specific interactions, and thoroughly investigated its property via stochastic simulation in 3D. Their study confirms that homolog-specific interactions are necessary for homolog pairing. They also tested the effect of time, interaction strength, initial inter-homolog distance, and button density. The authors went on to perform live imaging of pairing dynamics at two selected loci, using the fluorescent signal from nascent mRNA at the corresponding locus. They fitted the model to the experimentally quantified pairing probability of the selected loci over a 6-hour developmental window, and used the constrained model to predict the individual pairing dynamics. The predicted inter-homolog distance post pairing agrees very well with experimental observation.

      Their study supports a button mechanism for homolog pairing, where stable pairing is initiated by reversible random encounters that are propagated chromosome-wide. This work suggests that active processes are not necessary to explain pairing and paves the way for further investigating the molecular mechanism of such a pairing phenomenon.

    1. Reviewer #1 (Public Review):

      The authors have identified an entire set of genes for the synthesis of sulfated exopolysaccharides (EPS) in the cyanobacterial model Synechocystis 6803. They show convincingly that the respective gene products are involved in the production of these compounds and they have extensively characterized the regulation of these genes. Among the regulators they found a STAND protein. STAND proteins include animal and plant regulators of programmed cell death but were rarely characterized in bacteria. Last but not least they come up with an entirely new model for the buoyancy regulation of cyanobacteria (as light-dependent aquatic organisms it is important for cyanobacteria where they are in the water column). The authors suggest a mechanism in which EPS-entrapped cells together with extracellular gas bubbles derived from photosynthesis form multicellular complexes that float at certain depths. This would be a very important function and explain the extensive regulatory and signaling apparatus in controlling the synthesis of these sulfated EPS.

    1. Reviewer #1 (Public Review):

      Major Comments/Concerns

      On line 101 - The use of only the longest transcript for each gene could miss important functional sections of the genome. This could create bias against genes with many isoforms and miss exons that do not happen to lie in the longest transcript. How different would the resulting profiles of conservations be if all coding regions or exons of every gene were used?

      On line 106 - Does this approach create good specificity to our gene of interest rather than just broad functional similarity? For example, with this approach, are there any major neuronal function genes that have NPP very different from MeCP2? Could authors provide a more objective evaluation to baseline/null?

      Minor Comments/Concerns

      On line 132 - It seems fair to examine this set of genes first, but I am not sure this approach to filtering in particular moves us further towards finding a therapeutic for Rett. These genes could be all good potential targets, and your subset of focus are just the best ones for current validation.

      Figure 2C could be made with all 390 co-evolved genes to strengthen the argument that chr19p13.2 is an important region for MeCP2s role.

      Figure 3, 4, 5, 6 - Dynamite plots. While the stats tests are great for understanding the impact of different treatments, box plots or jittered dots would be even more clear.

    1. Reviewer #1 (Public Review):

      The energy released upon zippering of SNARE complexes from the N-terminus to the membrane-proximal C-terminus is widely believed to provide the driving force for membrane fusion, and the cis-SNARE complexes resulting after fusion are disassembled by formation of a 20S complex with Sec17 and Sec18, followed by ATP-hydrolysis by Sec18. This paper now shows that membrane fusion still occurs when the hydrophobic interactions that drive C-terminal zippering of the yeast vacuolar SNARE complex is completely prevented by C-terminal truncation of two of the SNAREs and replacement of the hydrophobic residues at the C-terminus of the SNARE domain of a third SNARE with polar residues, and that such fusion requires Sec17, Sec18 and non-hydrolyzable ATP homologues, in addition to the HOPS tethering complex, which mediates SNARE complex assembly. The results also show that Sec17 plays a key role in fusion through hydrophobic residues in an N-terminal loop that are known to interact with membranes. These results suggest that the core membrane fusion machinery is formed by the SNAREs, Sec17 and Sec18 rather than by the SNAREs alone, and that fusion is driven by a combination of SNARE C-terminal zippering and perturbation of the lipid bilayers by the hydrophobic loops of Sec17. These conclusions are strongly supported by a variety of membrane fusion experiments. FRET assays to SNARE complex assembly also support the conclusions but are less convincing.

    1. Reviewer #1:

      The authors investigate a role for a candidate new inhibitor of USP28 in destabilizing c-MYC to reduce the growth of lung squamous carcinomas. They demonstrate that c-MYC levels are higher in lung squamous cell carcinomas (LSCC) versus lung adenocarcinomas (LADC), and depletion of c-MYC reduces LSCC cell growth. The deubiquitinase USP28 is known to stabilize c-MYC; the authors show that depletion of USP28 also decreases c-MYC protein levels. USP28 action opposes that of a ubiquitin complex targeted by the FBXW7 tumor suppressor. The authors create a new mouse model in which FLP recombinase initially causes deletion of FBXW7 and activation of KRAS to cause tumorigenesis with LSCC and LADC, followed by tamoxifen-dependent CRE recombinase deletion of USP28. Loss of USP28 in this model reduced numbers of LSCC but not LADC, and led to decreased expression of c-MYC and other short-lived proteins such as c-JUN and deltap63. A limitation of the data shown is that tumor number calculations are shown for a relatively small number of mice. Deletion of USP28 also did not restrict LADC growth in a second mouse model, with tumors forming based on activation of KRAS and loss of TP53. The authors then describe a compound, FT206, which they show is a specific inhibitor of USP28 among other ubiquitinases. They demonstrate that this compound reduces expression of c-MYC, c-JUN, and deltap63, but do not demonstrate this effect is directly mediated through USP28. They also show FT206 reduces growth of LSCC but not LADC in the KRAS/FBXW7 tumor model, and in human LSCC xenografts. These latter data suggest the compound FT206 may be useful as a lead compound. However, the current data are not sufficient to demonstrate FT206 binding and biological effect is specific for USP28, as the compound may also bind and regulate other non-deubiquitinase proteins.

  2. Apr 2021
    1. Reviewer #1 (Public Review):

      In this manuscript, Ma, Hung and colleagues rewind the tape to explore the genetic landscape that precedes carbapenem resistance of Klebsiella pneumoniae strains. The importance of this work is underscored by the paucity of new drugs to treat CPO (carbapenemase producing organisms). 'Given the need for 35 greater antibiotic stewardship, these findings argue that in addition to considering the current 36 efficacy of an antibiotic for a clinical isolate in antibiotic selection, considerations of future 37 efficacy are also important.' And so I would say the major weakness of the paper is the aspirational nature of how this work could be used by clinicians in antibiotic selection or treatment of the patient.

      The strains selected for these experiments and the evolutionary in vitro models are both well considered. One idea that has stuck with me from the figures of a review article by Kishony (https://pubmed.ncbi.nlm.nih.gov/23419278/, figure 4) is the concept of constraining the evolutionary pathways or fitness landscape for antibiotic resistance. Are there any peaks that a microbial strain reaches that optimize resistance to one AbX but basically leave it inherently unable to evolve resistance to another AbX? This could have application for dual drug therapy or pulsed therapy. When you sequence the isolates that have increased their MIC do you find 'unrelated' mutations in genes that would control protein synthesis or other functions that might be compensatory mutations. Developing a clearer understanding of the rewiring of the bacterium's basic processes might also elucidate both integrated functions and potential weaknesses. You mention mutations in wzc, ompA, resA, bamD.

      Point of discussion. Classic ST258 carries blaKPC on pKpQIL plasmid. Your ST258 strain (UCI38) carries blaSHV-12 on pESBL. Am I to assume that pESBL is in lieu of pKpQIL? Transformation of CPO have many variables and in vitro data does not always mirror what is observed in vivo. So the findings of Fig 2f might need to be considered under different laboratory conditions (substrate, temperature) [https://pubmed.ncbi.nlm.nih.gov/27270289/].

    1. Reviewer #1 (Public Review):

      Mandal and colleagues identified novel functions of Relish in the hematopoietic niche development and its coordinative role in innate immunity. The authors found that Relish is expressed in the PSC, which is essential for various developmental functions, including the maintenance of hematopoietic progenitors, the number of PSC cells, expression of Wg, and the PSC actin cytoskeletal structure. Furthermore, Relish acts as an inhibitor of JNK signaling and functions downstream of the ecdysone pathway in the PSC. The authors moved on to find the developmental and physiological relevance of this phenomenon and discovered that Relish is downregulated upon bacterial infections to accommodate immune responses. These findings show that Relish plays a critical role in hematopoiesis as a downstream of hormonal control and in switching between the developmental and physiological mode of the PSC.

      Conclusions are well-supported by data, and experiments were carefully performed and analyzed. Given that most of the studies on Relish describe its function in innate immunity and that it is the first study showing critical roles of Relish in blood development, this study will draw broad attention and contribute to understanding insect hematopoiesis and immunity.

    1. Reviewer #1 (Public Review):

      Yan et al. take a comprehensive look at structural variants in the 1000 Genomes Project high-coverage dataset, using recent developments that can link short- and long-read data. Combined with genomic simulations, they identify and characterize the timing and origin of a likely selected region in Southeast Asian populations. The combination of multiple data types adds depth to the interpretation.

      The study is timely, combing recently released data and methods, and had interesting biological implications. Tree main areas would help interpretation and robustness of the paper:

      1) Further context and interpretation of the original SV set found is needed, for example comparisons to previous work to identify clearer "positive controls" or sanity checks on the method, and to understand what the contribution of the method/dataset/paper is.

      2) The above is particularly important across ancestries/populations which differ in their LD levels. How does population-specific LD patterns impact the ability to detect these SV patterns? and therefore to make cross-population comparisons or infer differences in frequency that are central to the selection scan and the 220 highly differentiated SVs of interest. Perhaps this is in the original methods paper, but is central to this paper so should at least be explained or analyzed.

      3) The genomic simulations to infer the strength selection was a nice addition, a step beyond common empirically-driven work. It would help to know how to interpret the ABC model in the context of the later finding that the region was introgressed from Neanderthals--the model seems to not include this aspect.

    1. Reviewer #1 (Public Review):

      This MS combines two-photon glutamate sensing (using the iGluSnFR fluorescent probe), two-photon glutamate uncaging, two-photon calcium imaging and electrophysiology to investigate whether synaptically released glutamate activates receptors outside the synapse of release, and at neighboring synapses. The data themselves are very impressive. The authors arrive at the revolutionary conclusion that synaptically released glutamate is able to activate both NMDA and even AMPA receptors at neighboring synapses, remarkably strongly. I say revolutionary, because previous modelling has yielded diametrically opposite conclusions. The reflex would be to prefer experiment over theory, yet the modelling was based upon quite strongly constrained physical parameters that would be quite incompatible with the interpretations reported here. However, I believe the authors have failed to take into account significant technical limitations inherent in the technologies they apply. These include spatial averaging of fluorescence, possible saturation of iGluSnFR and diffusive exchange of (caged) glutamate during uncaging. As a result, the conclusion is wholly unproven. Indeed, I believe it highly probable that all of the data in favor of distal activation will prove to be consistent with synapse specificity and the presence of technical artifacts related to spatial averaging of fluorescence signals and diffusive exchange of (caged) glutamate during uncaging.

    1. Reviewer #1 (Public Review):

      In this work the authors address inter-subunit interactions leading to ESCRT-III function during MVB sorting in a yeast model system. ESCRTs mediate function in multiple biological processes, however the fundamental question of how ESCRT-III mediates membrane remodeling is not understood. As such this represents a topic of considerable interest despite significant technical limitations surrounding the issue. Random and rational mutagenic strategies, including compensatory mutations, are combined with protein-protein interaction studies and in vivo functional assays to identify residues within Vps24 and Vps2 mediating associations with each other and Snf7. Based on these analyses the authors put forth a series of "rules" governing ESCRT-III assembly and function. While beneficial to our conceptual understanding of ESCRT-III these rules fall short in explicitly defining the structural basis of assembly and function including explaining requisite heterodimerization of Vps24-Vps2. This work represents a significant step forward in addressing this challenging question, the experimental design and implementation are convincing, however the limitations of this work could be conveyed more clearly.

    1. Reviewer #1 (Public Review):

      For bacteria, yeast and mammalian cells, energy depletion has been linked to a vitrification of cytosol and protein aggregation. Previous studies have postulated this is in part due to acidification and the shift in pH to match a large set of proteins pIs resulting in large-scale protein aggregation as well as changes in crowding of the cytosol. Additionally, a more direct role for ATP in protein aggregation has been proposed through its chemical properties as a hydrotrope. The appeal of this hypothesis is that the steady-state levels of ATP far exceed the Kd of most enzymes pointing to a potential non-enzymatic role for the high levels.

      In this study, the authors take advantage of a FRET-reporter for ATP that they developed previously called "Queen". They then manipulate ATP levels using mutants in AMP kinase(Snf1) or Adenyl kinase (Adk1) and find null mutants indeed have lower concentrations of ATP and experience sudden drops in ATP levels which the authors term ATP catastrophe. These mutants also show genetic interactions with protein folding/glycosylation pathways and are sensitive to conditions that generate proteotoxic stress. Hsp104 forms foci in the genetically induced lowered ATP levels as well as exogenous ectopic aggregation prone proteins such as alpha-synuclein. The authors attempt to show that the cause of aggregation is due to limiting ATP directly by adding excess adenosine to the media and showing this diminished the formation of foci, potentially due to the ability of increased exogenous to raise ATP levels according to previous reports.

      The issue of whether ATP levels play a direct or indirect role in preventing protein aggregation is extraordinarily challenging to address. While ATP can act as a hydrotrope, the formation of aggregates could be due to limitations of the activity of chaperones and helicases which would not be surprising role for ATP in the cell. While the experiments are carefully performed, well analyzed and fairly interpreted; questions still remain about the impact of these experiments on understanding how ATP impacts cytosol.

    1. Reviewer #1 (Public Review):

      Cucinotta et al. examine the widespread, transient transcription of genes that occurs within minutes of refeeding quiescent Saccharomyces cerevisiae cells, focusing on the role of the RSC remodeler complex in this process. A range of appropriate genomic approaches are used to characterize the initial burst of transcription, changes in localization of RSC and RNA Pol II, and changes in the occupancy and positioning of nucleosomes during the first minutes after nutrient repletion. Several new insights are reported including the role of RSC in maintaining promoters in state that is ready to respond rapidly to nutrient repletion, the relocalization of RSC into genes following initiation of transcription, a role for TFIIS in exiting quiescence that was not apparent in log phase, the timing of histone acetylation in response to transcription, changes in chromatin architecture during the exit from quiescence, and the effects of chromatin changes on transcription start site selection and repression of antisense transcription from downstream nucleosome depleted regions. Given how little is known about the emergences of cells from quiescence and how common and important this transition is in long-term viability, development, and carcinogenesis, these insights are certain to have broad impact. The data are of high quality and the manuscript is very clearly written, with good correlation between the level of support provided by the data and the strength of the conclusions drawn. Only minor issues remain to be addressed.

    1. Reviewer #1 (Public Review):

      In this study, Leydon et al., use an elegant multi-component genetic system to address the mechanisms of repression by the Arabadopsis TOPLESS (Tpl) protein. Taking advantage of the genetic tools and knowledge of the structure of the Tpl protein the authors determine two short alpha helical regions that act as independent repression domains. They provide evidence that the target of one of these domains is the N-terminal region of the Med21 subunit of the mediator complex. Chromatin immunoprecipitation experiments, anchor-away loss of function and co-immunoprecipitation assays indicate that Tpl mediated repression involves formation of a promoter complex comprising the mediator complex along with several general transcription factors, but lacking RNA polymerase II. The authors also show that Tpl-Med21 interactions are involved in Tpl mediated repression in plants.

    1. Reviewer #1 (Public Review):

      This paper was a pleasure to read. It is a tour-de-force study that is well-written, clear, and transparent. The study recounts how the HMA domain became integrated into the Pik NLRs and how it evolved higher affinity binding to a pathogen effector. Strikingly the authors demonstrate adaptability of distinct regions of the HMA:effector interface on two Pik NLRs, driving the convergent evolution of high-affinity binding to the effector. The study furthermore provides a framework for understanding protein evolution in the context of host-microbe interactions. The breadth and depth of the experiments that support the authors conclusions is extraordinary in my view.

    1. Reviewer #1 (Public Review):

      The study by Hendley et al takes advantage of duct-specific DBA-lectin expression to purify pancreatic ductal populations that were then subjected to scRNA-seq analysis. The ability to enrich for this relatively low abundant pancreatic cell population resulted in a more robust dataset that had been generated previously from whole pancreas analyses. The manuscript catalogs several different gene clusters that delineate heterogeneous subpopulations of three different pancreatic ductal subpopulations in mice: mouse pancreatic ductal cells, pancreatobiliary cells, and intra pancreatic bile duct cells. Additional comparisons of the resulting data sets with published embryonic and adult datasets is a strength of the study and allows the authors to subclassify the different ductal cell populations and facilitates the identification of potentially novel subpopulations. Pseudotime analysis also identified gene programs that led the authors to speculate the existence of an EMT axis in pancreatic ducts. Overall, the data analyses is strong, but the authors tend to draw conclusions that are not fully supported by the presented data.

      The second half of this study focuses on three candidate proteins that were identified in the transcriptome analysis - Anxa3, SPP1 and Geminin. Crispr-Cas9 was used to delete each gene in an immortalized human duct cell line (HPDE). Deletion of each gene resulted in increased proliferation; SPP1 mutant cells also displayed abnormal morphology. Additional functional studies of the cell lines or in mouse models suggested a role for SPP1 in maintaining the ductal phenotype and Geminin in protecting ductal cells from DNA damage, respectively. Although the provided phenotypic analysis suggest important functional roles for these proteins, follow up studies will be required to fully understand the role of these genes in homeostatic or cancer conditions.

      Strengths:

      1) Enrichment of pancreatic ductal populations enhanced the robustness of the scRNA-Seq dataset

      2) Quality of the sequencing data and extensive computational analysis is extremely good and more comprehensive than previously published datasets

      3) Comparative analysis with existing mouse and human data sets

      4) Use of human ductal cell lines and mouse models to begin to explore the function of candidate ductal genes.

      Weaknesses:

      1) There are many suppositions based on gene expression changes that are somewhat overstated.

      2) The conclusion that there is an EMT axis in pancreatic ducts is not fully supported by the gene expression and immunofluorescence data

      3) A good rationale for choosing Anxa3, SPP1 and Geminin for additional functional analysis is not provided. In addition, it isn't clear why Anxa3 function isn't pursued further.

      4) Although extensive models (transplanted cells for SPP1 and mouse conditional KOs for Geminin) were generated, the functional analysis for each gene is preliminary; additional longer term studies will be necessary to fully understand the role of these proteins in pancreatic duct development and cancer.

    1. Reviewer #1 (Public Review):

      The authors develop a mechanistic model for inferring infectiousness profile from data on times of symptom onset in pairs of infector-infectee. The novelty of their approach lies in assuming that infectiousness of an infected individual depends also on the whether or not they have symptoms. The authors fit a data set of time of symptom onset in 191 transmission pairs to a model that assumes that infectiousness varies along the incubation period. They compare the model fit to fits from models and find that their model of differential infectiousness explains the data better than the other models considered.

      This is a carefully constructed study, and the conclusions are well supported by the analysis carried out. My only concern is that the data used were obtained during the early stage of the pandemic (January to February 2020). As the pandemic was growing in most countries during this time, we are more likely to have observed shorter serial intervals. Similarly, as isolation of infected individuals would prevent them from transmitting further, longer serial intervals are likely to be under-represented in the data. Indeed, the longest serial interval in the data used was 5 days. It would be interesting to understand whether the conclusions about the proportion of onward transmissions averted by contact tracing and subsequent isolation still hold as the pandemic progresses, and we continue to observe longer serial intervals. If the authors are unable to find more recent data, this caveat should be clearly discussed.

    1. Reviewer #1 (Public Review):

      Stokes, et al. describe the effects of isoflurane on metabolism in post-natal day 7 mice, and older mice. They demonstrate that blood levels of glucose and ß-hydroxybutyrate fall quickly in response to isoflurane, and that the magnitude of the decrease increases with the length of the exposure. Mice 30 days post-natal do not exhibit these changes in response to isoflurane. The authors document the much higher circulating levels of ß-hydroxybutyrate in the post-natal day 7 mice, highlighting the importance of this substrate for supporting the energetics of the developing brain. Important control experiments, administering 100% oxygen without anesthetic to post-natal day 7 mice, as well as administering anesthetics to 30 day old mice on a ketogenic diet, did not result in significant decreases in glucose and ß-hydroxybutyrate blood levels. Remarkably, they observed significant decreases in response to very small, subanesthetic doses of isoflurane, halothane and sevoflurane in post-natal day 7 mice. Administration of bolus glucose corrects the glucose level for these mice under anesthesia, but not the level of ß-hydroxybutyrate, while administration of bolus ß-hydroxybutyrate corrects both levels.

      The authors then proceed to a series of measurements in an attempt to determine a direct target of volatile anesthetics on metabolism, focusing on hepatic metabolism. This is something of a Procrustean bed, given that the there is ample evidence that volatile anesthetics affect a large number of different membrane bound processes. Nonetheless, these experiments provided valuable data demonstrating anesthetic induced decreases in fatty acid oxidation. This reviewer finds the arguments regarding impairment of the citric acid cycle a bit unconvincing: 7 and 30 day old mice exhibit the same increase in citrate and isocitrate levels, yet only the 7 day old mice show elevated lactate levels. Rather than exhibiting increased metabolic flexibility, as the authors suggest, this finding seems to argue that 7 day old mice have less metabolic flexibility. The authors demonstrate that several perturbations of fatty acid metabolism can result in depression of ß-hydroxybutyrate, leading them to focus on carnitine palmitoyl transferase-1. They demonstrate that inhibition of this enzyme produces a decrease in ß-hydroxybutyrate; however, they also find that mice with a knockout of this enzyme do not have decreased ß-hydroxybutyrate levels.

      The authors are circumspect in their conclusions regarding the targets responsible for the metabolic changes observed in neonatal mice in response to anesthetics. They do correctly highlight the potential importance of these metabolic effects. It will be crucial for future research to determine whether these effects can be directly correlated to measures of cerebral function during anesthesia, e.g., EEG or evoked potentials, and to measures of neuropathological change. Of great interest to clinicians will be demonstration of whether co-administration of glucose or ß-hydroxybutyrate together with anesthetics can abrogate such changes.

  3. Mar 2021
    1. Reviewer #1 (Public Review):

      In this manuscript, Zilova et al. show that primary embryonic cells derived from blastula-stage Medaka and Zebrafish embryos can self-organize into retinal organoids. When aggregates of 1000-2000 primary embryonic cell are embedded in Matrigel addition, they form a neuroepithelium under the control of Rx3 which develops into a retinal organoid. The process mirrors some aspects of embryo development. Moreover, another interesting finding is that Rx3 expression is initiated in the absence of Matrigel at day 0, which indicates that the retinal fate occurs by default and is not dependent on extracellular matrix components. The authors compare the ability of cells from Mesaka and zebra fish and show that both are competent to form organoids, though each does it with the time scale of the embryo of origin. The authors show that by reducing the number of Medaka cells to aggregate (500-800 cells), Rx2 and Rx3 are expressed only in restricted regions of the small aggregates, presumably where they organize into discrete circular Rx2 and Rx3 positive neuroepithelial units that develop into structure resembling retinal epitjhelia with some diversity of retinal cell types including amacrine, ganglion, photoreceptor, bipolar and horizontal cells.

      This is a novel and original piece of work that reveals the capacity of fish primary embryonic pluripotent cells to behave like mammalian embryonic stem cells and organize optic cup organoids.

    1. Reviewer #1 (Public Review):

      The complexity of the infection model developed by the authors is to be praised as it allows the dissection of host-pathogen interactions with multiple players coming together, namely human epithelial cells, endothelial cells and neutrophils, UPEC, urine, antibiotics and mechanical forces at play during bladder filling and micturition. This is truly a tour de force and should provide the authors (and other labs potentially able to recapitulate it) with an unprecedented model to study UTIs and their response to antibiotics. Notably, authors have been able to document the formation of NETs in response to UPEC infection in this model. One small caveat was the choice of antibiotics used to treat the infection in their model. Is Ampicillin really a drug of choice, both because of its inability to reach intracellular niches and it not being a drug of choice in the clinic?

    1. Reviewer #1 (Public Review):

      In their manuscript, Lawrence et al. investigate the direct effects of the microtubule-associated protein, SSNA1, on microtubule (MT) dynamics and damage using purified proteins and TIRF microscopy. Prior work on this protein showed that SSNA1 self-assembles into higher-order filaments and binds longitudinally along stabilised MTs, inducing MT branching and nucleation. In this study, they find that SSNA1 promotes templated MT nucleation, consistent with prior results, but further define the effect of SSNA1 on MT dynamics. SSNA1 overall dampens MT dynamics by reducing both growth and shrinkage rates, suppressing catastrophe frequency, and increasing rescues. The authors also quantify SSNA1 on GMPCPP over a timecourse both at single-molecule and multi-molecule concentrations. On dynamic MTs, SSNA1 recognizes the growing end and promotes end curvature, but it did not recognize the curves of taxol-stabilised MTs, leading the authors to conclude that it likely induces curvature, rather than recognizes it. Perhaps this is the mechanism by which SSNA1 prevents catastrophe, a role which the authors demonstrate for SSNA1 after both tubulin dilution or stathmin sequestration of tubulin. The most interesting part of this study is found in Figure 4, where the authors show that SSNA1 prevents MT severing by spastin and also localizes to sites of lattice damage. The authors conclude that SSNA1 is a MT stabilizing protein and a sensor of MT damage. The results on MT dynamics do not provide much insight into the mechanism of this protein, which isn't even found to colocalize with MTs in vivo (SSNA1 instead accumulates at branchpoints in neurons). The role of SSNA1 in lattice damage recognition is the highlight of this paper, and also correlates well with its in vivo localization pattern, indicating this could be a true function of this protein. This damage recognition ability could potentially be the first step that leads to SSNA1-induced MT nucleation and branching from an existing MT.

    1. Reviewer #1 (Public Review):

      The authors interrogated an underexplored feature of CRISPR arrays to enhance multiplexed genome engineering with the CRISPR nuclease Cas12a. Multiplexing represents one of the many desirable features of CRISPR technologies, and use of highly compact CRISPR arrays from CRISPR-Cas systems allows targeting of many sites at one time. Recent work has shown though that the composition of the array can have a major impact on the performance of individual guide RNAs encoded within the array, providing ample opportunities for further improvements. In this manuscript, the authors found that the region within the repeat lost through processing, what they term the separator, can have a major impact on targeting performance. The effect was specifically tied to upstream guide sequences with high GC content. Introducing synthetic separator sequences shorter than their natural counterparts but exhibiting similarly low GC content boosted targeted activation of a reporter in human cells. Applying one synthetic separator to a seven-guide array targeting chromosomal genes led to consistent though more modest targeted activation. These findings introduce a distinct design consideration for CRISPR arrays that can further enhance the efficacy of multiplexed applications. The findings also suggest a selective pressure potentially influencing the repeat sequence in natural CRISPR arrays.

      Strengths:

      The portion of the repeat discarded through processing normally has been included or discarded when generating a CRISPR-Cas12a array. The authors clearly show that something in between-namely using a short version with a similarly low GC content-can enhance targeting over the truncated version. A coinciding surprising result was that the natural separator completely eliminated any measurable activation, necessitating the synthetic separator.

      The manuscript provides a clear progression from identifying a feature of the upstream sequences impacting targeting to gaining insights from natural CRISPR-Cas12a systems to applying the insights to enhance array performance.

      With further support, the use of synthetic separators could be widely adopted across the many applications of CRISPR-Cas12a arrays.

      Weaknesses:

      The terminology used to describe the different parts of the CRISPR array could better align with those in the CRISPR biology field. For one, crRNAs (abbreviated from CRISPR RNAs) should reflect the final processed form of the guide RNA, whereas guide RNAs (gRNAs) captures both pre-processed and post-processed forms. Also, "spacers" should reflect the natural spacers acquired by the CRISPR-Cas system, whereas "guides" better capture the final sequence in the gRNA used for DNA target recognition.

      A running argument of the work is that the separator specifically evolved to buffer adjacent crRNAs. However, this argument overlooks two key aspects of natural CRISPR arrays. First, the spacer (~30 nts) is normally much longer than the guide used in this work (20 nts), already providing the buffer described by the authors. This spacer also undergoes trimming to form the mature crRNA. Second, the repeat length is normally fixed as a consequence of the mechanisms of spacer acquisition. At most, the beginning of each repeat sequence may have evolved to reduce folding interactions without changing the repeat length, although some of these repeats are predicted to fold into small hairpins.

      Prior literature has highlighted the importance of a folded hairpin with an upstream pseudoknot within the repeat (Yamano Cell 2016), where disrupting this structure compromises DNA targeting by Cas12a (Liao Nat Commun 2019, Creutzburg NAR 2020). This structure is likely central to the authors' findings and needs to be incorporated into the analyses.

      Many claims could better reflect the cited literature. For instance, Creutzburg et al. showed that adding secondary structures to the guide to promote folding of the repeat hairpin enhanced rather than interfered with targeting. Liu et al. NAR 2019 further showed that the pre-processed repeat actually enhanced rather than reduced performance compared to the processed repeat. Finally, the complete loss of targeting with the unprocessed repeat appears represent an extreme example given multiple studies that showed effective targeting with this repeat (e.g. Liu NAR 2019, Zetsche Nat Biotechnol 2016).

      Relating to the above point, the vast majority of the results relied on a single guide sequence targeting GFP. While the seven-guide CRISPR array did involve other sequences, only the same GFP targeting guide yielded strong gene activation. Therefore, the generalizability of the conclusions remains unclear.

    1. Reviewer #1 (Public Review):

      This manuscript is a meta-analysis of literature, predominantly that from evolutionary psychology. The background seems well-explained, and the discussion and literature review well-written. The authors have done an impressive job of collating and synthesising a truly vast amount of literature that (as they demonstrate) is often pretty ambiguous in its results. The results are well-presented and well-reasoned, without overstating the evidence. The entire manuscript is clear and easy to read and follow. Table 1 makes it particularly easy to follow. I appreciate their emphasis that the various hypotheses about sexual dimorphism are not mutually exclusive, and that this study does not seek to explicitly test either one of them.

      There is enough evolutionary anthropology inserted here to see that the authors have a passing familiarity with it, although I would encourage them to dig much more deeply into this literature in framing their work. In short, there is a tension between evolutionary psychology and evolutionary anthropology that can be very fruitfully explored with the results of this analysis, and the authors only scratch the surface of this at the end of the manuscript.

      Something that seems crucial here, and in this literature more generally, is the likelihood that men have a number of different effective strategies. The background and discussion do a good job of discussing the various possibilities, and how combinations of possibilities that include both female choice or male-male competition could explain human mating behavior. However, it does not really dig into what the implications might be for how multiple, distinct strategies could impact different aspects of the data. What comes to mind is orangutans, in which the large, masculine males appear to obtain mating opportunities primarily through female choice, while the smaller males that have not developed the large body sizes and facial flanges may obtain additional mating opportunities through sexual coercion. In a large sample or meta-analysis like this, a combination of strategies in human males that are at odds with one another, yet both highly effective, may have results that tend to cancel one another out - is there any evidence of this? Getting more into the primate literature here could be useful.

      The authors point out that there could be a strong confounding effect with the way testosterone operates developmentally. Testosterone during adolescence translates well to the development of masculine characteristics, but does not necessarily predict testosterone later in life (hence, the expression of masculine features may not actually relate well to circulating testosterone that could be at least partially drive male-male competition). The authors did an excellent job of discussing these potential confounding effects, but I would have found potential issues like this (and like the one above) to be presented usefully in a table that lays out the different potential confounding issues, and then discusses what the predictions should be in the meta-analysis results for each one.

      The meta-analysis seems well-designed, and the methods appropriate. However, it did feel a bit like data mining with so many different variables run against one another. I do not think this is actually the case, and the authors do justify each of their decisions. In fact, one of the main outcomes of this work is that they show how few of these parameters actually relate strongly to one another. However, the authors might want to be aware that this study could be read as data-mining because of the search for significance amongst so many different variables, and offset this with explicit discussion and framing up front that they intend to examine how effective the various study parameters actually are at uncovering the relationships they seek to uncover. This is something the authors discuss very articulately at the end, but I would appreciate seeing this up front as one of the goals of the paper.

    1. Reviewer #1 (Public Review):

      Summary: The study by Steenkiste focuses on the formation of adaptor protein complexes at sites of integrin receptor adhesion in the modulation of in vitro membrane ruffling and cell movement. The authors are studying the role of BCAR3 (also termed AND34 or NSP1) protein regulation by post-translational mechanisms (ubiquitin degradation and tyrosine phosphorylation). This is one of many adaptor proteins localized to adhesion sites. Studies are being performed on MCF10A or Hela cells to knockdown (siRNA) or over-express tagged protein constructs. By proteomics, a new phosphorylation site was identified (BCAR3 Y117). Mutagenesis showed that BCAR3 Y117 is important for enhancement of in vitro cell movement under conditions where the cullin-5 E3 ligase has also been reduced by siRNA expression.

      Opinion: The authors provide support for a "co-regulatory" model whereby the recruitment of BCAR3 to adhesions acts in part to modulate another adaptor protein tyrosine phosphorylation, p130Cas. This is associated with enhanced cell migration. The data presented are generally supportive of the conclusions and consistent with previous studies of BCAR3 and p130Cas. However, an unresolved issue is why cell phenotypes are dependent on cullin-5 knockdown or otherwise investigated by BCAR3 mutant over-expression. Cul5 loss can alter multiple aspects of cell signaling and the transient knockdown or inducible over-pression assays are a limited primary means of investigation. As multiple protein domains and post-translational modifications modulate the BCAR3-p130Cas complex, the authors did not establish a strong mechanistic linkage between newly-identified BCAR3 Y117 phosphorylation, SOCS6 binding, and a CUL5-dependent cell phenotype. Additionally, some of the experimental conditions (+/- EGF in growth media) are difficult to connect to EGF receptor activation and or signaling.

    1. Reviewer #1 (Public Review):

      In the current manuscript, Frey et al. describe a convolutional neural network capable of extracting behavioral correlates from wide-band LFP recordings or even lower-frequency imaging data. Other publications (referenced by the authors) have employed similar ideas previously, but to my knowledge, the current implementation is novel. In my opinion, the real value of this method, as the authors state in their final paragraph, is that it represents a rapid, "first-pass" analysis of large-scale electrophysiological recordings to quickly identify relevant neural features which can then become the focus of more in-depth analyses. As such, I think the analysis program described by the authors is of real value to the community, particularly as it becomes more commonplace for labs to acquire multi-site in vivo recordings. However, to maximize its utility to the community, several aspects of the analysis need clarification.

    1. Reviewer #1 (Public Review):

      Pulgar et al. describe an interesting mechanism explaining how directed motion of group of cells maintain their migratory path as a group of cells. Incomplete delamination allows here to maintain coordinated cell movements amongst the DFC. The story is self-contained, logical, well-written and just in general very nice. The mechanism described belongs to the so-called mechanical drag which is a new type of multicellular locomotion and may be a general feature involved in many morphogenetic systems.

      The major strength of the study is the extensive use of live imaging and analysis of dynamic events. The study provides a nice cellular mechanism in the process they described. The molecular mechanism would be the only weakness of the study.

      An overall very exciting study.

    1. Reviewer #1 (Public Review):

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

      The study takes advantage of the interesting plant genus Leucadendron to compare gene expression between male vs. female in species with more or less sexual dimorphism. This question was addressed in a somewhat comparable manner in only one previous paper by Harrison et al. 2015 across six bird species. The overarching question is the role of natural selection in sexual dimorphism.

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

      -Beside the genus-wide comparison of whole transcriptomes across related species, which makes in itself a strong dataset, the major strength of the analysis is the phylogenetic framework that allows the authors to track the evolution of sex bias through several tens of million years of evolutionary history. Despite ancestral dioecy in the genus, very few genes show consistent sex bias across several species, with sex-bias being mostly species-specific. Two striking negative results will be of special interest to the community : 1) species with more pronounced sexual dimorphism at the morphological level do not tend to exhibit more pronounced sex-biased gene expression 2) the few genes that do show sex-biased expression were apparently recruited among those with the highest expression variance to begin with, strongly suggesting that sexual selection has not been the main force driving their expression divergence.

      -In my view, the main limitation of the work is the use of leaf rather than reproductive tissues, making the comparison to other studies less straightforward to interpret. It is especially important that the expectations for somatic vs gonadic tissues be made a lot clearer in the text. Also, the fact that a single leaf phenotype is measured (specific leaf area) seems arbitrary : one could imagine sexual dimorphism on many other characteristics, yet they are not considered here. The text on p.324 mentions "striking convergence in aspects of morphological dimorphism across the genus", but there is no way for the reader to appreciate the extent of this convergence. Finally, it would be useful to at least make some mention of the sex-determination system in these species, since the expectations would differ if some of the sex-biased genes were linked to sex chromosomes.

      *An appraisal of whether the authors achieved their aims, and whether the results support their conclusions.

      The analysis is mostly sound, but I am a bit concerned by the arbitrary threshold used to define SBGE. The text on p.305 says that "This result is extremely robust to the choice of threshold", but 1) the results are not reported so it is impossible for the readers to evaluate the basis of this assertion and 2) it is not clear whether robustness of the other results has been evaluated at all. This aspect clearly deserves more attention.

      *A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community.

      This work will be of interest to the community, as rapid rates of expression evolution would generally be interpreted as the consequence of sex bias, whereas the phylogenetic analysis presented here instead supports the idea that the expression of genes that end up being sex biased were instead intrinsically less constrained to begin with.

    1. Reviewer #1 (Public Review):

      This manuscript by Lauer et al follows up on previous articles that ask the question whether there are funding disparities at the National Institutes of Health for African American or Black (AAB) investigators. The investigators breakdown the analysis by race, topic of proposal, and NIH institute-Center (IC) to which an application was assigned. They conclude that the most important factor in determining funding is the Institute assignment with lower funding rates related to the funding capacity of a particular Institute (e.g National Eye Institute vs Minority Health and Health Disparities). The present study is a welcome addition to this debate since if biases do exist, NIH needs to address these. The strengths of this manuscript are the detailed breakdown of the available data in order to evaluate for biases, the availability of data for multiple years (2011-2015) and the consideration of alternate explanations (e.g new applications vs resubmissions; single vs multi PI, etc). A weakness of the data is that if their conclusion is that Institute assignment was the main determinant of funding rates, why wasn't the approach for Institute assignment discussed? Are there possible biases in this assignment besides keyword searches? There is also the question of whether there is circular logic operating here. The Minority Health and Health Disparities received the most AAB applications but had one of the lowest funding rates. Wouldn't this Institute be expected to be one in which AAB applicants would try to direct their application to? This manuscript is sure to generate additional discussion on this topic which is an important step in trying to address the issue of potential funding disparities. However as the authors point out the fact that only 2% of the applications submitted to the NIH were from AAB investigators is of concern.

    1. Reviewer #1 (Public Review):

      Ekeng et al. have sequenced and analyzed 46 Vibrio cholerae whole genome sequence data. The authors demonstrated a predominant lineage (T12) where all isolates from 2018-2019 fall. Their analysis suggest continuous transmission through repeated reintroduction of the same lineage back into the population. The work is interesting and the conclusions of this paper are mostly well supported by data. The present study reinforce the need of more genome sequence data and a strong surveillance network to interpret the data.

      This is a successful model of regional coordination to have genomic surveillance data from a region where surveillance data was inadequate. The manuscript should be modified to focus on strengthening of genomic surveillance further.

    1. Reviewer #1 (Public Review):

      Halliday et al. developed a framework to disentangle the total effect of environment on disease into a direct effect and indirect effects by environment-induced change of host community and by modifying the relationships between host community and disease.

      Applying this framework, the authors studied the direct and indirect effects of elevation on plant leaf disease in the Swiss Alps. They focused on host community structures as mediator of indirect effects. Host community structures were measured by host species richness, phylogenetic diversity, and community pace of life. One important finding is that the positive effect of host community pace-of-life on disease weakened as elevation increased, suggesting an important, but less appreciated, mechanism on how elevation can indirectly influence plant disease. However, since the major findings were based on the analyses with elevation but not specific environmental variables, it does not have that strong implications about the influence of global climate change on disease as the authors stated.

      The developed framework on environmental effects on disease, the well-designed filed study and the large-scale dataset would all make this paper an important contribution to the field.

      Overall, the statistical analyses were reasonable. However, accurate interpretations of some results would require more clarifications on the analyses.

    1. Reviewer #1 (Public Review):

      The authors of this paper seek to understand how HIV infects cells. HIV is a retrovirus that harbors a core of RNA nucleic acid in complex with important replication enzymes such as reverse transcriptase. After infection, reverse transcriptase converts the RNA into DNA, which is then integrated into the chromosome. The authors used advanced imaging techniques to visualize the DNA that is made by reverse transcription. They used fluorescent readout markers of proteins to also look at the viral proteins that are brought into the cell and track with the viral DNA during the virus infection.

      From this work the authors conclude that reverse transcription is completed in the cell nucleus, that intact or nearly intact cores are the substrate for nuclear import, and that virus core uncoating likely occurs in the nucleus, immediately preceding the integration step. Moreover, by using electron tomography, they drill down to the sub-micron level to glean an ultrastructural view of the viral complexes that are performing these important HIV infection steps. Some of these complexes appear to be novel, and thus the work will be of interest to other scientists in this field.

      Weaknesses of the study include insufficient control samples for some of the experiments and also clarifying some of the approaches used and some of their interpretations of the data (detailed below). The authors of this paper could have also done a better job of citing papers published by other scientists who came up with very similar conclusions and/or used very similar techniques.

    1. Reviewer #1 (Public Review):

      The centrality of RAS proteins in human malignancies has long been established, but many issues regarding their regulation and functions remain unresolved. The results of this paper provide strong supporting evidence for an emerging model that posits that activated KRAS can only be tolerated by cells up to a certain point, after which the stress it imposes outweigh its transforming potential. These restrictions impose limits on the amount of KRAS expressed in tumor cells and are also consistent with the frequent coupling of KRAS mutations with loss of the tumor suppressor p53, as the latter relieves the stress signals induced by KRAS.

    1. Reviewer #1 (Public Review):

      Summary and Strength:

      Single-cell RNA sequencing is the most appropriate technique to profile unknown cell types and Koiwai et al. made good use of the suitable tool to understand the heterogeneity of shrimp hemocyte populations. The authors profiled single-cell transcriptomes of shrimp hemocytes and revealed nine subtypes of hemocytes. Each cluster recognizes several markers, and the authors found that Hem1 and Hem2 are likely immature hemocytes while Hem5 to Hem9 would play a role in immune responses. Moreover, pseudotime trajectory analysis discovered that hemocytes differentiate from a single subpopulation to four hemocyte populations, indicating active hematopoiesis in the crustacean. The authors explored cell growth- and immune-related genes in each cluster and suggested putative functions of each hemocyte subtype. Lastly, scRNA-seq results were further validated by in vivo analysis and identified biological differences between agranulocytes and granulocytes. Overall, conclusions are well-supported by data and hemocyte classifications were carefully performed. Given the importance of aquaculture in both biology and industry, this study will be an extremely useful reference for crustacean hematopoiesis and immunity. Moreover, it will be a good example and prototype for cell-type analysis in non-model organisms.

      Weaknesses:

      The conclusions of this paper are mostly well supported by data, but some aspects of data analysis QC and in vivo lineage validation need to be clarified.

      1) It is not a trivial task to perform genome-wide analyses of gene expression on species without sufficient reference genome/transcriptome maps. With this respect, the authors should have de novo assembled a transcriptome map with a careful curation of the resulting transfrags. One of the weaknesses of this study is the lack of proper evaluation for the assembly results. To reassure the results, the authors would need to first assess their de novo transcripts in detail and additional data QC analysis would help substantiate the validity.

      2) The authors applied SCTransform to adjust batch effects and to integrate independent sequencing libraries. SCTransform performs well in general; however, the authors would need to present results on how batch effects were corrected along with before and after analysis. In addition, the authors would need to check if any cluster was primarily originated from a single library, which could be indicative of library-specific bias (or batch effects).

      3) Hem6 cells lack specific markers and some cells in this cluster are scattered throughout the other clusters (Fig. 1 & 2). Based on the pattern, it is possible that these cells are continuous subsets of other clusters. It would be good if the authors could group these cells with Hem7 or other clusters based on transcriptomic similarities or by changing clustering resolution. Additionally, they may also be a result of doublets, and it is unclear whether doublets were removed. Hem6 cells require additional measures to fully categorize as a unique subset.

      4) The authors took advantage of FACS sorting, qRT-PCR, and microscopic observation to verify in silico analyses and defined R1 and R2 populations. While the experiments are appropriate to delineate differences between the two populations, it is not sufficient to determine agranulocytes as a premature population (Hem1-4) and granulocytes as differentiated subsets (Hem5-9). To better understand the two groups (ideally nine subtypes), additional in vivo experiments would be essential. For example, proliferation markers (BrdU or EdU) could be examined after FACS sorting R1 and R2 cells to show R1 cells (immature hemocytes) are indeed proliferating as indicated in the analyses.

      5) FACS-sorted R1 or R2 population does not look homogeneous based on the morphology and having two subgroups under nine hemocyte subtypes may not be the most appropriate way to validate the data. The better way to prove each subtype is to use in situ hybridization to validate marker gene expressions and match with morphology.

    1. Reviewer #1 (Public Review):

      This manuscript, which follows on from a recent eLife paper documenting the relevance of the multi-basic cleavage site (MBCS) in the spike (S) protein of SARS-CoV-2, shows that growing SARS-CoV-2 on relevant epithelial cell lines or differentiated stem cell-derived culture systems prevents the emergence of MBCS mutations than impact on properties of S that contribute to cell tropism and the viral entry mechanism.

      The paper builds on the authors previous work and that of others, and in some respects the results are not surprising. Nevertheless, the paper sets out a number of important findings. 1) That SARS-CoV-2 grown in Vero cells rapidly acquire MBCS mutations, where as virus grown in airway epithelial cells or Vero-TMPRSSR2 cells do not; 2) that deep sequencing is necessary to see mutations that are not apparent in consensus sequence reads, 3) that factors such as the addition of fetal calf serum can influence the selection of mutant phenotypes and 4) that cultures derived from differentiated stem cells can provide reproducible systems for virus culture. Together, the work sets out clear guidelines for the production of SARS-CoV-2, and potentially other viruses, avoiding the pitfalls that can arise from growing viruses in permissive transformed cell lines.

      The data and manuscript are clearly presented, and my concerns are minimal. Overall, the paper will make a useful addition to the SARS-CoV-2 literature and will be of value to researchers working not just of SARS-CoV-2 but on many other viruses.

    1. Reviewer #1 (Public Review):

      Mature mammalian hair cells in the cochlea do not regenerate after damage. The outer hair cells of the cochlea, which function to amplify sound, are particularly susceptible to damage. Ectopic activation of two key transcription factors for outer hair cell formation, Atoh1 and Ikzf2, in damaged adult cochlea is sufficient to convert supporting cells into hair cells expressing Prestin, which is an essential protein mediating outer hair cell functions. Although there is no functional recovery in these transgenic mice based on auditory brainstem response, this study paves the way for future design of models for hearing recovery. The main concern is the identity of the OHC-like cells drawn from the small sample size in the scRNA-seq experiments.

    1. Reviewer #1 (Public Review):

      The presented manuscript takes a comprehensive and elaborated look at how T cell receptors (TCR) discriminate between self and non-self antigens. By extending a previous experimental protocol for measuring T cell receptor binding affinities against peptide MHC complexes (pMHC), they are able to determine very low TCR-pMHC binding affinities and, thereby, show that the discriminatory power of the TCR seems to be imperfect. Instead of a previously considered sharp threshold in discriminating between self and non-self antigen, the TCR can respond to very low binding affinities leading to a more transient affinity threshold. However, the analysis still indicates an improved discrimination ability for TCR compared to other cell surface receptors. These findings could impact the way how T cell mediated autoimmunity is studied.

      The authors follow a comprehensive and elaborated approach, combining in vitro experiments with analytical methods to estimate binding affinities. They also show that the general concept of kinetic proofreading fits their data with providing estimates on the number of proofreading steps and the corresponding rates. The statistical and analytical methods are well explained and outlined in detail within the Supplemental Material. The source of all data, and especially how the data to analyze other cell surface receptor binding affinities was extracted, are given in detail as well. Besides being able to quantify TCR-pMHC interactions for very low binding affinities, their findings will improve the ability to assess how autoimmune reactions are potentially triggered, and how potent anti-tumour T cell therapies can be generated.

      In summary, the study represents an elaborated and concise analysis of TCR-pMHC affinities and the ability of TCR to discriminate between self and non-self antigens. All conclusions are well supported by the presented data and analyses without major caveats.

    1. Reviewer #1 (Public Review):

      The authors demonstrate applications including fluorescent marking of membranes with GFP or monomeric RFP, reporter alleles for convenient assessment of differentiation status based on fluorescence, and targeted gene knockout. They also demonstrate conditional gene knockdown and induction with tight control achieved by engineering a protein destabilizing domain. The design of the constructs is clever and imparts the ability to leverage iterative FACS to enrich successfully targeted cells, particularly useful when targeting alleles that are not actively expressed by the progenitors. The work is well done and clearly presented.

    1. Reviewer #1 (Public Review):

      In this study, Moncla et al. used genomic data to analyse a mumps outbreak in Washington, in order to draw inferences about the epidemiological factors driving the outbreak. Some important strengths of the analysis include sophisticated sequencing and modeling techniques to reconstruct chains of transmission during the outbreak, which support the conclusions that the mumps virus was introduced several times in Washington from other North American regions during the outbreak, and that the Washington Marshallese community was particularly at risk of mumps infection and transmission during this time. Limitations of the analysis include potential for sampling bias, where the sample may not be entirely representative of mumps outbreak cases, and a sample size that is too low to allow sufficient statistical power to assess the impacts of age and vaccination status on transmission. The work has potential public health impacts in terms of identification of a vulnerable community and points to social networks as the primary risk factor for potential future respiratory virus outbreaks. The analysis methods could be potentially applied for the phylodynamic analysis of other infectious disease outbreaks.

    1. Reviewer #1 (Public Review):

      It is difficult to overestimate the importance of this paper. The full connectome of the Drosophila central complex is both the beginning and the end of an era. It provides the first comprehensive dataset of arguably the most enigmatic brain region in the insect brain. This endeavor has generated ground truth data for years of functional work on the neural circuits the connectome outlines, and constitutes an unparalleled foundation for exploring the structure function relations in nervous systems in general. This will be of great importance far beyond work on the Drosophila brain, and will have far reaching implications for comparative research on insect brains and likely also smoothen the path toward understanding navigation circuits in vertebrate nervous systems. Based on presented data, the paper develops overarching ideas (at exquisite detail) of how sensory information is transformed into head direction signals, how these signals are used to enable goal direction behavior, how goals are represented, and how internal state can modulate these processes. The connectome enables the authors to base these ideas and their detailed models on actual biological data, where earlier work was forced to indirectly infer or speculate. While significantly going beyond models of central-complex function that existed previously, the authors have to be much credited for incorporating huge amounts of existing knowledge and data into their interpretations, not only work from Drosophila, but also from many other insects. This makes this paper not only an invaluable resource on the connectome of the Drosophila central complex, but also a most comprehensive review on the current state of the art in central-complex research. This unifying approach of the paper clearly marks a reset of central-complex research, essentially providing a starting point of hundreds of new lines of enquiry, probably for decades to come.

      Given the type and amount of data presented, the paper is clearly overwhelming. That said, it also clearly needs to be presented in the way it was done, mostly because no single aspect of the function of this neuropil makes as much sense in isolation as it makes sense when viewed in conjunction of all its other functions. The complexity of the neural circuits discussed is clearly reflected in the enormous scope of the paper. Nevertheless, the authors have done a fantastic job in breaking the circuits and their function down into digestible bits. The manuscript is very systematic in its approach and starts with sensory pathways leading to the CX, covering the clearly delineated head direction circuits and then moving on to the more complex and less understood parts, always maintaining a clear link between structure and function. As function is necessarily based on previous work, including that from other species, the results part is interwoven with interpretation, but this is clearly necessary to keep the text readable. The authors have made considerable efforts to provide additional introductions and summaries whenever needed, almost creating nested papers embedded within the overall paper.

      The figures are equally overwhelming as the text at first sight, but when taking the time to digest each one in detail, they present the data in a rich and clear manner. The figures are often encyclopedic and will serve as reference about the central complex for years. The summary graphs that are presented in regular intervals are welcome resting places for the reader, helping to digest all the detailed information that has preceded or that will follow.

      The analysis performed in the paper is excellent, comprehensive and should set the standard for any future work on this topic. Also, the text is very honest about the limits of the conclusions that can be reached based on this kind of data, which is important in generating realistic and feasible hypotheses for future experiments.

    1. Reviewer #1 (Public Review):

      Brascamp and colleagues address pupil-size changes around perceptual switches in perceptual multistability. Several previous studies have found pupil dilation around or after the switch and some have found pupil constriction, though the latter was typically less robust. Moreover, while most previous studies included some controls for the effect of reporting and for the physical stimulus change, to my knowledge, so far, no study has fully crossed the factors report/no-report and endogenous/exogeneous switch. In the present study, this gap is filled using a binocular-rivalry stimulus and an OKN-based no-report paradigm. This allows the authors to isolate the constriction component from the dilation component and interestingly they find the constriction more robustly tied to the perceptual switch, while the dilation component is mostly related to the response. Experiments are soundly conducted and analysed and results are interpreted with appropriate care. Since the results challenge frequent interpretations as to why perceptual switches in multistability may cause pupil-size changes, the paper is of high relevance to the fields of pupillometry and multistability, but also to other areas where pupillometry is used as index of perceptual and cognitive processes. I only have some minor questions and requests for clarification with regard to result presentation and interpretation.

    1. Reviewer #1 (Public Review):

      Strengths and Weaknesses. The authors did quite a lot to establish gene expression and function of the annelid's trunk cells and compare them to photoreceptors of the annelid's eye. They isolated the cells with FACS and characterized gene expression in detail, they knocked down r-opsin with TALEN in the trunk and found a significant difference in a crawling response, and they express the opsin in cell culture to confirm wavelength and G-protein sensitivity. As a potential link between light sensitivity and mechano-sensitivity, they report r-opsin function and light intensity influence expression of atp2b2, a gene that modulates neuronal sensitivity in other organisms. Wavelength and G-protein activation data are valuable because I can think of few or no other organisms in the entire group of lophotrochozoan animals, where this level of experimental manipulation could be done. In short, a strength of this manuscript is the detailed characterization of the trunk receptor cells, which express r-opsins. The authors have brought much evidence to the claim that these TRE cells have both light and mechano-sensitive gene expression and function. Based on these findings in an annelid worm, I believe the paper is a significant advance, and of interest to a broad audience by adding to a growing set of discoveries of similar hybrid sensory cells.

      If a hybrid mechano/photo-receptor is indeed an ancient cell type in bilaterians, this would bring many evolutionary implications for sensory biology. However, in these evolutionary interpretations is where I find a weakness of the manuscript. Namely, with only a handful of species shown thus far to have the hybrid cell type - and many differences in detail about these cell types in different organisms - we can not yet make firm conclusions about whether the multi-functional cells were ancestral. I believe other interpretations are equally valid (and still interesting) and should be given more consideration. Namely, it seems possible that photo- and mechan- sensory processes "joined forces" (e.g. through separate co-option events) in new cell-types, multiple times during evolution. The current manuscript loosely indicates ancestral multi-functionality is more parsimonious. However, no detail is given about that. I suppose the authors mean a single origin of hybrid cell types requires fewer evolutionary transitions than multiple origins. However, such a parsimony count does not count the transitions requiring loss of phototransduction in mouse hearing and do not count transitions to loss of mechanosensitivity in eye photoreceptor cells.

    1. Reviewer #1:

      The authors have acquired a substantial multimodal dataset and have used careful statistical approaches throughout. The data are acquired and analysed using appropriate methods.

      Overall, this is an impressive body of work that aims to answer an interesting question. However, a number of questions over the methods and interpretation make the authors' conclusions difficult to justify.

      When comparing between older and younger adults it would also be helpful to know the amount of grey matter in the voxels of interest. It might be expected that older adults might have more atrophy and therefore lower GABA+, than younger adults and this should be controlled for in the statistical models. The authors have put assumptions into their quantification, which are reasonable but are still assumptions. It would be helpful to directly test for a difference in grey matter fraction in the voxel between the two groups, and include this in the model if necessary.

      The authors then look at behaviour, where they use a previously described task which consists of bimanual tapping, with switching between two patterns. The results are complex as there are a number of behavioural metrics, and no clear pattern emerges. While older adults produced more errors in continuation, they also produced more fully correct switching transitions. Older subjects were slower than younger adults in all trials. While this task produces a very rich dataset, which is helpful for analysing complex behaviour, it is not clear how each metric should be interpreted in terms of the underlying neural mechanisms, and how they can be usefully combined, could be given.

      In terms of connectivity, the authors found no significant group X task difference between in-phase and anti-phase conditions. They therefore look at the groups and tasks separately. They show different changes in connectivity between age groups in different frequency bands, for example between left and right M1 in the alpha/mu and beta, between EMG and left M1 in the theta band. I am not sure that describing EEG-EMG connectivity as cortico-spinal is strictly accurate - there may be a number of other factors in this -corticomuscular would seem to be more precise. The frequency bands used are not typical, and it would be helpful to have an a priori explanation of which are being tested and why - as well as details about correction for multiple comparisons across these bands.

      Finally, the authors bring their GABA, behaviour and connectivity metrics together in a number of mediation analyses. They demonstrate a relationship between cortico-cortical connectivity and behaviour, which is mediated by age.

      The authors describe their finding of higher GABA+ in the occipital cortex as a posterior-anterior gradient, which I think is not justified by the results - there could be a number of other reasons for this, for example that different functional networks have different GABA+ levels, which is not related to their anatomical position. With only three voxels it is difficult to make a general claim such as this, and this should probably be reworded.

      The authors state that higher GABA+ indicated neural system integrity and better functioning in the older group. This seems to be rather over-interpreting their results - there are many other metrics of integrity and functioning that have not been assessed here. I would suggest rewording.

    1. Reviewer #1 (Public Review):

      This paper presents the exciting statement that increasing viral loads within a community can be used as an epidemiological early-warning indicator preceding increased positivity. It would be interesting to support this claim to present both Ct and positivity on the same graph to demonstrate that indeed, declining Ct can be used as an early marker of a COVID-19 epidemic wave. Percentage of positive test data should not only include the ones obtained in the present study but should be compared with "national data" as the present study design includes a bias in patients selection that might not reflect the "true" situation at the time. Only with this comparison, we could claim that the present study design could predict COVID-19 epidemic waves. A correlation of Ct with clinical evidence to rank the confidence of positive results is also included and further support the high specificity of the RT-PCR for detecting SARS-CoV-2 (99.995%).

      In a serological investigation, it was observed that some of these RT-PCR-positive cases do not appear to seroconvert and that possible re-infections might occur despite the presence of anti-spike antibodies. Although, reported on few individuals and therefore to be taken with extreme caution, this add some piece of information to the current unknown of the serological response of COVID-19 patient and would be of uttermost importance in the context of the current vaccination campaign.

    1. Reviewer #1 (Public Review):

      Sensorimotor integration is required for the accurate execution of volitional movements, but the neural circuits underlying sensorimotor integration are still not fully understood. The whisker system of the rodent has emerged as one model of sensorimotor integration with many recent studies focused on the synaptic organization of the underlying circuitry. Here, Yamawaki et al report results regarding the synaptic organization of the ascending sensory pathways related to mouse forelimb somatosensory and motor cortex. Using anatomical and functional approaches, they elucidate the circuitry from the cuneate nucleus through thalamus to forelimb S1 and M1. This work complements recent studies in the mouse of other aspects of the forelimb sensorimotor pathways and leads to informative comparisons to the circuit organization of the whisker system. The studies are well executed and well explained. The use of multiple approaches compensates for the limitations of each individual technique, although some limitations such as any effects of viral tropism are difficult to overcome. Overall, this work contributes to a better understanding of the wiring diagram of sensorimotor circuits in the mouse.

    1. Joint Public Review:

      Worker bees perform specialised tasks: young workers nurse larvae, older ones forage for either nectar or pollen. Behaviours - including these specialist ones - arise when a stimulus (nectar, pollen or larvae) exceeds a certain 'response threshold' of the organism. This threshold can be modulated by neuropeptides to alter behaviour.

      The study first shows that response thresholds to task-related stimuli differ among nurse bees, nectar and pollen foragers. Pollen foragers are most responsive to sucrose and pollen, and nurse bees most responsive to chemical stimuli of larvae. Then, taking a proteomic approach, they identify a neuropeptide, Tachykinin related protein (TRP), to be expressed in a task-specific pattern: low in nurse bees and highest in the nectar foragers.

      This work provides valuable resource information on the abundance of brain neuropeptides in two species of bees. The study is exceptional in its breath of techniques used and the addition of manipulative experiments which are difficult to do in honey bees. Through their studies the authors identify a neuropeptide that modulates response thresholds of bees.

      The study would have been exceptional if the authors had included studies on the expression of the tachykinin receptor. The level of tachykinin expression increases between nurse bees and foragers, but does not involve changes in spatial expression (Takeuchi et al., 2004 ref. 56). So, it is likely that the specificity of the effects of tachykinin are due to differences in the spatial expression of the receptor.

    1. Reviewer #1 (Public Review):

      The authors developed a very interesting tool, named NICEdrug.ch, used it to identify drug metabolism and toxicity, and finally predicted druggability of disease-related enzymes and reposition drugs. Comprehensive integration effort based on publicly available datasets and several previous methods developed by the authors (e. g. BridgeIT, BNICE.ch, ATLAS of Biochemistry) results with a resource named NICEdrug.ch. The idea is interesting and addresses a very important problem in the field. The manuscript is clearly written, provides enough analysis of overall challenges and an overview of the most important results. Also, it presents figures that are remarkable.

    1. Reviewer #1 (Public Review):

      Kinetochores are huge protein assemblies on chromosomes which are used as attachment point for microtubules and allow microtubules to pull chromosomes into daughter cells during cell division. The proteins that form the kinetochore are well known, but the temporal regulation of the assembly of all these proteins into functional kinetochores is less understood.

      In this paper the authors have identified phosphorylation sites in the 'CCAN' of budding yeast, the 'inner', i.e. chromatin-proximal, part of the kinetochore. They characterize in detail the function of phosphorylation of Ame1 (CENP-U in humans), which is part of CCAN. The data support the idea that a cluster of phosphorylation sites in Ame1 is phosphorylated by mitotic CDK1 and serves as phospho-degron for the E3 ligase SCF/Cdc4.

      The authors show phosphorylation of these CDK1 consensus sites in vivo and their phosphorylation by CDK1/Clb2 in vitro. Genetic experiments and molecular dynamics simulations support the idea that phosphorylation sites on Ame1 can serve as phospho-degron for SCF/Cdc4. Even the non-phosphorylatable mutant of Ame1 is stabilized in an SCF mutant background, though, suggesting that this phospho-degron is not the only way in which SCF influences kinetochore protein levels.

      Mutants in the characterized phosphorylation sites do not impair budding yeast growth. This suggests that the degron characterized in this paper may be important for fine-tuning, but is not essential for the proper execution of mitosis. The observations overall add to prior evidence that kinetochore assembly can be regulated by phosphorylation and/or ubiquitination.

      Interestingly, the authors find that phosphorylation of Ame1 by CDK1 in vitro is impaired when Ame1 binds Mtw1, another kinetochore protein. The fact that Mtw1 seems to shield these sites from phosphorylation leads the authors to put forward an interesting model: they propose that cell cycle-dependent phosphorylation and SCF-dependent degradation of kinetochore subunits allows for excess subunits during kinetochore assembly in S-phase (which will speed up assembly) while depleting any excess subunits after assembly, when the kinetochore needs to be functional.

      This is an interesting model. The in vivo evidence is still limited, though. For now, it remains unknown whether the phosphorylation status of kinetochore-bound and free Ame1 is indeed different, whether more soluble Ame1 exists in S-phase, whether too early degradation of Ame1 (or possibly other kinetochore proteins) indeed impairs kinetochore assembly, or whether a failure to remove the soluble pool after assembly leads to mitotic defects. It is an attractive proposal, though, that can now be further explored experimentally.

      In addition to the specific characterization of Ame1 sites, the paper also includes comprehensive data on CCAN phosphorylation sites obtained by mass spectrometry which can serve as basis for future studies.

    1. Reviewer #1 (Public Review):

      The manuscript is somewhat readable but the many acronyms for the cell types in model and biology make it difficult to follow. Is there a reason why the biological neuron names cannot be used in the model? The presentation of data in figures can be more powerful. In many cases, the data in figures and the supplemental videos show apparently different results. This can be an artifact of how the videos were made and if yes, these can be improved. Tail tip coordinates can be plotted to show the behaviors in much better detail.

      Especially for beat and glide swimming, the points regarding burst firing, inhibition, etc. have not been robustly made.

    1. Reviewer #1 (Public Review):

      The study presents relatively high and robust sensitivity of Abbott ID NOW for the detection of SARS-CoV-2 (COVID-19) in an ambulatory population, utilizing the RT-PCR methodology as a comparative correlation. The study was well designed and enrolled both symptomatic and asymptomatic populations to provide sufficient statistical power for the comparative analysis of the methodologies, as well as to represent accurately the patient populations. This is a useful and timely study that has a great impact in clinical setting for the rapid detection of COVID-19.

    1. Reviewer #1 (Public Review):

      The goal of this manuscript is to develop gene-agonistic approaches for promoting cone survival in retinal degenerative diseases. Based on their previous studies, the authors tested a total of 20 genes by subretinal delivery using an AAV vector which utilized a cone-specific promoter. Most of these genes augmented glucose utilization. Interestingly, only Txnip showed a positive result by prolonging cone survival (tested up to 50 days in rd1 retina). Txnip therapy also appears to be effective in rd10 and rho-/- retina. Additional strength of this study is the use of Txnip C247S allele that blocks its association with thioredoxin. Furthermore, additional work on how Txnip may contribute to cone survival by better utilization of lactate for energy is well presented though the conclusion on "heathier" mitochondria require additional data. This manuscript is potentially of great interest. The data are extensive and biological implications of the study are clear. However, the broad conclusions with respect of Txnip therapy for RP (or even AMD) are less than justified based on the data. Two weaknesses are apparent: the first is related to the method of quantification using whole mount retina, and the second related to the duration of the study. Immunostainings of retinal sections (and even TEMs) are critical to elucidate the structure of surviving cone photoreceptors (specially in the absence of rods) and their relationship to other cells (e.g., RPE, bipolar cells, glia). Similarly, Prusky's OMR can't be equated to visual acuity. The authors need to show cone structure/function at P50 and beyond (how long do the cones survive?) in rd1 and other models before claiming the potential benefit of Txnip for retinal and macular degeneration.

    1. Reviewer #1 (Public Review):

      Oon and Prehoda report pulsatile contraction of apical membrane in the process of Par protein polarization in Drosophila neuroblasts. This explains how/why actin filament was required to localize/polarize Par complex. Specifically, using spinning disc confocal microscopy with high temporal resolution, they found the directed actin movement toward the apical pole, which nicely correlates with concentration of aPKC. They also show that myosin II is involved in this pulsatile movement of actin filament. This very much resembles the observation in C. elegans embryos, and nicely unifies observations across systems. Although descriptive in nature, I think this is an important observation and indicates a universal mechanism by which cells are polarized. I think this is a well executed study.

    1. Reviewer #1 (Public Review):

      The primary objective of this manuscript was to examine if multi-kinase inhibitor YKL-05-099 can inhibit salt inducible kinases (SIKs) with the goal to examine a new class of bone anabolic agents for the treatment of osteoporosis. They found that YKL-05-099 was successful in increasing anabolism and, surprisingly, decreasing bone resorption, leading them to investigate why this inhibitor differed from the effects of deletion of SIK2 and SIK3. They found that YKL-05-099 also inhibited the CSF1 (M-CSF) receptor, thus, inhibiting osteoclast activity. This is an interesting manuscript but there are some flaws in the conduct of the experiments and in the analyses which lessen its impact. Nevertheless, it opens the way for another possible oral therapeutic for osteoporosis.

    1. Reviewer #1 (Public Review):

      One of the most consistent and thus surprising patterns revealed by experimental evolutionary studies is the observation of a very predictable pattern of increase in fitness of replicate populations. The fitness increase tends to be very rapid at the beginning and then slows down but continues to increase for tens of thousands of generations (e.g. the Lenski LTEE). The studies from the Desai group specifically two: one by Kryazhmisky et al and one by Jonnson et al further established that the pattern of decrease in the fitness gain is due to really counterintuitive patterns of global epistasis. In particular it is not due to the evolution running out of adaptive mutations but rather to the fact that the same adaptive mutations are less beneficial on fitter backgrounds (Kryazhmisky et al). Johnson et al further found that the fitter backgrounds are more fragile with deleterious mutations being more deleterious on fitter backgrounds. All of this is rather bizarre at first glance as the microscopic epistasis is known to be highly idiosyncratic.

      This paper, along with one by Lyons et al (Nat Ecol Evol 2020), resolves this paradox and shows that the observed pattern of global epistasis is in fact directly dependent on microscopic epistasis being widespread, involving multiple loci - with most parts of the organisms being connected in an "everything affecting everything" pattern, and being idiosyncratic. The Lyons et al paper focused on the data showing the epistasis is in fact idiosyncratic - their key observation - and provided an intuition for why such widespread idiosyncrasy would result in the observed pattern of global epistasis. Although neither set of authors seems to use this term, this should fit the notion of the Anna Karenina principle: "All happy families are alike; each unhappy family is unhappy in its own way." That is, in order for the right things to happen, most things need to go right, but in order for things to fail, anyone of many such things can go wrong. The more adapted systems are more fragile and more difficult to improve, because in both cases it is easier to disrupt what is already working.

      The Reddy and Desai paper takes this notion and develops a very simple and transparent quantitative theory of this principle that generates specific quantitative predictions about the dynamics of adaptation that we, as a field, will spend considerable time now testing. The work has the potential to become a seminal paper in the field.

    1. Reviewer #1 (Public Review):

      This study builds upon previous findings by the authors and others that the Hedgehog (Hh) co-receptor Ihog not only binds Hh to trigger Hh signal transduction, but also engages trans-homophilic interactions in cell-cell adhesion. Using experimental manipulation and mathematical modeling, the authors assessed the role of Ihog trans-homophilic binding in stabilizing cytoneme structure and the relative strengths of Ihog-Ihog and Hh-Ihog binding. These findings led to a model whereby the weaker Ihog-Ihog trans interaction promotes direct membrane contacts along cytonemes and that Hh-Ihog binding releases Ihog from trans Ihog-Ihog complex. The studies are well designed and executed, and the findings are convincing.

    1. Reviewer #1:

      In this manuscript, Fernandez et al examine the impact of defective telomere length maintenance on type II alveolar epithelial cells, which are thought to be central to the pathogenesis of pulmonary fibrosis in dyskeratosis congenita (DC) and related telomere biology disorders. Murine models have been used to address how telomere dysfunction in AT2 cells drives pulmonary fibrosis however these models have limitations. Therefore, the investigators' study of human AT2 cells/organoids derived from induced pluripotent stem cells (iAT2 cells) in the presence and absence of a known DC pathogenic variant provides an exceptional model. In addition, the investigators use expression profiling to uncover decreased canonical WNT signaling in iAT2 cells with telomere dysfunction and then demonstrate rescue of telomere dysfunction and iAT2 cell growth with the addition of a GSK3 inhibitor, a canonical WNT agonist. The data appear to be of high quality, the approaches and interpretation appropriate, with some noted exceptions below. Given the importance of the problem (dysfunctional telomere-induced pulmonary fibrosis) and the apparent benefit of GSK3 inhibition of iAT2 cell growth and telomere dysfunction, which extends the work published by this group previously on intestinal organoids (might enhanced canonical WNT signaling more broadly affect other tissues with telomere-induced senescence?), this work is significant.

      A few aspects of the studies dampen the ability to draw certain conclusions. For example, the authors use iPSCs that are 5 vs 25 passages after introduction (or not) of the DKC1 A386T mutation for the generation of iAT2 cells. They then show iAT2 DKC1 mutant organoids generated from the later passage iPSCs have an apparent growth defect as early as Day 50 but that those generated from the earlier passage iPSCs do not at Day 70 [with caveats the images are of different quality (comparing Fig. 1B and Fig. S3D) and quantitative data (similar to Fig. 1C) are lacking for the iAT2 organoids generated from the early passage iPSCs]. They argue that progressive telomere shortening is the cause of the growth defects. If this is the case, then the iAT2 cells generated from the earlier passages should eventually show growth defects with progressive telomeres shortening, which was not shown.

      The telomere length analysis of the iAT2 cells at Day 50 and Day 70 are not markedly different, and neither the % p21 + nor TIF+ cells is shown for Day 50. Therefore, the conclusion that it is the accumulation of short uncapped telomeres in the DKC1 mutant iAT2 cells that alters gene expression and induces senescence at Day 70 ignores the extent of these changes at Day 50.

      The statement that CHIR99021 (when present in the medium from Day 49-70) rescued the growth defect seems generous; the effect is partial and the assay is for organoid formation efficiency only. Moreover, it is most likely prohibiting the further accumulation of senescent cells rather than rescuing cells that were not previously growing.

      It is striking that prolonged CHIR99021 treatment (ie, through to Day 70) resulted in increased telomerase activity, and more so in mutant compared to wild type cells. First, how reproducible was this effect? I appreciate that the authors have not explored this for this manuscript, however, TERT expression does not rescue DKC1 mutants but TERC does. Were TERC levels increased? Also, given this robust increase, it is striking that no difference is detected in TeSLA assays given the proportion of very short detected telomeres that would presumably be substrates for telomerase. It is noteworthy that, in the protocol to derive iAT2 cells, CHIR99021 is present in the media prior to Day 28. This raises the question of whether there is rescue of telomerase in the cells exposed to CHIR99021 in the interval of iAT2 specification?

    1. Reviewer #1 (Public Review):

      The authors used a CRISPR screen to investigate the basis of metastasis of ovarian cancer (OC) cells. Overall, they identified two key genes, IL20RA, one of which was studied in detail. They identify an IL20/IL20RA communication between ovarian cancer cells and peritoneal mesothelial cells to promote M1 macrophages and prevent dissemination of the cancer cells. IL-20 mediated crosstalk is blocked in metastasized OC cells by decreased expression of IL-20RA. Interestingly, IL20RA is also decreased in cells from OC patients with peritoneal metastasis, and reconstitution of IL20RA in metastatic OC cells suppresses metastasis. Moreover, OC cells induce mesothelial cells to produce IL20 and IL24.

      Overall, this is a nice study. It is well-written, and the data are clear. A range of methodologies are used that support the conclusions, with both over-expression and under-expression related studies supporting some key conclusions.

      The overall model is that there is crosstalk between disseminated OC cells and mesothelial cells and macrophages. OC cells when disseminated into the peritoneal cavity stimulate mesothelial cells to produced IL20 and IL24, which via IL20RA trigger STAT3 to produced OAS/RNase L and production of IL-18, to promote an M1 phenotype. The M1 phenotype lowers metastasis. Highly metastatic cells block this pathway by decreasing IL20RA expression.

      These findings are interesting, with potential therapeutic ramifications.

    1. Reviewer #1 (Public Review):

      In this work, Panigrahi et. al. develop a powerful deep-learning-based cell segmentation platform (MiSiC) capable of accurately segmenting bacteria cells densely packed within both homogenous and heterogeneous cell populations. Notably, MiSiC can be easily implemented by a researcher without the need for high-computational power. The authors first demonstrate MiSiC's ability to accurately segment cells with a variety of shapes including rods, crescents and long filaments. They then demonstrate that MiSiC is able to segment and classify dividing and non-dividing Myxococcus cells present in a heterogenous population of E. coli and Myxococcus. Lastly, the authors outline a training workflow with which MiSiC can be trained to identify two different cell types present in a mixed population using Myxococcus and E. coli as examples.

      While we believe that MiSiC is a very powerful and exciting tool that will have a large impact on the bacterial cell biological community, we feel explanations of how to use the algorithm should be more greatly emphasized. To help other scientists use MiSiC to its fullest potential, the range of applications should be clarified. Furthermore, any inherent biases in MiSiC should be discussed so that users can avoid them.

      Major Concerns:

      1) It is unclear to us how a MiSiC user should choose/tune the value for the noise variance parameter. What exactly should be considered when choosing the noise variance parameter? Some possibilities include input image size, cell size (in pixels), cell density, and variance in cell size. Is there a recommended range for the parameter? These questions along with our second minor correction can be addressed with a paragraph in the Discussion section.

      2) Could the authors expand on using algorithms like watershed, conditional random fields, or snake segmentation to segment bacteria when there is not enough edge information to properly separate them? How accurate are these methods at segmenting the cells? Should other MiSiC parameters be tuned to increase the accuracy when implementing these methods?

      3) Can the MiSiC's ability to accurately segment phase and brightfield images be quantitatively compared against each other and against fluorescent images for overall accuracy? A figure similar to Fig. 2C, with the three image modalities instead of species would nicely complement Fig. 2A. If the segmentation accuracy varies significantly between image modalities, a researcher might want to consider the segmentation accuracy when planning their experiments. If the accuracy does not vary significantly, that would be equally useful to know.

      4) The ability of MiSiC to segment dense clusters of cells is an exciting advancement for cell segmentation algorithms. However, is there a minimum cell density required for robust segmentation with MiSiC? The algorithm should be applied to a set of sparsely populated images in a supplemental figure. Is the algorithm less accurate for sparse images (perhaps reflected by an increase in false-positive cell identifications)? Any possible biases related to cell density should be noted.

      5) It is exciting to see the ability of MiSiC to segment single cells of M. xanthus and E. coli species in densely packed colonies (Fig. 4b). Although three morphological parameters after segmentation were compared with ground truth, the comparison was conducted at the ensemble level (Fig. 4c). Could the authors use the Mx-GFP and Ec-mCherry fluorescence as a ground truth at the single cell level to verify the results of segmentation? For example, for any Ec cells identified by MiSiC in Fig. 4b, provide an index of whether its fluorescence is red or green. This single-cell level comparison is most important for the community.

    1. Reviewer #1 (Public Review):

      In this paper, authors did a fine job of combining phylogenetics and molecular methods to demonstrate the parallel evolution across vRNA segments in two seasonal influenza A virus subtypes. They first estimated phylogenetic relationships between vRNA segments using Robinson-Foulds distance and identified the possibility of parallel evolution of RNA-RNA interactions driving the genomic assembly. This is indeed an interesting mechanism in addition to the traditional role for proteins for the same. Subsequently, they used molecular biology to validate such RNA-RNA driven interaction by demonstrating co-localization of vRNA segments in infected cells. They also showed that the parallel evolution between vRNA segments might vary across subtypes and virus lineages isolated from distinct host origins. Overall, I find this to be excellent work with major implications for genome evolution of infectious viruses; emergence of new strains with altered genome combination.

      Comments:

      I am wondering if leaving out sequences (not resolving well) in the phylogenic analysis interferes with the true picture of the proposed associations. What if they reflect the evolutionary intermediates, with important implications for the pathogen evolution which is lost in the analyses?

      Lines 50-51: Can you please elaborate? I think this might be useful for the reader to better understand the context. Also, a brief description on functional association between different known fragments might instigate curiosity among the readers from the very beginning. At present, it largely caters to people already familiar with the biology of influenza virus.

      Lines 95-96 Were these strains all swine-origin? More details on these lineages will be useful for the readers.

      Lines 128-132: I think it will be nice to talk about these hypotheses well in advance, may be in the Introduction, with more functional details of viral segments.

      Lines 134-136: Please rephrase this sentence to make it more direct and explain the why. E.g. "... parallel evolution between PB1 and HA is likely to be weaker than that of PB1 and PA" .

      Lines 222-223: Please include a set of hypotheses to explain you results? Please add a perspective in the discussion on how this contribute might to the pandemic potential of H1N!?.

      Lines 287-288: I am wondering how likely is this to be true for H1N1.

    1. Reviewer #1 (Public Review):

      In this paper, the authors tried to investigate complex roles of immune cells during acute myocardial infarction (AMI) by examining immune cells in blood samples from acute coronary syndrome (ACS) patients. They found an increase in the circulating levels of CD14+HLA-DRneg/low monocytes and CD16+CD66b+CD10neg neutrophils in the blood of ACS patients compared to healthy people, all of which were correlated with elevated levels of inflammatory markers in serum. Those findings were then further explored at a mechanistic level by using in vitro and in vivo experiments. Interestingly, the researchers also found that high cytomegalovirus (CMV) antibody titers could affect the immunoregulatory mechanisms in AMI patients. Taken together, the findings of the researchers could potentially contribute to the development of a more effective strategy to prevent cardiac deterioration and cardiovascular adverse events after AMI.

      Strengths:

      This paper contains novel insight regarding role of neutrophil and monocyte subset in pathophysiology of AMI. Although the increased level of CD10neg subsets of neutrophils in AMI patients has recently been reported (Marechal, P., et al. 2020. Neutrophil phenotypes in coronary artery disease. Journal of Clinical Medicine), the current paper aptly complemented the previous findings obtained by using its in vitro and in vivo mice model. This study also has robust methods to support their conclusion.

      Weakness:

      To further improve the strength of their conclusion, the experiments investigating the effects of immunoregulatory function of immature neutrophils and HLA-DRneg/low monocytes subsets would be advised.

    1. Reviewer #1 (Public Review):

      The manuscript by He et al. reveals a novel role for PKC-theta, following T cell receptor (TCR) stimulation, in regulating the nuclear translocation of several key activation-dependent transcription factors by regulating the assembly of key components of the nuclear pore complex (NPC). The authors make use of T cell lines and primary T cells to show that following TCR stimulation, PKC-theta phosphorylates RanGAP1 to promote its interaction with Ubc9 and increase the sumoylation of RanGAP1, which, in turn, enhances assembly of the RanBP2 subcomplex of the NPC that then promotes the nuclear import of AP-1, NFAT and NFB. These conclusions are well supported by a rigorous experimental approach, which included the use of PKC-theta deficient, sumoyltion-defective, kinase-dead, and constitutively active mutants, and RanGAP1-deficient cells.

    1. Reviewer #1 (Public Review):

      This paper uses a large breeding colony of guppies to measure genetic correlations between hormonal stress responses and behavior in an open-field test. Although we know a lot about the mechanisms of hormone-mediated behavior, we know less about variation in hormonal systems, particularly genetic variation. Understanding how hormones relate genetically to the behaviors they mediate is particularly important because it helps us understand how the entire hormone-behavior system evolves. A priori, we would expect genetic correlations between hormones and the behavior they underlie, such that selection on the hormone would lead to a response in the behavior and vice versa. However, evidence for this pattern is rare.

      Here, the authors show that stress-induced levels of cortisol are repeatable and heritable. Interestingly, they also show that individuals show a lower stress response to later stress and slightly less variation, indicating a G X E interaction. There was a significant genetic correlation between the hormonal response and one of the behaviors measured in the open field test, and the hormone loaded positively in the first genetic principal component along with all the behaviors. This is evidence of an correlated suite of traits that would evolve together in response to selection.

      This is an important study, because evidence of genetic variation in hormonal systems, not to mention genetic covariation with hormone-mediated traits, is rare. The results presented here provide insight into how a hormone-behavior complex might adapt to a changing environment. They are also relevant to ideas about the maintenance of variation in coping styles in natural populations.

    1. Reviewer #1 (Public Review):

      Using various voltage and concentration protocols in a heterologous expression system, the authors provide compelling evidence for strong block of GluR1 AMPA receptors by intracellular NASPM, and unlike spermine, the block is independent of auxiliary subunit expression. The authors also show that intracellular NASPM provides a more complete block than spermine of synaptic currents in GluR2-KO neurons.

      Overall the manuscript contains high quality data that is clearly presented. It seems likely that this approach will be useful for assessing the contribution of CP-AMPARs in various scenarios. However, currently the authors have fallen short of providing a comprehensive analysis of the use of NASPM to differentiate between CP and CI AMPARs in intact systems containing multiple AMPAR subunits and auxiliary proteins.

    1. Reviewer #1 (Public Review):

      In "Asymptomatic Bordetella pertussis infections in a longitudinal cohort of young African infants and their mothers", the authors analyze longitudinal data from a cohort in Zambia of infant/mother pairs to investigate the evidence for subclinical and asymptomatic infections in both pairs as well as the use of IS481 qPCR cycle threshold (CT) values in providing evidence for pertussis infection. Overall, the manuscript lacks substantial statistical support or clear evidence of some of the patterns they are stating and would require a substantial revision to justify their conclusions. The majority of the manuscript relies on 8 infant/mother pairs where they have evidence of pertussis infection and rely on the dense sampling to investigate infection dynamics. However, this is a very small sample size and further, based on the results displayed in Figure 1, it is not obvious that the data has a very pattern that warrant their assertions.

      Major comments:

      The main results and conclusions are highly reliant on details from eight mother/infant pairs. However, Figure 1 does not show a clear picture of the fade-in/fade-out. The authors go into great detail describing each of these 8 pairs, however based on the figure and text there does not appear to be clear evidence of an underlying pattern. While there are some instances with a combination of higher/lower IS481 CT values, it does not appear to have a clear pattern. For example, what are possible explanations for time periods between samples with evidence of IS481 and those without (such as pair A, C, D, E, F and H)? There also does not appear to be a clear pattern of symptoms in any of these samples (aside from having fewer symptoms in the mothers than infants). Further, it is not obvious how similar these observed (such as a mixture of times of high or low values often preceded or followed by times when IS481 was not detected) is similar to different to the rest of the cohort (in contrast to those who have a definitive positive NP sample during a symptomatic visit). The main results are primarily a descriptive analysis of these 8 mother/infant pairs with little statistical analyses or additional support.

      The authors do not provide evidence or detail about what is known about the variability in IS481 CT values, amongst individuals, or over time, or pre/post vaccination. Without this information, it is not clear how informative some of this variability is versus how much variability in these values is expected. I think particularly in Figure 1, how many of the individuals have periods between times when IS481 evidence was observed when it was not observed, is concerning that these data (at this granular a level) are measuring true infection dynamics. Adding in additional information about the distribution and patterns of these values for the other cohort members would also provide valuable insight into how Figure 1 should be interpreted in this context. As it stands, the authors do not provide sufficient interpretation and evidence for having relevant infection arcs.

      It appears that Figure 2A was created using only 8 data points (from the infant data values). If so, this level of extrapolation from such few data points does not provide enough evidence to support to the results in the text (particularly about evidence for fade-in/fade-out population-level dynamics). Also, in Figure 2, it is not clear to me the added value of Figure 2C and the main goal of this figure.

      The authors have created a measure called, evidence for infection (EFI), which is a summary measure of their IS481 CT values across the study. However, it is not clear why the authors are only considering an aggregated (sum) value which loses any temporality or relationship with symptoms/antibiotic use. For example, the values may have been high earlier in the study, but symptoms were unrelated to that evidence for infection - or visa versa. This seems to be an important factor - were these possible undiagnosed, asymptomatic, or mild symptomatic pertussis infections? It is not clear why the authors only focus on a sum value for EFI versus other measures (such as multiple values above or below certain thresholds, etc.) to provide additional support and evidence for their results.

      It is not clear why the authors have emphasized the novelty and large proportion of asymptomatic infections observed in these data. For example, there have been household studies of pertussis (see https://academic.oup.com/cid/article-abstract/70/1/152/5525423?redirectedFrom=PDF which performed a systematic review that included this topic) that have also found such evidence. While cross-sectional surveys may be commonly used in practice, it is not clear that there is no other type of study that provides any evidence for asymptomatic infections. Further, it is not clear why the authors refer to widespread asymptomatic pertussis when a large proportion of individuals with evidence for pertussis infection had symptoms. Would it not be undiagnosed pertussis if it is associated with clinical symptomatology?

    1. Reviewer #1 (Public Review):

      Galdadas et al. applied a combinatorial approach of equilibrium and nonequilibrium molecular dynamics methods to study two important members of the Class A β-lactamase enzyme family in detail. Authors carefully chose two representative enzymes from this family, TEM-1 and KPC-2 in this study. Understanding of the nature of the communication pathways between allosteric ligand binding site and the active site has been the main focus of this study. Another very interesting finding of this study was the position of clinical variants that was precisely mapped along the allosteric communication pathway. This approach certainly has broad utility as it can be applied to study long-range communications in enzymes that are triggered by binding of a ligand (drug candidate) to an alternative/remote site, and also in cases where certain mutations occur far away from the active site but lead to drug resistance.

      Overall, the manuscript is well written, and the conclusions are mostly well supported by data.

    1. Reviewer #1 (Public Review):

      The authors aimed to survey a large transfusion database in Sweden to catalog associations between ABO/RhD blood group antigens and a wide variety of clinical phenotypes in a systematic, unbiased and comprehensive manor. They succeed at surveying over 1200 phenotypes in over 5 million people and identify 49 statistically significant associations for ABO blood group and point out a couple novel associations. Their statistical methods are appropriate and help eliminate potential false positive associations. The strengths of this study are the unbiased survey of a large database and the appropriate corrections for multiple observations which allow the authors to explore a large number of associations without loosing site of what is really a significant association.

      This study sheds light on a topic of interest to many scientists. The ABO gene encodes a glycosyltransferase enzyme that has 4 major haplotypes in human populations and results in a specific pattern of posttranslational modification of plasma proteins and blood cells including erythrocytes. Proteins decorated with an H antigen can receive additional carbohydrate antigens from ABO transferase intracellularly. The common A allele transfers UDP-GalNAc while the B allele transfers UDP-Gal. The A2 allele is hypomorphic compared to the A allele and transfers lower amounts of UDP-GalNAc and the common O allele is a null resulting in no transferase activity.

      The allele frequencies of these common alleles varies by ancestry and has geographic differences. Variation at ABO is unconstrained with many rare variants contributing to the four common haplotypes at ABO. Interestingly, geographically specific selective pressures may have led to allele frequency differences. For example. ~40-50% of individuals are homozygous for the null (type O) allele. These null haplotypes are more common in individuals of Latino or African ancestry while 'A' haplotypes are slightly more common in individuals of European origin and 'B' alleles are more common in individuals of Asian and African ancestry. Overall, O is more common than A or B alleles. An unbiased survey of phenotype frequencies by blood type allows for confirmation of previous associations and discovery of novel associations. In this largely European ancestry cohort, blood type A is the most common (45-47%) while blood type O is second most common at 38-39%.

      Limitations of Phenome-wide Association Studies (PheWAS) like the one presented in this manuscript should be noted. Associations with complex phenotypes or those with small effect size will not be detected even in a large cohort such as the SCANDAT. This study is also biased toward associations with phenotypes more common in the Scandinavian population. This may present associations related to the population substructure and not a direct association with ABO. In genome-wide association studies this can be addressed through multiple methods but it is not clear how the authors correct for population structure in this study. Likewise, the insight into the mechanistic reasons for ABO associations is not a strength of this study and will await subsequent studies for many phenotypes. Mechanistic insight might be particularly interesting for the novel associations uncovered by this study.

    1. Reviewer #1 (Public Review):

      In this manuscript, the authors use single cell RNA sequencing to investigate cell-type specific eQTL within C. elegans. This relies on the well known ability to genotype individuals via their transcriptome allowing the authors to generate both phenotypes and genotypes from single cell transcriptomes. This identifies a blend of cis and trans-eQTL that are cell type specific and starts to provide numerical observations to the communities expectation of cell type specificity.

      The use of simultaneous single cell sequencing on a diversity of individuals is a unique method that is absolutely essential to get around the vast scale issues that are presented when contemplating single cell eQTL within multicellular organisms. However, an unfortunate outcome of this approach that the cell-autonomy of the eQTL cannot be studied. Instead the cell types have to be considered completely independent of each other.

      The authors conduct an analysis of eQTL per each cell type to get at specificity. This identifies a number of eQTL found in only a single cell type but these binary tests can have an ascertainment issue that may be over-estimating the cell type specificity. Optimally, this would be conducted by incorporating the different cell types as different environments within a single eQTL model but given the different sample sizes, this may not be feasible. Alternatively an investigation of how eQTLs specific to one cell type are or are not found by shifting the detection threshold in the other tissues could test this possibility.

    1. Reviewer #1 (Public Review):

      This manuscript describes the curation of a training dataset, CEM500K, of cellular electron microscope (EM) data including STEM, TEM of sections, electron tomography, serial section and array tomography SEM, block-face and focused-ion beam SEM. Using CEM500K to train an unsupervised deep learning algorithm, MoCoV2, the authors present segmentation results on a number of publically available benchmark datasets. They show that the standard Intersection-over-Union scores obtained with the CEM500K-trained MoCoV2 model, referred to as CEM500K-moco, equal or exceed the scores of benchmark segmentation results. They also demonstrate the robustness of CEM500K-moco's performance with respect to input image transformations, including rotation, Gaussian blur and noise, brightness, contrast and scale. The authors make the remarkable discovery that MoCoV2 spontaneously learned to use organelles as "landmarks" to identify important features in images, simulating human behavior to some degree.

    1. Reviewer #1 (Public Review):

      The submitted manuscript 'Distinct higher-order representations of natural sounds in human and ferret auditory cortex' by Landemard and colleagues seeks to investigate the neural representations of sound in the ferret auditory cortex. Specifically, they examine the stages of processing via manipulating the complexity and sound structure of stimuli. The authors create synthetic auditory stimuli that are statistically equivalent to natural sounds in their cochlear representation, temporal modulation structure, spectral modulation structure, and spectro-temporal modulation structure. The authors use functional ultrasound imaging (fUS) which allowed for the measurement of the hemodynamic signal at much finer spatial scales than fMRI, making it particularly suitable for the ferret. The authors then compare their results to work done in humans that has previously been published (e.g. Norman-Haignere and McDermott, 2018) and find that: 1. While human non-primary auditory cortex demonstrates a significant difference between natural speech/music sounds and their synthetic counterparts, the ferret non-primary auditory cortex does not. 2. For each sound manipulation in humans, the dissimilarity increases as the distance from the primary auditory cortex increases, whereas for ferrets it does not. 3. While ferrets behaviorally respond to con-specific vocalizations, the ferret auditory cortex does not demonstrate the same hierarchical processing stream as humans do.

      Overall, I find the approach (especially the sound manipulations) excellent and the overall finding quite intriguing. My only concern, is that it is essentially a null-result. While this result will be useful to the literature, there is always the concern that a lack of finding could also be due to other factors.

      Major points:

      1) What if the stages in the ferret are wrong? The authors use 4 different manipulations thought to reflect key elements of sound structure and/or the relevant hierarchy of the processing stages of the auditory cortex, but it's possible that the dimensions in the ferret auditory cortex are along a different axis than spectro/temporal modulations. While I do not expect the authors to attempt every possible axis, it would be beneficial to discuss.

      2) For the ferret vocalizations, it is possible that a greater N would allow for a clearer picture of whether or not the activation is greater than speech/music? While it is clear that any difference would be subtle and probably require a group analysis, this would help settle this result/issue (at least at the group level).

      3) Relatedly, did the magnitude of this effect increase outside the auditory cortex?

      4) It would be useful to have a measure of the noise floor for each plot and/or species for NSE analyses. This would make it easier to distinguish whether, for instance, in 2A-D, an NSE of 0.1 (human primary) vs. an NSE of 0.042 (ferret primary) should be interpreted as a bit more than double, or both close to the noise floor (which is what I presume).

    1. Reviewer #1 (Public Review):

      In this study, the authors set out to address the interesting question of how activating septal cholinergic neurons affects learning and memory of reward locations. The work provides compelling evidence showing that activation of septal cholinergic cells at reward zones suppresses sharp wave-ripples and impairs memory performance in freely behaving animals. The data are properly controlled and analyzed, and the results support the conclusions. The results shed new light on the functional significance of cholinergic projections in reward learning. Future follow-up studies designed to selectively activate cholinergic projections specifically at times when sharp wave-ripples occur will be interesting to determine the importance of cholinergic sharp wave-ripple suppression for these effects.

    1. Reviewer #1 (Public Review):

      Here, Houri-Ze'evi, et al. treated progeny of parents that had inherited small RNA response (silencing of an artificial, single-copy GL-expressed gfp with anti-gfp dsRNA) with 3 stresses – heat shock, hyperosmolarity, or starvation – starting at L1, and examined gfp silencing. All three treatments reduced silencing (visible as increased GFP fluorescence) in subsequent generations (F1-F3). The authors tested resetting of endogenous (endo-siRNA) and piRNAs using an endo-siRNA sensor target sequence and piRNA recognition sites, respectively. Again, all 3 stressors reset both in the same generation, but did not reset the effect transgenerationally, suggesting that exogenous RNAi resetting functions through a different mechanism than endogenous.

      Next, they tested adults, which also led to resetting. However, only the F1 generation, not F2, is susceptible to resetting (how? Why?), revealing a critical period for resetting susceptibility. Reversal of the stress with RNAi treatment does not result in resetting, nor does simpy changing conditions. The authors then went on to examine mutants that might be defective in stress responses or in resetting; MAPK genes and skn-1 are required for resetting. Small RNA-seq from stressed worms and their progeny showed a decrease overall with stresses, and reveals some potential classes of genes, including targets of the mutator genes, and overlap with classic stress response pathways (dauer, IIS). Overall, this work presents some interesting phenomena and moves towards explaining how it might work through the identification of a critical period and some genes that are required.

      In this version, the authors have added more information regarding the relationship between MAPK and SKN-1, and transcriptional targets. Most importantly, they have performed tissue-specific rescue of sek-1; in neurons, this rescues, but intestine did not.

      These data add to prior work from the Rechavi lab and others in the field, which together address the interplay of small RNAs, response to stress, and transgenerational inheritance.

    1. Reviewer #1:

      Single molecule localization microscopy (SMLM) has become an important method for understanding the subcellular distribution of fluorescently labelled biomolecules at length scales of a few tens of nanometers. A critical challenge has been to find out, whether and to what extent biomolecular clustering occurs. While methods have been published which address the problem of identifying biomolecular clusters in SMLM images, they still suffer from many user-defined parameters, which - if selected inappropriately - influence the obtained results substantially. The StormGraph-3D method proposed here addresses these issues, based on a comprehensive mathematical framework which reduces the number of user-defined input parameters. The method was evaluated using comprehensive simulations of data, which show its robustness compared to alternative approaches.

      The methods part of the paper would benefit, however, from more realistic data of single molecule blinking behavior, and the evaluation of the consequences on the performance of the method. As the authors acknowledge, overcounting due to blinking has challenged data analysis previously, and gave rise to artifactual localization clusters that do not represent the underlying protein distribution. It would be of particular interest, which results in the method yielded for a random biomolecular distribution.

    1. Reviewer #1:

      The authors of Working Memory Gates Visual Input to Primate Prefrontal Neurons studied how working memory influences information transmitting from V4 to frontal eye field via extracellular recording and electrical stimulation on behaving primate. They found that V4 neurons target FEF neurons with both visual and motor properties, and its synaptic efficacy of V4 to FEF was enhanced by working memory. These findings are interesting and important to our understanding about how our brain acts during daily WM-related activity.

      1) In classical working memory tasks, the task periods usually consist of fixation, cue, delay and then a response period. The neural activity during the delay period is typically considered to be a working memory-related signal. However, in the current study, the authors didn't point out whether only delay period activity was included in analysis when they compared synaptic efficacy between stimulation and non-stimulation trials, in Figure 4a. Because the differences of neuronal response during fixation period cannot be viewed as relevant to information held in working memory, it may be better if only neuronal activity in the delay period was included in their analysis.

      2) Did the 96 visual-recipient FEF neurons exhibit working memory-related activity in their memory guided saccade task? The example neuron in Figure 3a didn't show significant difference between In and Out trials during the delay period. If the visual-recipient neuron didn't present working memory related activity, how could the authors say enhanced synaptic efficacy from V4 to FEF was caused by working memory?

      3) Did the two example neurons in Figure 4c show adjusted values (subtracting the same measure during non-stimulated trials)? The authors mentioned in Method that Figure 4 showed adjusted values, but it may not be applicable for raster plot in Figure 4c. It may be helpful that using adjusted values show stimulation effects on evoked spike counts during memory In and Out trials.

      4) Did the authors find some FEF cells showing elevated firing during delay period in outside-RF trials compared with baseline firing? These elevated firing was not caused by RF cue, may underlying working memory signal.

      5) The sample size should be indicated in Figure 3b Venn diagram.

      6) It's better to indicate electrical stimulus protocol in Figure 1.

    1. Reviewer #1:

      The present study sought to better characterize how listeners deal with competing speech streams from multiple talkers, that is, whether unattended speech in a multitalker environment competes for exclusively lower-level acoustic/phonetic resources or whether it competes for higher-level linguistic processing resources as well. The authors recorded MEG data and used hierarchical frequency tagging in an unattended speech stream presented to one ear while listeners were instructed to attend to stories presented in the other ear. The study found that when the irrelevant speech contained structured (linguistic) content, an increase in power at the phrasal level (1 Hz) was observed, but not at the word level (2 Hz) or the sentence level (0.5 Hz). This suggests that some syntactic information in the unattended speech stream is represented in cortical activity, and that there may be a disconnect between lexical (word level) processing and syntactic processing. Source analyses of the difference between conditions indicated activity in left inferior frontal and left posterior parietal cortices. Analysis of the source activity underlying the linear transformation of the stimulus and response revealed activation in the left inferior frontal (and nearby) cortex. Implications for the underlying mechanisms (whether attentional shift or parallel processing) are discussed. The results have important implications for the debate on the type and amount of representation that occurs to unattended speech streams.

      The authors utilize clever tools which arguably provided a unique means to address the main research question, i.e., they used hierarchical frequency tagging for the distractor speech, which allowed them to assess linguistic representations at different levels (syllable-, word-, phrase-, and sentence-level). This technique enabled the authors to make claims about what level of language hierarchy the stimuli are being processed, depending on the observed frequency modulation in neural activity. These stimuli were presented during MEG recording, which let the authors assess changes in neurophysiological processing in near real time--essential for research on spoken language. Source analyses of these data provided information on the potential neural mechanisms involved in this processing. The authors also assessed a temporal response function (TRF) based on the speech envelope to determine the brain regions involved at these different levels for linguistic analysis of the distractor speech.

      Critiques:

      Speech manipulation:

      In general, it is unclear what predictions to make regarding the frequency tagging of the unattended distractor speech. On the one hand, the imposed artificial rhythmicity (necessary for the frequency tagging approach) may make it easier for listeners to ignore the speech stream, and thus seeing an effect at higher-level frequency tags may be of greater note, although not entirely plausible. On the other hand, having the syllables presented at a consistent rate may make it easier for listeners to parse words and phrasal units because they know precisely when in time a word/phrase/sentence boundary is going to occur, allowing listeners to check on the irrelevant speech stream at predictable times. For both the frequency tagging and TRF electrophysiological results, the task-irrelevant structured speech enhancement could be interpreted as an infiltration of this information in the neural signal (as the authors suggest), but because the behavioral results are not different this latter interpretation is not easily supported. This pattern of results is difficult to interpret.

      Behavioral Results:

      Importantly, no behavioral difference in accuracy was observed between the two irrelevant speech conditions (structured vs. non-structured), which makes it difficult to interpret what impact the structured irrelevant speech had on attentive listening. If the structured speech truly "infiltrates" or "competes" for linguistic processing resources, the reader would assume a decrease in task accuracy in the structured condition. This behavioral pattern has been observed in other studies. This calls into questions the face validity of the stimuli and task being used.

      Attention:

      In this study activation of posterior parietal cortex was found, that could be indicative of a strong attentional manipulation, and that the task was in fact quite attentionally demanding in order for subjects to perform. This may align with the lack of behavioral difference between structured and non-structured irrelevant stimuli. Perhaps subjects attempted to divide their attention which may have been possible between speech that was natural and speech that was rather artificial. The current results may align with a recent proposal that inferior frontal activity may be distinguished by language selective and domain general patterns.

      Lack of word level response:

      A major concern is that the results do not seem to replicate from an earlier study with the same structured stimuli, i.e., the effects were seen for sentence and word level frequency tagging. As the authors discuss, it seems difficult to understand how a phrasal level of effect could be obtained without word-level processing, and so a response at the word level is expected.

      Familiarization phase:

      The study included a phase of familiarization with the stimuli, to get participants to understand the artificial speech. However it would seem that it is much easier for listeners to report back on structured rather than unstructured stimuli. This is relevant to understanding any potential differences between the two conditions. It is unclear if any quantification was made of performance/understanding at this phase. If there is no difference in the familiarization phase, this might explain why there was no difference in behavior during the actual task between the two conditions. Or, if there is a difference at the familiarization phase (i.e. structured sequences are more easily repeated back than non-structured sequences), this might help explain the neural data result at 1 Hz, given that some higher level of processing must have occurred for the structured speech (such as "chunking" into words/phrasal units).

    1. Joint Public Review:

      Eisele et al. evaluated the direct action of erythropoietin (EPO) on hematopoietic stem and progenitor cells that included hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). They used cellular barcoding to enable in vivo tracking of cellular output and then used scRNA-seq to corroborate their findings. They observed the transiently promoted output of Myeloid-Erythroid (ME)-biased and Myeloid-B-cell (MB)-biased clones. Single-cell RNA sequencing analysis revealed that EPO acted mostly on MPP1 and MPP2. Based on these data, the authors concluded that EPO acts directly on MPPs and transiently modulates their output. Although the conceptual advance brought by this study is incremental as similar findings have been presented by previous studies, the integration and use of both barcoding and scRNA-seq adds strength to the conclusions reached in the present study.

    1. Reviewer #1 (Public Review):

      In this work, the authors set out to better understand the mechanisms by which the nematode C. elegans responds to bacterial pathogens.

      Using behavioral assays and genetic manipulations, the authors find that C. elegans can rapidly learn to avoid the pathogen E. faecalis (E.f.). While recent studies from other groups have shown that small RNAs (sRNAs) produced by some pathogenic bacteria can trigger aversive learning, the authors find that this seems not to be the case for E. faecalis. Instead, they provide evidence that E. faecalis causes abdominal distention, and that this may provide the trigger for learning. Because the evidence for this is largely correlative, alternative explanations may still be possible. Further, the authors identify two TRPM-class ion channels whose function appears to be necessary for learned avoidance of E.f. The authors propose that one or both of these may mediate detection of abdominal distention, an interesting idea that merits further study. While the paper's title indicates that these channels "mediate" this function, this remains speculative.

      The authors also find that wild-type C. elegans prefer olfactory stimuli from E.f. to those of their regular diet, E. coli, but that this pattern is reversed after exposure to E.f. This plasticity involves the function of the chemosensory neurons ASE, AWC, and AWB, as well as the cyclic-nucleotide-gated channel TAX-2/TAX-4. This finding provides important insight into the nature of the changes in neural circuit function that are triggered by pathogen exposure, leading to pathogen avoidance.

      The paper also examines a role for the neuropeptide receptor npr-1 in learned E.f. avoidance. Animals lacking npr-1 function are known to strongly avoid high (ambient) oxygen concentrations, and instead prefer the lower-oxygen environment of a bacterial lawn. The authors find that this oxygen avoidance overcomes any avoidance of E.f.; thus, npr-1 mutants do not avoid E.f. when tested with ambient oxygen, but they do avoid it in a low-oxygen environment. This indicates that npr-1 is not required for pathogen avoidance per se. Although the authors suggest that npr-1 may be a target of the learning process, this is not well justified by the data and it may be more likely that oxygen avoidance and pathogen avoidance are separate processes.

      Together, these findings demonstrate that the mechanisms underlying learned pathogen avoidance in C. elegans differ substantially depending on the nature of the pathogen, and that worms likely use a combination of strategies to deal with these threats in the wild.

    1. Reviewer #1 (Public Review):

      In this manuscript the authors demonstrate that acute systemic inflammation induces a new system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors but not other progenitors or stem cells from BM to lymphatic capillaries. This traffic is mediated by Ccl19/Cccr7 and is NfkB independent but Traf activation dependent. This type of trafficking is anti-inflammatory with promotion of early survival.

      Specifically, authors work shows the traffic of DC-biased myeloid progenitors through direct transit from BM to bone lymphatic capillaries. This type of trafficking is highly activated in endotoxic inflammation. Giving LPS to mice results in massive mobilization of myeloid progenitors from the BM to lymph and retention in LN takes place. This happens rapidly and before the appearance of these cells in PB. This type pf LPS challenge induces Ccr7 expression on GMPs as well as secretion of CcL9 in the LN. Importantly, loss of CcL9 or neutralizing Ccr7 inhibits GMP/MDP migration to the LN and inflammation induce mortality.

      The studies are well performed and the data supports the conclusions. The role of this signaling axis in the recruitment of GMPs/MDPs has not been investigated in this detail.

    1. Reviewer #1 (Public Review):

      The manuscript by Tindle et al describes generation of adult lung organoids (ALO) from human lung biopsies and their use to study the changes in gene expression as a result of SARS-CoV-2 infection. The main advantage of the use of organoids is the ability to generate many cell types that make up the lung. In this particular case the authors report the presence of AT1, AT2 cells, Basal cells, Goblet cells, Ciliated cells and Club cells. The authors were able to cultivate the cells at the air-liquid interface and to establish cultures of predominately proximal and predominately distal airway cells. The main finding is that proximal cells are more prone to viral infection, while distal cells are governing the exuberant inflammatory response, with both cells required for the exuberant response to occur. A useful information provided by the paper is the analysis gene signatures of various cellular models, in comparison to the infected human lung.

    1. Reviewer #1 (Public Review):

      This manuscript by Taylor et al. carefully investigates (1) ParB-ParA and (2) ParB-ParB interactions in the F Plasmid SopABC system using microfluidics, TIRF microscopy and magnetic tweezers.

      (1) The work shows that the activation of ParA ATP hydrolysis requires a dimer of ParA to bind to two protomers of ParB. Surprisingly, ParB can bind to ParA either using the two protomers of a single dimer or two protomers from distinct dimers. The former occurs in the absence of ligands, the latter upon addition of either CTP or parS DNA, thus presumably corresponding to the state of ParB found in the cells near a parS site. The authors suggest that this is crucial for the precise timing of ParA-ParB anchoring and release.

      (2) Magnetic tweezer experiments demonstrate nucleotide-dependent compaction of DNA by ParB. This compaction is strictly parS-sequence dependent and robust even at elevated DNA extension force (5 pN) and at relatively low ParB concentrations. This implies ParB dimer-ParB dimer interactions exclusively on parS DNA.

      The conclusions are generally well supported by the data. Few control experiments are suggested.

    1. Reviewer #1 (Public Review):

      The paper "Insights from a Pan India Sero-Epidemiological survey (Phenome-India cohort) for SARS-CoV2" reports a longitudinal survey of about 10000 subjects from laboratories of the CSIR (India) who consented to be tested for antibodies to SARS-CoV-2, across August and September 2020. The methodology is a standard one, using the Roche kit to test for antibodies to the nucleocapsid antigen with a followup to detect neutralising antibodies using the GENScript Kit. A questionnaire for all participants asked about age, gender, pre-existing conditions and blood group, among other questions.

      The principal results of the study were:

      1) An overall seropositivity of 10.14% [95% CI: 9.6 - 10.7] but a large variation across locations

      2) Virtually all of the seropositive exhibited neutralising activity

      3) Seropositivity correlated with population density in different locations

      4) A weak correlation was seen to changes in the test positivity across locations

      5) A large asymptomatic fraction (~75%) who did not recall symptoms

      6) Of those symptomatic, most reported mild flu-like symptoms with fever

      7) A correlation with blood group, with seropositivity highest for AB, follow by B, O and A

      8) A vegetarian diet correlated with reduced seropositivity

      9) Antibody levels remained constant for 3 months across a sub-sample white neutralising activity was lost in ~30% of this subsample. Over a longer period, in a still smaller subsample of those tested at 3 months, anti-nucleocapsid antibody levels declines while neutralising antibody levels remained roughly constant

      10) There is a reasonable agreement with the results of the second Indian serosurvey which obtained a seroprevalence of about 7% India-wise, although excluding urban hotspots.

      The deficiencies of this study are:

      1) This is a very specific cohort, largely urban, with - presumably - relatively higher levels of education. It is hard to see how this might translate into a general statement about the population

      2) The presentation of Figure 1 was quite confusing, especially the colour coding

      3) It is surprising that the state of Maharashtra shows only intermediate to low levels of seropositivity, given that the impact of the pandemic was largest there and especially in the city of Pune. There have been alternative serosurveys for Pune which found much higher levels of seropositivity from about the same period.

      4) The statement "Seropositivity of 10% or more was associated with reductions in TPR which may mean declining transmission": For a disease with R of about 2, this would actually be somewhat early in the epidemic, so you wouldn't expect to see this in an indicator such as TPR. TPR is also strongly correlated with amounts of testing which isn't accounted for.

      5) The correlation with vegetarianism is unusual - you might have argued that this could potentially protect against disease but that it might protect against infection is hard to credit. Much of South Asia is not particularly vegetarian but has seen significantly less impact

      6) On the same point above, it is possible that social stratification associated with diet - direct employees being more likely to be vegetarian than contract workers - might be a confounder here, since outsourced staff seem to be at higher risk.

      7) There may be correlations to places of residence that again act as confounders. If direct employees are provided official accommodation, they may simply have had less exposure, being more protected.

      8) The correlations with blood group don't seem to match what is known from elsewhere

      9) The statement that "declining cases may reflect persisting humeral immunity among sub-communities with higher exposure" is unsupported. What sub-communities?

    1. Reviewer #1 (Public Review):

      In the manuscript "Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted heart", the authors isolated epicardial stromal cells (EpiSC) and cardiac interstitial/stromal cells (termed active CSCs) from the same I/R heart and identified transcriptionally distinct subpopulation of EpiSCs via 10x genomics technology. They also performed transcriptome profile comparison between EpiSCs and aCSCs. This manuscript shows rigorous scientific investigation. Their isolation protocol is supported by their previous publication. Method section documented in detail of step-wise QC process of bioinformatics analysis. In summary, the analysis identified 11 clusters of EpiSC, some of which overlap with the well-established epicardial marker WT1 with confirmed in situ anatomical localization. When compared to aCSC, the two groups showed clear different function/states as expected. In the lineage tracing model, RNA velocity predicts cell hierarchy, cell-cell communication between populations, as well as cell cycle activity. Overall this manuscript provides a significant degree of information that can be helpful to the field.

    1. Reviewer #1 (Public Review):

      The work by Lutes et al. addresses how thymocytes undergo positive selection during their differentiation into mature T cells. The authors make use of several in vitro and in vivo model systems to the test whether developing thymocytes at the critical preselection CD4+CD8+ stage, expressing T cell receptors (TCRs) with different levels of putative self-reactivity, undergo different or similar differentiation events, in terms of migration, thymic epithelial cell engagement and temporal kinetics, and gene expression changes.

      The authors selected three TCR-transgenes, which have increasing levels of self-reactivity, TG6, F5 and OT1, respectively, to test their hypothesis, that TCR signals during positive selection are not only sensed differently but lead to different outcomes that then define the functional status of mature T cells. The author's conclusions that thymocytes with low self-reactivity differentiate with distinct kinetics (migration, engagement and temporal) and express a different suite of genes than thymocytes that experience high self-reactivity is well supported by several elegant approaches, and convincing findings.

      The authors clearly established that low to high TCR signaling outcomes affect the timing of positive selection, which is beautifully illustrated in Figures 3-6, and extend that work to non-TCR transgenic mice as well. Lastly, their findings from RNA-seq analyses shed light into the different genetic programs experienced by high-reactivity fast differentiating CD8 T cells as compared to low-reactivity slower differentiating cells, which appear to retain the expression a unique set of ion channels during later stages of their differentiation process.

      However, what the expression of these ion channels means in terms of either supporting the slow progression or perhaps responsible for the slow progression is not directly addressed, and likely beyond the scope. Nevertheless, the authors posit as to the potential role(s) for the differently expressed gene subsets. Overall, the work is crisply executed, and the findings reveal new aspects as to how positive selection can be achieved by thymocytes expressing very different TCR reactivities.

    1. Reviewer #1 (Public Review):

      Nielsen and colleagues describe a large new multi-ome database containing combinations of absolute mRNA quantities, proteome and amino acid concentrations in a set of 14 yeast populations grown in various conditions in chemostats. Apart from being a valuable resource for colleagues, analysis of the data confirms the results of several previous seminal studies.

      For example, the authors confirm the relatively high correlation between transcript and corresponding protein abundance. Moreover, it is shown that for most genes, changes in transcript abundance related to manipulated changes in growth rate largely reflected the availability of RNA polymerase II. Interestingly, this was not the case for genes involved in central carbon metabolism, suggesting that these are regulated separately, likely to maintain the cells' ATP levels. Similarly, manipulation of growth through the use of different nitrogen sources led to changes in transcription that correlated with certain amino-acid-derived metabolites (including nucleotides), but not with RNAPolII levels. Genes involved in central carbon metabolism are again an exception to this rule.

    1. Reviewer #1 (Public Review):

      This paper describes the development of a suite of viral vectors that allow expression (either on or off) of genes of interest depending on both Cre and Flp expression. They demonstrate that their system can solve the problem encountered with the other approach and use it for mapping axonal projections of the glutamatergic, LepR-expressing neurons and the consequences of chronic activation of these neurons on food intake and energy expenditure. The results are significant and clearly presented. The failure of the other system (INTRSECT) for their application is not clearly understood, but authors say that it may be due to low expression of Cre or Flp in these neurons; however, Supp Fig. 1 shows that it Lepr-Cre and Slc17a6-FLPo were sufficient to activate a transgenic reporter (Supp Fig. 1). The authors reveal that they probably could have used Nr5a1-Cre mice manipulate the activity of these VMH neurons. Nevertheless, it is worthwhile having multiple methods to attack a specific problem because of unforeseen complications with particular methods.

    1. Reviewer #1 (Public Review):

      The zebrafish has a rich history of enabling innovative microscopy techniques, and is also a well established system to model inflammation and infection by human pathogens. Consistent with this, Miskolci et al use zebrafish to test a novel imaging approach that has great potential to significantly impact the field of immunometabolism. Fluorescence lifetime is a label-free, non-invasive imaging approach to detect metabolic changes in situ, at the level of the single cell. In this report, Miskolci et al use fluorescence lifetime imaging of NAD(P)H and FAD to detect metabolic changes in zebrafish macrophages (with temporal and spatial resolution) in response to inflammatory and infectious cues.

      Miskolci et al (eLife 2019) have previously characterized inflammatory and wound healing responses to distinct caudal fin injuries (tail wound, infection and tail wound, thermal injury). In this report, authors use these different injury models to show that fluorescence lifetime imaging can detect variations in macrophage metabolism. Although many interesting results are presented and future directions are proposed, the study in its current state is descriptive and lacks validation across different modalities. As a result, the reliability of fluorescence lifetime imaging in zebrafish macrophages is not yet convincing. Moreover, any metabolomic changes in macrophages are not functionally linked to zebrafish phenotypes (eg inflammation, bacterial burden, caudal fin regeneration).

    1. Reviewer #1 (Public Review):

      The manuscript presents a very nice and very detailed approach to illustrate the anatomical hierarchies and also some differences of signal transmission in the olfactory vs. thermosensory-/hygrosensory systems.

      The authors first provide a complete description of the Drosophila olfactory system, from first, second and third-order neurons in the lateral horn. Using a generally applicable analysis methods, they extract information flow and layered organisation between olfactory input and descending interneurons. Among the results is the interesting finding that downstream of the mushroom body and lateral horn, output neurons converge to presumably regulate behavior. In an additional set of analyses, Schlegel et al. describe and quantify inter- vs. intraindividual stereotypy of neurons and motifs. They actually compare neurons from three hemispheres of two brains and show an astounding degree of similarity across brains. This is somewhat reassuring and helpful to the field of Drosophila connectomics.

      While the many details and data make the manuscript a somewhat strenuous read, and the sheer flood of data could be a bit overwhelming, the data and findings are impressive and important.

      1) The work is very complementary to the data presented by Li et al. on the mushroom body.

      2) The structure and the step-by-step approach to showing increasingly complex circuitry and by defining different layers of the circuitry is very helpful for the reader to get an impression of the complexity of this brain.

      3) Of significant importance and of use for the community are, in addition to the data, the described methods tools for data analysis.

      4) Using this type of analysis, the authors test hypotheses and prevailing assumptions in the field. For instance, they find that in early layers of the olfactory system neurons tend to connect to the next higher layer, whereas neurons in higher layers interconnect or even connect back to earlier layers. This is a very interesting finding that might have important implications regarding top-down feedback and recurrent loops in olfactory processing.

      5) Analysis of connectivity in the antennal lobe suggests that the system is highly lateralized. This finding also has important implications and helps to explain why flies might be able to discern left from right odor sources.

      6) The manuscript shows many examples of what other scientists/readers of the manuscript could extract from the raw anatomical data. This will be very useful for the community beyond the data that is actually already shown in the manuscript.

      7) The authors also compare their findings to the connectome/motifs identified for the larval olfactory system. There are many similarities as expected.

    1. Reviewer #1 (Public Review):

      Claudi et al. present a new tool for visualizing brain maps. In the era of new technologies to clear and analyze brains of model organisms, new tools are becoming increasingly important for researchers to interact with this data. Here, the authors report on a new tool for just this: exploring, visualizing, and rendering this high dimensional (and large) data. This tool will be of great interest to researchers who need to visualize multiple brains within several key model organisms.

      The authors provide a nice overview of the tool, and the reader can quickly see its utility. What I would like to ask the authors to add is more information about computational resources and computing time for rendering; i.e. in the paper, they state "Brainrender uses vedo as the rendering engine (Musy et al., 2019), a state-of-the-art tool that enables fast, high quality rendering with minimal hardware requirements (e.g.: no dedicated GPU is needed)" - but would performance be improved with a GPU, runtimes, etc?

      I would also be happy to see the limitations and directions expanded. For example, napari is a powerful n-dimensional viewer, how does performance compare (i.e. any plans for a napari plug in, or ImageJ plug in, or is this not compatible with this software's vision?). How does brain render compare (run time, computing wise) to Blender, for example, or another rendering tool standard in fields outside of neuroscience?

      The methods are short (maybe check for all open source code citations are included, as needed), but they have excellent docs elsewhere; it would be nice to have minimal code examples in the methods though, i.e. "it's as easy as pip install brainrender" ... or such.

      Lastly, I congratulate the authors on a clear paper, excellent documentation (https://docs.brainrender.info/), and I believe this is a very nice contribution to the community.

    1. Reviewer #1 (Public Review):

      The manuscript by Schrieber et al., explores whether inbreeding affects floral attractiveness to pollinators with additional factors of sex and origin in play, in male and female plants of Silene latifolia. The authors use a combination of spatial sampling, floral volatiles, flower color, and floral rewards coupled with the response of a specialized pollinator to these traits. Their results show that females are more affected by inbreeding and in general inbreeding negatively impacts the "composite nature" of floral traits. The manuscript is well written, the experiments are detailed and quite elaborate. For example., the methodology for flower color estimation is the most detailed effort in this area that I can remember. All the experiments in the manuscript show meticulous planning, with extensive data collection addressing minute details, including the statistics used. However, I do have some concerns that need to be addressed.

      Core strengths: Detailed experimental design, elaborate data collection methods, well-defined methodology that is easy to follow. There is a logical flow for the experiments, and no details are missing in most of the experiemnts.

      Weaknesses: A recent study has addressed some of the questions detailed in the manuscript. So, introduction needs to be tweaked to reflect this.

      Some details and controls are missing in floral scent estimation. Flower age, a pesticide treatment of plants that could affect chemistry..needs to be better refined. While the study is laser-focused on floral traits, as the authors are aware inbreeding affects the total phenotype of the plants including fitness and defense traits. For example, there are quite a few studies that have shown how inbreeding affects the plant defense phenotype. This could be addressed in the introduction and discussion.

    1. Reviewer #1 (Public Review):

      In this study, Zhang et al. systematically analyze the effect of xanthohumol (XN) and TXN, a xanthohumol derivative, in a model of high-fat diet (HFD) feeding to mice, inducing several pathologies related to the metabolic syndrome. They authors convincingly show that XN and TXN attenuate HFD-induced weight gain, hepatic steatosis and lipid accumulation in adipose tissues. Furthermore, they newly show that XN and TXN bind to the PPARgamma ligand-binding domain pocket and that this inhibitory effect on PPARgamma is at least in part responsible for the observe beneficial effects.

    1. Reviewer #1 (Public Review):

      This manuscript describes a set of biochemical studies on the substrate and reaction specificity of PARP1, an important drug target and component of DNA damage response. The focus of the work is on the specific role of HPF1, and how PARP1's numerous activities are altered by complexation with it and with a variety of substrates. There are many important findings described in this paper, which will be of great interest to the researchers studying PARP1 and issues related to NAD+ metabolism. Perhaps the most significant finding is that HPF1 binding to PARP1 causes a shift from primarily PARylation activity to that of hydrolytic activity, yielding a large pool of free ADPR. The paper is very well written. Addressing the following issues would provide clarity.

      1) The kcat enhancement from employing nucleosome substrates is exceedingly small, and probably will not ever be clearly correlated to a specific structural feature. However, more concerning is a possible uncontrolled variable when examining the nucleosome substrates. Specifically, the nucleosome substrates which yield a distinctly higher kcat (Table 1) are the larger, trivalent nucleosomes. It seems prudent to show that simply adding more potential binding sites, or perhaps just adding more protein itself is not causing these small increases in kcat (relative to DNA alone).

      2) Concerning the assignment of E284 of HPF1 as the catalytic base in the deprotonation of the Ser hydroxyl, I'm wondering if there might be a dynamical explanation for its role instead. E284A causes a significant decrease in the KD for HPF1 binding, and an elimination of the observed PARylation activity, suggesting that it may play an allosteric role. Also, we see from Table 2 that H303Q also produces a large reduction in the activity and large reduction in the KD; the standard error on the H303Q binding data is very large, but does suggest that some observations were quite low (similar to E284A). Additionally, H303Q almost eliminates enzymatic activity as well. Overall, this set of data gives me pause about certainty of the assignment of E284 as the catalytic base, as there may be a more complex origin of the loss of enzymatic activity.

      3) It may be that the reason that there is no apparent PARylation at the standard carboxylate residue sites (in the presence of HPF1) is that they are forming transient ester bonds with the anomeric carbon, which are labile to hydrolysis. I feel that a better development of the treadmilling effect would enhance the paper (e.g., mutation of the orthodox carboxylate nucleophiles and examination of changes in HPF1-induced hydrolytic activity). I'm not sure that it can be quantitatively shown that the shorter PAR chains in the presence of HPF1 account for the pool of free ADPR.

    1. Reviewer #1 (Public Review):

      This is an interesting manuscript which does a lot - both building and validating an epigenetic clock for the Amboseli baboons, and then looking to see which factors predict deviations in epigenetic age relative to chronological age. This is an important study, and perhaps the first of its kind from a free-ranging primate population. I believe it will be influential and well-cited.

      In particular, it is extremely thorough in the data and analyses that it presents. It is also clearly structured and easy to follow, despite covering some dense material.

      In sum, this manuscript is a high-quality and important manuscript that I believe will be influential.