Evaluation Summary:

Gene regulation triggered by recognising N6-methyladenosine (m6A) by RNA-binding proteins emerged as a fundamental process for development and adaptation in most eukaryotes. This paper is of interest to scientists within the field of post-transcriptional gene regulation and RNA biology. In a deep and thorough analysis, the authors resolved previously contradictory reports of sequence motifs associated with methylation sites in messenger RNAs and shed light on the proteins that recognise this modification.

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This manuscript was co-submitted with: https://www.biorxiv.org/content/10.1101/2021.08.01.454660v1

Evaluation Summary:

Approaching the search of novel viruses while in an endogenized stage, rather than as free virions, the study by Hackl et al. reveals a large diversity of complete and fragmented virophage genomes - termed EMALEs- scattered throughout the genomes of four strains of the marine protist Cafeteria. Given that the activation of the integrated virophage mavirus during infection by the giant virus, CroV, has been shown to have a protective effect on the Cafeteria population, this study provides a tantalizing window into the traces of virophage-giant virus¬-protist interactions in the marine environment. Given the enormous diversity of virophages and giant viruses that have been found in metagenomes with no known hosts, this study is a step towards deciphering the biology of these viruses.

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Evaluation Summary:

This paper is of potential interest to an audience of biochemists, cell biologists, and structural biologists working in the area of chromosome organization and segregation. A wide range of in vitro methods is used to provide compelling biochemical evidence for the interaction of MatP and ParEC at the hinge of MukB, the condensin of Enterobacteria. However, the evidence supporting the significance of these interactions in vivo is less strong, limiting the biological implications of the elegant biochemical findings.

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Evaluation Summary:

This work combines elegant experimental approaches with modelling predictions to study of metabolic adaptations in the bacterium Cupriavidus necator, a microorganism of interest given its metabolic versatility and potential industrial applications. This manuscript will be interesting for microbiologists and systems biologists who want to understand how protein production and economy and enzyme utilization differs in a versatile microorganism in different conditions.

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Evaluation Summary:

This manuscript will be of interest to immunologists studying mechanisms of thymic central tolerance. The study elegantly makes use of multiple genetic mouse models to generate data supporting the conclusion that different dendritic cell subsets in the thymus capture self-antigens from distinct subsets of thymic epithelial cells. However, some key conclusions are not entirely novel, and the final model, as currently presented, draws from only selective analyses and thus may not accurately reflect the antigen transfer between thymic epithelial cell and dendritic cell subsets that promote central tolerance.

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Evaluation Summary:

The authors report continuum electrostatics, molecular dynamics and QM/MM simulations to probe the protonation pattern of key residues in anion channelrhodopsin (GtACR1). The findings provide new mechanistic insights into the gating mechanism of GtACR1, and the study will be of potential interest for the community focused on biophysical chemistry, protein simulations and optogenetics. More generally, the study helps highlight that due to potential compensatory effects, care needs to be exercised when interpreting absorption spectra of mutant proteins.

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Evaluation Summary:

This study describes the addition of peptide immunoprecipitation by means of SISCAPA technology to the Sars-Cov2 mass spectrometry-based diagnostics toolbox. While MS-based viral detection has been described by several other groups, the presented method enriches SARS-CoV-2 peptides from clinical samples, thus enhancing the sensitivity and addressing one of the major limitations of this new method for viral diagnostics. The work is straightforward and of potentially great importance to the field. It shows beyond any doubt that mass spectrometry can become a clinically applied diagnostic instrument to detect (viral) infection.

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Evaluation Summary:

This manuscript introduces a novel method for the monitoring of immune dynamics from blood, based on a measurement of the burden of cell-free DNA derived from seven key immune cell types in blood. The authors demonstrate the possibility to quantify the specific amounts of cell-free DNA by assaying cell-type specific CpG methylation marks via a targeted DNA sequencing assay. Assays of specific immune cell counts in blood are commonplace in modern diagnostic medicine. Rather than counting cells, the assay reported in this manuscript measures the burden of DNA released by dying immune cells, and this new assay may provide information about the immune system beyond what is possible with mere cell counting. The authors test their assay in three different settings (vaccination, inflammation and cancer), and provide significant support for the utility of their assay for immune monitoring in health and disease.

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Evaluation Summary: 

In this interesting study, the authors combined Brillouin microscopy with Optical Diffraction Tomography and epi-fluorescence imaging to investigate physical properties of biological materials including nucleoplasm, cytoplasm, phase-separated organelles, and adipocytes. The results are largely convincing and offer interesting insights into the material properties of these subcellular structures. 

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Evaluation Summary: 

The goal of this study was to test whether multiple attempts at suppressing the retrieval of an (emotional) memory is associated with degradation of the representation of information in the brain about such memories. A combination of sophisticated computational modelling in fMRI reveals that neural representations of previously suppressed memories are sustainably weakened during memory retrieval attempts. This manuscript is of interest for neuroscientists in the field of motivated forgetting and memory control. 

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Evaluation Summary:

In large nervous systems such as mammalian cortex excitatory synapses are stochastic and the probability of release of neurotransmitter can be modulated by plasticity and neural activity. This paper presents a simple biologically plausible mechanism that regulates the probability of release during learning. Using network simulations the authors show that this can result in more energy efficient processing of learned stimuli by enhancing the reliability of important connections, with lower expected rates of transmission at less important synapses.

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Evaluation Summary:

This paper investigates the evolutionary path of Escherichia coli resistance to the antibiotic trimethoprim. The authors show that adaptive mutations that accumulate early are often not in the drug target itself, but rather mutations that lead to transcriptional up-regulation of the drug target. Higher-level resistance can then evolve due to the addition of mutations in the drug target; however, at lower drug concentrations, cells are more likely to accumulate mutations that reverse the fitness defect associated with the initially acquired mutations. Overall, this study shows that regulatory mutations can play a major role in the evolution of antibiotic resistance in bacterial populations, and that the evolutionary path is influenced by the level of drug exposure.

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Evaluation Summary:

This work examines how METTLL18-mediated RPL3 histidine methylation on 245 position regulates translation elongation and protects cells from cellular aggregation of Tyr-rich proteins. The study hints at the existence of a "ribosome code" and how posttranslational modification of ribosomal proteins could affect translation.

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Evaluation Summary:

This is an innovative and important paper with interest to basic and translational neuroscientists that demonstrates the power of experimental models to advance our understanding of human disease. The authors focus on early-life epilepsy, a devastating and common disorder, and specifically on genetic epilepsies generated via pathological sequence variations in the hyperpolarization-activated nonspecific cation (HCN) channel subtype 1. They delineate the epileptic phenotype and demonstrate some of the potential mechanisms leading to the generation of spontaneous seizures in genetically engineered mice.

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Evaluation Summary:

Brodbeck and colleagues make a strong contribution to the field of neurolinguistics by asking whether speech comprehension uses local (e.g., sublexical) or global (e.g., sentences) contextual probabilities. To tackle this, they recorded participants with magnetoencephalography while they listened to a story. The authors assessed which of three possible speech models best explained brain activity using state-of-the-art analyses and information-theoretic measures. The authors report strong and valuable evidence for both local and global contextual analyses supporting the coexistence of both hierarchical and parallel speech processing in the human brain.

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Evaluation Summary:

When a cell undergoes rapid shrinking, excess plasma membrane becomes available. The authors show that excess plasma membrane forms very small bleb-like evaginations that disappear after a few minutes. They show a new role for the I-BAR protein IRSp53 and Arp2/3-dependent actin polymerization which surprisingly leads to the flattening of the bud instead of its growth, as it is the case in filopodial protrusions. This manuscript will be of general interest to cell biologists working on membrane-cortex interactions.

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Evaluation Summary:

The authors investigate the contribution of a CTCF binding site located 3' of the beta-globin locus to the relative expression of the genes within the cluster. Their results serve to further clarify a longstanding model for how relatively high levels of fetal beta-globin are observed in some individuals harboring a deletion of this region, and contribute to existing models of the function of CTCF binding sites in genome organization.

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Evaluation Summary:

All-trans retinoic acid (atRA) is a potent regulator of synaptic function known to be critical for certain forms of homeostatic plasticity. Previous work by the Vlachos group established that atRA also modulates synaptic function in human cortex and linked the synaptic effects of atRA to the spine apparatus protein synaptopodin. As a follow up study, the present work investigated the effect of atRA in the hippocampus. The authors found that atRA can play a key role in modulating enduring forms of synaptic plasticity in the dentate gyrus of the hippocampus even when it does not seem to drive overt changes in basal synaptic function, and this "metaplasticity"-related effects also require synaptopodin. Together, these studies establish a critical role of atRA in modulating synaptic transmission and plasticity at multiple regions of the human brain.

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Evaluation Summary:

The manuscript by Yanofsky et al. describes the high-resolution structure of the CIII/CIV super-complex from Mycobacterium smegmatis bound to the anti-tuberculosis drug Q203 which is currently in clinical trials. The authors also provide biochemical data for inhibition of purified CIII/CIV and add important new information regarding the mechanism of Q203. This is an important contribution that will be of broad interest to the field of bioenergetic and tuberculosis, as telacebec represents one of few novel drug classes with potential for treatment of tuberculosis and other mycobacterial infections.

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Evaluation Summary:

This paper is of broad interest to virologists, immunologists, and structural biologists as it provides new insights into how the biophysical properties of viruses shape the development of immune responses. Overall, the data presented support key claims of the paper, and the strengths and limitations of the approach are properly acknowledged. However, the working model and its implications could be clarified and extended.

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Evaluation Summary:

This interesting genetics study shows that two well known tumor suppressor genes, ATR and Chk1, have a new function in sensing oxidative stress agents. The study is good quality and the results generally support the rather novel conclusions. It should be of interest in the fields of cancer genetics and cell biology.

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Evaluation Summary:

The authors demonstrate that selective inactivation of carnitine acetyltransferase (Crat) – a key metabolic enzyme – in AgRP neurons attenuates the response of AgRP neurons to peanut butter (PB) chips, the release of dopamine in the nucleus accumbens, and the motivation to work for food when mice are fasted. The strength of this study is the demonstration that metabolic sensing by AgRP neurons is somehow linked to dopamine release in the nucleus accumbens, but a weakness is that it is unclear how the lack of Crat in AgRP neurons affects their responsiveness to PB chips or how AgRP neurons regulate dopamine release.

Evaluation Summary:

In mammals the cellular retinaldehyde binding protein, CRALBP, is expressed in the pigment epithelium (RPE) and in Müller glial cells (MGCs) in the retina. Zebrafish has two copies of the gene, each expressed in one of the cell types. By knocking out each gene with CRISPR/Cas9, the authors could show that it is the copy expressed in the RPE that is essential for turnover of retinal and for cone function. Thus, the zebrafish gene duplication suggests that the RPE role of CRALBP is the important one also in humans, implying the RPE as target for future gene therapy in humans with mutations in CRALBP.

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Evaluation Summary:

This manuscript is of considerable interest to neuroendocrinologists and other neuroscientists because it provides important insights into mechanisms controlling the synchronous activity of a specific subpopulation of hypothalamic neurons. Luteinizing hormone is secreted from the pituitary gland in pulses which vary over the estrous cycle. The pulses arise by patterned secretion of a hypothalamic 'releasing factor', the secretion of which is itself governed by a population of hypothalamic neurons that express the neuropeptide kisspeptin. The paper by Voliotis et al. combines novel experimental evidence from transgenic mice with an elegant mathematical model to analyze how the kisspeptin neurons generate the varying pulsatile patterns.

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Evaluation Summary:

This technical paper describes a novel strategy for conditional mutagenesis in zebrafish. The method employs the authors' previously reported GeneWeld CRISPR/Cas9 targeted integration strategy to allow target genes to be "turned off" or "turned on" in a tissue-specific manner. Once fully validated, the approach would provide a valuable new addition to the "zebrafish genetic toolkit" that is likely to be widely used for assessing cell- and tissue-specific gene function in this model organism.

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Evaluation Summary:

This paper will be of interest to the large class of neuroscientists who perform network analyses and are interested in the processing of visual information. It sets a new standard in connectomic analysis because is combines EM data of a whole fly brain with fluorescent labeling of specific neurons. The key claims of the manuscript are well supported by the data, and the approaches used are thoughtful and rigorous.

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Evaluation Summary:

This manuscript investigates the plastic properties of synapses impinging on pyramidal neurons in the piriform cortex from the lateral olfactory tract (LOT) and intracortical inputs. Their findings uncover some of the location and pathway-dependent plasticity rules and challenge the notion that LOT inputs (carrying direct odor information from the bulb) become "hardwired" after the critical period. The results provide novel information about how activity and experience alter synaptic communication in the olfactory circuit in a synapse-type specific manner.

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Evaluation Summary:

This paper investigates the role of common human genetic variation in explaining the relationship between host genetics, red blood cell physiology, and susceptibility to Plasmodium falciparum (the parasite responsible for malaria). It finds evidence that common variants in a small set of red blood cell proteins predict parasite invasion and growth rates. Contrary to hypotheses about ancestry-associated malaria selection, these variants are not more common in African ancestry populations. The approach used to select host factors that impact parasite fitness is pragmatic especially in the context of a small sample size, but the high predictive accuracy (despite moderate within-subject assay replicability) and the uncertain influence of including closely related family members in the analysis, raises some concerns about generalizability beyond the study sample.

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Evaluation Summary:

The Codetta method developed by Shulgina and Eddy is a powerful approach for inferring codon reassignment by comparative analysis of bacterial and archaeal genomes. Given the rapid advances of genomic and metagenomic sequencing, this will be an important tool for elucidating the genetic codes employed by prokaryotes.

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Evaluation Summary:

Attempts to produce effective vaccines against HIV have not yet been successful, with the lack of understanding correlates of protection being a significant limitation. This paper analyses gene expression in a number of human and non-human primate vaccine trials and identifies a profile that appears to correlate with protection from infection. This profile is linked primarily to monocytes and the ability of these cells to mediate antibody dependent cellular phagocytosis. The work has implications for ongoing attempts to generate effective vaccines against HIV and perhaps other viral diseases.

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Evaluation Summary: 

This paper will be of interest across psychology and ecology. It attempts to replicate influential findings that imply Theory of Mind in food-caching decisions of Eurasian Jays. The authors' approach to both attempting to expand on and replicate earlier findings is both rigorous and thoroughly contextualized. The failure to reproduce earlier findings raises important questions for the field. 

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Evaluation Summary: 

This manuscript describes a novel tool for tracking synaptic plasticity at the single synapse resolution with a SEP-tagged GluA1 receptor expressed in a transgenic mouse. The authors rather convincingly demonstrate that this tool does not disturb synaptic physiology or mouse behavior. They also show that this tool can be used to measure the distribution of synaptic weights and its variation during a plasticity protocol in barrel cortex. This tool is useful for more quantitative measurements of synaptic strength in vivo, although some revisions would help making a convincing case of the usefulness of this tool with respect to previous methods. Genetic specificity of the expression of the construct is also a concern. 

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Evaluation Summary: 

This study investigates the important question of how vasopressin neurons, which are critical for fluid balance, are rapidly activated or inhibited when mice begin to eat or drink. The study presents useful anatomic data on connectivity between these neurons and other structures and tests a broad range of possible inputs that could mediate these effects. The conclusions are largely supported by the data. 

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Evaluation Summary:

Hwang et al. address the important question about whether specific thalamic sub-regions serve as essential "hubs" for interconnecting diverse cognitive processes. Using a group of patients with isolated thalamic lesions (n=20), and a group of size-matched lesions outside the thalamus (n=42), they report that lesions to the anterior-medio-dorsal thalamus are most likely to cause widespread cognitive deficit. Evidence from existing task-based and resting-state fMRI data sets, as well as data sets on gene expression further provide evidence the importance of these regions as a network hub for domain general cognitive functions.

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Evaluation Summary:

This is an interesting resource paper, describing the response of hematopoietic stem and progenitor cells to a few drugs commonly used to stress hematopoiesis. Transcriptomic analyses reveal interesting patterns, and the authors use ATAC-seq to investigate whether stem and progenitor subpopulations may be primed to respond in specific ways based on their chromatin accessibility. This turns out not to be the case when directly responsive genes are analyzed, and rather differences can be found in the promoter accessibility of genes further downstream.

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Evaluation Summary:

How pollen apertures are formed is not well understood. By studying Arabidopsis mutants producing pollen with aberrant aperture numbers, the authors identify proteins from the ELMOD protein family as important regulators for aperture formation. They use genetics, transgenic constructs, and site-directed mutagenesis to pinpoint important residues for protein function in this process, and show that changes in expression levels of one protein can have dramatic effects on patterning. This paper will interest scientists interested in cell polarity, patterning, and evolution of diverse morphologies. This is also the first study of the ELMOD protein family, whose potential GTPase activating activities have not yet been investigated in plants.

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Evaluation Summary: 

Using single cell RNA seq, the authors characterize the sensory neuron microenvironment in dorsal root ganglia after sciatic nerve crush, dorsal root crush and dorsal column transection spinal cord injury 3 days after injury. The data revealed differentially expressed genes and pathways with sciatic nerve and dorsal root crushes co-clustering, whereas spinal cord injury largely co-clusters with uninjured. The results reveal influences of the tissue microenvironment and neuron extrinsic factors on axonal regeneration and also provide new insights into the role of PPARa signaling in regeneration after dorsal root crush. This is an impressive data collection effort across multiple cell types that will be of importance for generating new hypotheses in the field. The impact could be further broadened by increased attention to functional validation of the findings.

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Evaluation Summary:

This work presented in this manuscript paper may advance our understanding of an important class of anti-viral drugs (nucleoside analogs) that target polymerase enzymes by being directly incorporated into the product strand. This class of drugs is known to be quite diverse in their precise mechanisms of action, yet many of the particular details have remained elusive, often due to experimental limitations. The current study employs a single-molecule magnetic-tweezers platform to provide a new paradigm for the mechanism of drug remdesivir against the SARS-CoV-2 polymerase target. The authors propose that remdesivir does not prevent the complete viral RNA synthesis but causes an increase of polymerase pausing and back tracking. This paper is of broad interest to readers and scientists working on SARS-CoV-2 and general nucleotide inhibitors. The work includes the single molecule characterization of other NAs.

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Evaluation Summary:

This paper will be of broad interest to chemists and biologists studying complex coacervate systems, including biomolecular condensates. Its model provides a new way of obtaining diffusion properties inside and outside the condensates without the necessity of nontrivial assumptions. The model's capability is well presented by applying to experimental data and through further investigating the model through simulations.

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Evaluation Summary:

This manuscript describes an analysis of cell type-specific alternative splicing using 10x scRNA-seq data. This work shows that in spite of the challenges associated with the analysis of such datasets, it is possible to identify alternative exons with differential splicing between tissue compartments and to some extent reveal cell types by splicing profiles of single cells. This work is informative regarding what can be done to analyse alternative splicing using 10X data and fills in a gap in the field.

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Evaluation Summary:

This study proposes the identification of "bivalent chromatin" in genes associated with the biosynthesis of secondary metabolites in Arabidopsis and describes an investigation into the role of chromatin states in the regulation of the major Arabidopsis phytoalexin. Perturbation of either H3K27me3 or H3K18ac levels using mutants were used to show that there were effects on the expression of these metabolic genes. It has previously been shown that H3K27me3 and H3K18ac colocalize in the Arabidopsis genome and that genes targeted by PRC2/H3K27me3 in Arabidopsis are enriched for genes that respond to the environment and/or developmental cues. Therefore, the reported changes to the regulation of these genes in defective mutants are as expected, although the finding of this study will still be of interest to those working on pathogen-induced changes to plant metabolism.

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Evaluation Summary:

Many algae, such as Chlamydomonas, form a pyrenoid under certain conditions to enable high photosynthetic rates during inorganic carbon limitation. The data presented here support that hydrogen peroxide, a common by-product of hyperoxia and CO2 limitation, induces pyrenoid formation in Chlamydomonas, even when CO2 levels are high. Although the underlying genetic mechanisms remain unresolved, these observations offer an exciting starting point to dissect the molecular components that drive pyrenoid formation. Therefore, this paper is of interest to a broad audience of scientists working in the areas of photosynthesis, synthetic biology of agriculture, and algal biotechnology.

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Evaluation Summary:

This paper will be of great interest to researchers interested in cognitive modulations of sensory processing as well as in the brain mechanisms of pain. It shows that attentional modulations of pain are associated with changes in neural communication between cortical areas, brainstem and spinal cord which are sensitive to opioidergic but not to noradrenergic modulations. These findings are conclusively supported by state-of-the-art simultaneous pharmacological fMRI of the brain and the spinal cord.

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Evaluation Summary:

This manuscript describes a very extensive set of experiments charting the origin and fate of various cell populations in the Drosophila ovary that is a powerful system to explore interactions between adult stem cells and their niches. The authors put forward a new view of how different cell types acquire their fates during development. This is a more nuanced view than extant models, involving common progenitors from which different cell fates (stem cell, progeny and niche cells) arise gradually and relying on spatiotemporal cues.

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Evaluation Summary:

This study by Wu et al., examined in a mouse model of Alzheimer how the lifespan and kinetics of both border-associated myeloid cells and microglia are affected. Taken together, these data provide evidence on the replenishment of CNS-associated myeloid cells under both steady-state and pathology.

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Evaluation Summary: 

Nguyen et al. examine how helminth co-infection alters shedding from a respiratory bacterial infection (Bordetella bronchiseptica), fitting a model to data from experimentally infected rabbits to link the presence/absence of two helminth species with immune responses (neutrophil and two antibody classes) and bacterial shedding. The authors find a larger frequency of intense bacterial shedding-supershedding events-among helminth-infected rabbits, and model results suggest that triple infection may be associated with faster bacterial replication in the respiratory tract and more rapid shedding of bacteria. Linking immune responses with infection outcomes is of enormous practical interest, as is identifying why certain hosts are superspreaders, but there are some limits to what can be gained from this data set and model framework. 

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Evaluation Summary: 

This paper reports a methodologically rigorous investigation into the neural mechanisms supporting encoding and retrieval of specific and general information in the context of memory schemas for events, or "scripts." Its findings will be of general interest to neuroscientists and cognitive psychologists who work both with typical young adults (as studied int he present works) and in particular populations (e.g., development and/or aging; patients with brain damage). The work is particularly comprehensive in how it links both specific and general narrative representation at both encoding and retrieval with later memory behavior, which is a notable strength. 

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Evaluation Summary: 

Kaanders et al. investigate how the sampling of visual information by human subjects is biased toward their previous choice. The novel experiments and rigorous analyses largely support the presence of a 'confirmation bias' when information sampling is under the subjects' control. After ruling out some remaining alternative explanations of the observed behavior the paper will be of broad interest in cognitive neuroscience. 

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Evaluation Summary: 

This paper establishes a behavioural paradigm to study concepts developed in the field of economic decision making using the tractable model organisms C. elegans. It is therefore interesting to scientists studying the neuronal mechanisms of decision making and animal cognition. 

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Evaluation Summary:

This manuscript provides an innovative merging of biophysical models with imaging data to explain the physical structure of microbial communities in the gut of zebrafish. Using imaging data to examine cluster sizes for eight different bacterial strains in the larval zebrafish gut, the authors report a common family of size distributions and show that these distributions arise naturally from a simple biophysical model of aggregation that tends to condense the system to a single massive cluster, reminiscent of gel formation observed in non-living systems. Within-host microbial dynamics represent an area of tremendous interest, as the microbiome is increasingly recognized to play a role in host physiology. This work contributes to a new perspective by elucidating physical mechanisms driving spatial segregation of these communities, opening the door to future studies that incorporate traditional genomic and microbiological insight with the physical and mechanical dynamics of microbial communities in living hosts.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

Evaluation Summary:

Koolschijn and colleagues present a novel and timely investigation of the balance between excitation and inhibition to explore the role of glutamate and GABA during memory retrieval. The innovative use of rapidly interleaved fMRI and fMRS provides a compelling link between successful retrieval effects in hippocampus and inhibitory/excitatory dynamics in visual cortex. The study itself is well-motivated and well executed, complementing prior cross-species work, and provides an intriguing set of results to support the major claims. This paper will be noteworthy to those interested in hippocampus-mediated cortical dynamics during memory retrieval. The rigorous methodology also demonstrates the utility of fMRS in investigating complex cognitive processes.

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Evaluation Summary: 

This is a well-executed study describing multiple new findings related to the association between enhancer activity as measured by massively parallel reporter assay (MPRA), chromatin features, and underlying TF binding profiles in the context of different transcriptional states of cutaneous melanoma. There are several technical and conceptual advances including the refinement of MPRA assays and the understanding of regulatory mechanisms controlling cell state-specific enhancer activity that will be valuable for other investigators to adapt the experimental design and data analysis strategies. This work will be of broad interest to those seeking to understand cell type- or cell identify-specific gene regulation at the level of transcription and epigenetic control using these and related approaches. 

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Evaluation Summary: 

This study, which makes a connection between several proteins known to regulate endothelial function in pulmonary arteries, may be of interest to vascular, pulmonary and ion channel physiologists. The study provides compelling evidence that ATP released from pulmonary artery endothelial cell (EC) pannexin1 channels activates TRPV4 channels via an EC P2Y2R-PLC-DAG-PKC alpha pathway that is facilitated by the scaffolding protein Caveolin-1 and that this pathway helps to maintain low pulmonary vascular resistance and pulmonary artery pressure. Identification of this pathway provides new drug targets to improve pulmonary endothelial function in disease states such characterized by impaired endothelial function. What remains to be established or understood is the physiological stimulus for activation of the pannexin1 channels and ATP release and also the potential dark-side of overactivity of EC TRPV4 channels, which appear to have negative effects on EC function.

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Evaluation Summary: 

This paper investigates the stability of the somatotopic organization of somatosensory cortex after spinal cord injury that results in tetraplegia. The findings suggest that somatotopic maps are preserved, degrading very slowly over decades, but that the amount of spared function is a poor predictor of somatotopic stability. These findings contribute to a developing story on how sensory representations are formed and maintained and has implications for the development of brain-machine interfaces for individuals with tetraplegia. The study is interesting and the manuscript is well-written. There remain concerns about some choices taken in the analyses. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

Evaluation Summary: 

This paper will be of significant interest to immunologists interested in understanding the determinants of antibody responses to vaccination. It uses tetramers to specifically identify and track CD4 T cell responses to influenza vaccination in younger and older adults, in an effort to understand why older individuals tend to have lower antibody responses to immunisation. The combination of tetramers, RNA sequencing and TCR clonotype tracking provides a powerful dataset to address fundamental questions of CD4 T cell immunology. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary: 

This paper describes the crystal structure of two key components of the autophagy system, the PI3P-binding protein WIPI2d in complex with its interaction region in the hATG8 E3 ligase scaffold component ATG16L1. The paper provides interesting new data and demonstrates the requirements for association of WIPI2d with membranes. Functional studies in cells provide evidence that mutation of residues at the interface for ATG16L1 binding affects function, although additional studies would support loss of function versus a dominant negative effect. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary: 

The theoretical study by Ecker et al. designs a minimal yet biologically plausible spiking neural network model for hippocampal region CA3 in order to pinpoint the mechanistic sources of important features of population activity (namely sharp wave ripples and replay) observed in vivo. It reproduces many properties of these network events and offers explanations for the observed dynamics. In doing so it demonstrates that the synaptic connectivity patterns formed during spatial exploration may be crucial to the occurrence of these phenomena. The study will be of interest primarily to theoretical researchers because of the many innovative approaches fielded to design the network and analyse its dynamics, and potentially also to experimentalists investigating the hippocampus. 

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Evaluation Summary: 

This study examined the mechanism underlying the development of prediction-error related responses of neurons in the primary visual cortex (V1) evoked by the mismatch between self-generated locomotor movement and visual feedback. The authors show that unilateral gene knockout of NMDA receptors or CaMKII in the primary visual cortex impaired the mismatch-related responses in V1. The experiments are well thought out and the paper is well presented. The results suggesting a role for local plasticity in V1 will be of great interest to those researchers interested in neural circuit development as well as cortical functions. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary: 

The conductance of CO2 into the chloroplast from the intercellular airspace is a key limitation to rates of net photosynthesis. Despite its importance, past work has been contradictory in what does and does not affect this mesophyll conductance. This paper takes a unique and multi-pronged approach to resolving the mechanisms of mesophyll conductance and proposing a transgenic approach for increasing it in C4 plants.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

Evaluation Summary: 

This study presents evidence supporting population and sex-specific selection and an epistatic interaction between variants in the genes ADCY9 and CETP of pharmacogenetic importance. The confluence of evidence from population genetics, gene expression, functional experiments, and phenotypic association lends support to the paper's claims beyond what may be achieved by a single analysis. All three reviewers and I agreed that this work is of high interest in medical and population genetics and addresses a challenging topic in an impactful way. 

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Evaluation Summary:

This manuscript presents a descriptive audit on statistically treatment, reporting and interpretation of the effects of sex as a biological variable (SABV) on the studied outcomes in articles published across nine scholarly disciplines. The manuscript highlights and provides data on prevalence of several inconsistencies and inaccuracies in the literature regarding treatment of SABV as an important moderator of the effects of an intervention on a considered outcome and how such inconsistencies could lead to biased conclusions regarding the effects of SABV. As such, the manuscript may inform not only funding agencies and grant reviewers, but also researchers in most scientific disciplines regarding the importance of adhering to rigorous methodological standards when examining the effects of SABV.

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Evaluation Summary:

This manuscript reports on two separate investigations. In the first, the authors provide novel evidence from two anaesthesia challenges that the slope of the 1/f structure of the power spectrum of the EEG fluctuates in a manner that tracks the presumed excitation : inhibition (E:I) balance of the tissue generating the EEG signal. Next they show that fluctuations in this slope also covary in systematic and modality- and stimulus-specific ways with behavioural performance on a multimodal attention task. These observations have potential foundational implications for how this previously unappreciated component of the EEG can be interpreted in terms of brain physiology and function. While the methodology employed is novel and interesting, the data as they stand, do not yet support the strong conclusions proposed by the authors.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

Evaluation Summary:

Wang et al. explores the relationship between the vigor of behavioral escape and cholecystokinin-expressing neurons in the premammillary nucleus of the hypothalamus that project to the periaqueductal gray. The experiments presented use a range of complementary approaches and techniques to perform a comprehensive examination of the circuitry underlying a particular aversively motivated behavior. However, a lack of clarity and specificity in how experiments and results are described, as well as a missed opportunity to relate this work to recent publications by many of the same authors, soften the conclusions that can be drawn from the manuscript in its current form.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

Evaluation Summary:

Using a range of cutting-edge techniques, the authors of this manuscript explore the functional consequences of developmental changes in dendritic morphology and synapse distribution in a GABAergic interneuron. The experiments are carefully performed and represent a thorough investigation of the structural and functional changes in synaptic inputs and postsynaptic dendritic branching patterns, an important topic for both developmental neurobiologists and synaptic physiologists. The data support most conclusions, but alternative interpretations remain possible and should be further considered.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary:

This report by LeClair et al. shows the importance of considering social dominance rank and history of winning/losing rank to define susceptibility to stress in mice. It has many strengths, including an elegant experimental design, including experiments in both males and females, and carefully considering two models of social defeat in females, and an excellent writing and representation of the data.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

Evaluation Summary:

Nordén et al. examine feather iridescent color diversity across bird species. Their findings show how key modifications in feather melanosomes, pivotal nanophotonic structures, underlie the brilliant colors of iridescent feathers, broadening feather color range approximately twofold. In a next step, the authors evaluate the function of feather melanosomes by performing optical modelling of nanostructure diversity, evaluating up to 4500 distinct nanostructure combinations, which are then contrasted with the observed (color) spectral data from 120 plumage regions across 80 (diverse) bird species. This meticulous integration of diverse methods across a comprehensive dataset will not only inform biologists studying structural color biodiversity, but it may also inspire engineers designing nanophotonic systems.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary:

Sleep serves vital functions for the body and particularly the brain, and accordingly these functions are impaired by sleep deprivation, as has been repeatedly shown for different cognitive processes. However, the neural mechanisms of such effects of sleep loss are still poorly understood. This manuscript is of interest to both sleep researchers and cognitive neuroscientists looking for insights into the effects of sleep deprivation across a broad range of methods and measures. The reported studies comprehensively investigate cortical excitability and plasticity with non-invasive brain stimulation, as well as electrophysiological markers and behavior. The studies confirm and extend previous findings, stating that, in general, sleep deprivation results in higher cortical excitability as well as a negative impact on cognitive processes.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary: 

This paper presents the results of a model for cortical plasticity and resulting increase in neuronal responses to unexpected stimuli. This is an elegant study that provides a number of interesting, experimentally testable, hypotheses and develops a prediction for a mechanism for novelty response generation. However, a number of concerns were raised about the model, including how it relates to certain experimental data, that should be addressed. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary: 

This manuscript applies the framework of information theory to lectin-glycan signaling modulating the NF-kappaB response. The paper suggests that the information transfer capacity and information flow through the signaling pathway may be affected by a combined action of two distinct receptors having different distributions across a cell population, with possible implications for the immune response. The paper can have an impact on our understanding of signaling through multiple receptors converging on the same output, and will be of interest to experts in cellular signaling, particularly those with interest in innate immune response. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary: 

This paper reports careful work using auxin-mediated degradation to manipulate the DAF-2 insulin/IGF-1 receptor in specific tissues and at different ages of the nematode C. elegans. Since its initial discovery as a gene that could dramatically alter lifespan in this organism, daf-2 has been extensively studied. The authors make excellent and thorough use of their novel reagents to successfully add important new findings to our understanding of this broadly conserved aging pathway, including a finer dissection of the spatial and temporal requirements for DAF-2 in multiple processes, such as the decision window for entering dauer arrest and that altering the levels of this protein very late in life can still have dramatic effects on lifespan. Overall the data are convincing, but the presentation and clarity of the text could be improved. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

Evaluation Summary: 

This study investigates the neural underpinnings of the bias property of timing, namely an overestimation for short and underestimation for long intervals, during an interval reproduction task in the medial prefrontal cortex of gerbils. The key novel result is that only neural populations with mixed responses, including ramping activity with linear increasing and slope-changing modulations as a function of reproduced durations, can encode the bias effect. Overall, experiment and data analysis are technically sound, and the conclusions are mostly well supported. However, the interpretation is too broad, and the manuscript would benefit of a more focused framing. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary: 

The ribonucleoprotein YBX1 is required for sorting of miR-233 to exosomes. Here, the authors demonstrate that YBX1 undergoes liquid liquid phase separation (LLPS) both in vitro and in vivo and YBX1 droplets specifically partition miR-233 and mediate its packaging into exosomes. The authors also demonstrate a possible connection between YBX1 condensates and P-bodies during cargo sorting into EVs. Overall, this is an elegant study and with a few additional experiments to clarify the involvement of P bodies, the story will be of broad impact to those interested in LLPS and RNA packaging into exosomes. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary: 

This study reports on the synaptic impact of basal forebrain stimulation on a population of olfactory bulb interneurons in acute mouse brain slices. The author reveals that optogenetic stimulation of GABAergic basal forebrain afferents by and large inhibits the discharge of periglomerular cells, whereas cholinergic afferents evoke a prolonged, M1 receptor-mediated depolarization and increase in firing in a subpopulation of periglomerular cells. The current study would further our understanding of the olfactory neural circuit and how different co-released neurotransmitters shape postsynaptic neuronal responses. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary: 

This manuscript has the potential to be of broad interest to scientists seeking to understand the evolutionary dynamics of plants during past periods of rapid climate change. Specifically, within the target genus of Cochlearia, the results indicate increased rates of speciation and diversification in response to pronounced glacial cycles. Future work to establish more direct mechanistic links between the results and conclusions will improve our understanding of adaptation and speciation. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

Evaluation Summary: 

Monitoring changes in pathogenic bacterial populations after bloodstream infection are poorly understood and in this work the authors develop new genetic analytic tools to dissect bacterial population dynamics at sites of infection. By harvesting bacteria from different sites and times of infection and performing deep sequencing to define the distribution of specific tagged-strains the authors provide a highly detailed snapshot of populations, with discriminatory power superior to prior studies. The work paves the way to future such studies in other organisms that cause persistent infection in humans. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary: 

This rigorous computational study simulates the sampling of the visual image by cone photoreceptors in the human eye, and explains how the image content can be reconstructed from those cone signals. The authors show that a number of properties of the human retina and of human perception are predicted from these simulations. The manuscript could be further improved by analysis of how these conclusions compare to those reached by alternate theoretical approaches, and by a consideration of human eye movements. 

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Evaluation Summary: 

In this study, the authors highlight a role for de novo biosynthesis of Poly-unsaturated Fatty Acids and the consequence effect of these metabolites on the production of arachidonic acid. The increased bio-availability of arachidonic acid seemingly promotes mycobacterial growth whilst inhibition of arachidonic acid formation, and its resultant downstream eicosanoid products, affect macrophage function but somewhat surprisingly do not affect growth of M. tuberculosis in macrophages or in mice. The uptake of the different classes of fatty acids in axenic culture as well as in macrophages is explored and the authors demonstrate that the Mce1 transporter is largely responsible for their uptake during in vitro growth but only plays a partial role in their uptake during growth of the pathogen in host cells. This work will be of interest to bacteriologists and those studying infectious diseases. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

Evaluation Summary:

This study addresses the structural basis of the ability of fatty acyl-AMP ligases (FAAL) to exclude condensation of activated fatty acids with coenzyme-A and facilitate the reaction with other 4-phosphopantetheine linked acceptors. This issue is of significant interest with regard to understanding how certain fatty acids are channeled to specific metabolic fates. The structural question at hand is the apparent discrimination of the CoA moiety (adenosine 3',5'-bisphosphate) versus a holo-ACP tethered to the 4-phosphopantethein head group. This work will contribute significantly to our current knowledge of how distinct classes of enzymes divert fatty acids to virulent lipids in mycobacteria, and it will be more broadly of interest for metabolic engineering.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

Evaluation Summary:

The paper presents a method for inferring the growth dynamics of bacteria from noisy single-cell data and applies this to C. glutamicum. Applying the method to experiments three phases of growth during the cell cycle are identified, including a dominant period of linear growth, which is interpreted in terms of a growth mode that is limited by polar cell wall synthesis.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary:

This research will be of interest to neuroscientists who want to understand how visual systems are tuned to and encode natural scenes. It reports that rats share phenomenology with humans in sensitivity to spatial correlations in scenes. This work shows that an earlier paper's hypothesis about efficient coding may be broadly applicable, but it is perhaps most interesting in opening up the possibility of studying this sort of visual tuning in an animal where invasive techniques can be used to study this sensitivity and its development.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary

This study uses non-invasive imaging to look at the coupling within and between layers and regions of the human visual cortex during the modulation of attention. The results presented here are a re-analysis of a previously recorded dataset, but the novelty is the analytic technique used to relate laminar connectivity to rhythms. This in principle promises to advance the field of both oscillations and laminar fMRI and could deliver valuable insights. The work provides a non-invasive window on how feedback and feedforward circuitry in the human brain operates. We deem the work of potential interest to a broad audience as it aims to provide direct links between the animal invasive electrophysiology and human neuroimaging fields. However, in its current form, major reservations with respect to the hypothesis space being explored here as well as important analytic and technical caveats remain.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

Evaluation Summary: 

Hülle cells, a type of cells formed by fungal species of the genus Aspergillus, are specialized cells that surround the sexual fruiting bodies of ascomycete fungi and are thought to nurse the fruiting bodies during fungal development. In this work, Liu et al. suggest that these cells have a strong ecological impact because they contain specific secondary metabolites that help the fungus to "withstand" the attack by fungivorous animals, like springtails, and also inhibit sexual reproduction of other fungi. This work will likely have a major impact on our view on the development and ecology of fungi as well as on the ecological functions of secondary metabolites. 

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Evaluation Summary: 

This work serves as an independent confirmation and expansion of previous work, suggesting that certain domains in fungal adhesins can form amyloid-like structures that are essential for cell-cell aggregation, but less so for cell-surface adhesion or invasive growth. Specifically, the expression of different adhesin constructs offers insight into the functional role of the different adhesin domains. Together, the results provide further insight into the molecular mechanisms underlying fungal adhesion, which does only further our understanding of basic fungal physiology, but also offers insight into fungal pathogenesis. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary: 

The unexpected key finding is that T-cell-independent B cell activation engages the intracellular protein kinase PKC-delta to induce the necessary machinery of altered gene transcription culminating in IgG production. The methodology and the experimental setting are well-controlled, although how exactly B-cell stimulation by T-cell-independent antigens triggers PKC-delta remains to be elucidated. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

Evaluation Summary: 

This paper by Piccolo and collaborators provides a general introduction to the common workflow language (CWL) with working examples taken from bioinformatics. The authors also introduce the ToolJig web application intended as a way of interactively creating CWL documents. This work should be of interest not only to beginner bioinformaticians but also to more experienced professionals that do not routinely make use of the latest developments in reproducible research. 

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Evaluation Summary: 

Poly-ADP-ribosylation (poly-ADPr) is a major histone modification that plays critical roles in DNA damage. However careful mechanistic dissection of the role of poly-ADPr has been challenging as the modification is found on multiple proteins and there is heterogeneity in terms of poly-ADP-ribosylation chain length and amino acid location of attachment. The PARP1-dependent semi-synthetic strategy developed by the authors allows generation of nucleosomes with mono ADP ribose and defined lengths of poly-ADPr chains at specific histone serine residues. The utility of this method is clearly demonstrated by the authors' findings that ALC1, a chromatin remodeler that recognizes poly-ADPr is stimulated substantially by the presence of poly-ADPr on H2A and H3. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary: 

The authors find that long-range interhemispheric synapses are selectively weakened following loss of function of the gene mediating fragile X syndrome, the most common inherited form of intellectual disability. Using clever genetic and physiological approaches in mice, the authors show that the effect is cell autonomous and occurs postnatally by impeding the normal developmental strengthening of these synapses. The results convincingly enhance our understanding of the complex pathophysiology of neurological dysfunction in this developmental disorder. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary:

This study describes a novel algorithm to perform fully automated sleep staging in humans. It is well validated and performs at the level of other state of the art algorithms although it does not yet include comparisons against the existing tools available in the field. Given the efforts made by the authors to ensure ease of use and accessibility it may help extend the use of automated methods in the study of sleep.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary:

The authors performed a systematic literature review and meta-analysis to develop a dataset of respiratory viral loads (rVLs) for SARS-CoV-2. Focus was on finding the relation between individual case characteristics (e.g. disease severity, age and sex) and lower and upper respiratory tract viral loads. The study appears robust and comprehensive, and the results are valuable and contribute to the scientific knowledge in this field.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

Evaluation Summary:

This study demonstrates for the first time that the single annotated fucosyltransferase (TbFUT1) in the parasitic protist, Trypanosoma brucei is specifically targeted to the mitochondrion, rather than in the secretory pathway or cytoplasm, as in other eukaryotes. TbFUT1 utilizes glycan substrates in vitro and conditional down-regulation of TbFUT1 expression causes a severe growth defect in the two major developmental stages of these pathogens, indicating that it is essential for pathogenesis and a potential drug target.

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Evaluation Summary: 

This paper examines the visual-ocular response in participants when exposed to the static magnetic field of a 3T MRI system. Historically, this problem has been approached from a safety perspective. In the present study, the authors ask about the behavioral consequences of this field given that it induces a response in the vestibular system, hypothesized to mimic that of a caloric vestibular stimulation event. As such, one should anticipate a biased vestibulo-ocular reflex in the static field as well as biases in spatial attention. These predictions were confirmed, with the attentional bias manifest in eye movements during a visual search task. This is an important finding because it reveals functional "artifacts" that may arise during fMRI studies, effects that may need to be considered by those conducting research in the MR environment (especially functional studies). 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

Evaluation Summary:

The question investigated - to understand the computational significance of different types of local interneurons in neural circuits - is an important and significant problem. Here authors elucidate the role of the two types of LNs, by combining whole-cell patch clamp recordings with calcium imaging via single cell dye injection. This method enables them to monitor calcium dynamics of the different axons and branches of single LNs in identified glomeruli of the antennal lobe, while the membrane potential can be recorded at the same time. They conclude that non-spiking LNs with graded responses show glomerular restricted responses to odorants and spiking LNs show similar responses across glomeruli.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary: 

This work, which will be of value to evolutionary and clinical microbiologists, uses a well-designed experimental evolution strategy to tease apart contributions of history, chance, and selection on the evolution of antibiotic resistance in A. baumannii, an important microbial pathogen. While relevant, the work will benefit from further clarification regarding some of the concepts and procedures used and revision of some of the interpretations. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

Evaluation Summary: 

This work will interest researchers who want to explore the functional potential in metagenomes. The authors present a new computational method, MetaGPA, for performing enrichment analysis on cohorts of metagenomes. They then use this approach to identify an enzyme that can modify cytosines in DNA from natural bacteriophage populations. Though successful, the approach needs to improve in clarity and methodology to be both reproducible and of broader impact, as claimed. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary:

Calcium-calmodulin (CaM) signaling plays an essential role within and outside of the nervous system. Moreover, it is conserved from plants to humans. While a lot is known about the mechanisms of cellular calcium level fluctuations, how CaM levels are regulated is less clear. In this manuscript, Vuong-Brender and colleagues characterize a, likely, conserved role of the transcription factor CAMT-1 in the homeostatic regulation of CaM levels and show how it impacts animal behavior and nervous system function. The paper is a tour-de-force across multiple techniques and model systems. The data is of a very high quality and supports most of the authors' claims strongly and convincingly.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary:

This paper will be of interest to neuroscientists in the fields of spinal motor control and spinal cord injury. The finding that silencing of spared lumbar to cervical long ascending propriospinal neurons after spinal cord injury enhances locomotor coordination is unexpected and well-supported by the data. This has potential implications for strategies aimed at promoting recovery of function after SCI.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary: 

In the study presented here, the authors used the fruitfly Drosophila melanogaster to identify a new molecular regulator - the E3 ubiquitin ligase Thin - of presynaptic homeostatic plasticity (PHP), a process where synaptic signaling between motorneurons and muscle cells is dynamically adapted to compensate exogenously imposed changes in synapse strength. Based on a very substantial set of high quality data, the authors propose that Thin functions presynaptically during PHP, that presynaptic thin negatively regulates neurotransmitter release under baseline conditions by limiting the number of release-ready synaptic vesicles, and that Thin controls transmitter release by regulating Dysbindin, a schizophrenia-susceptibility gene required for PHP. The authors' conclusion is that Thin links protein-degradation-dependent proteostasis of Dysbindin to homeostatic regulation of neurotransmitter release. The major claims of the paper are well supported by the data, but alternative hypotheses cannot yet be unequivocally excluded. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

Evaluation Summary: 

This manuscript describes a framework for rapidly quantifying relative extracellular vesicle (EV) yield and purity across isolation methods, with a focus on using size exclusion chromatography (SEC) for EV isolation from small volumes of pooled plasma and cerebrospinal fluid (CSF) samples. The authors used single molecule array (Simoa) assays for the quantification of EVs using three tetraspanins (CD9, CD63, and CD81), and report the outcomes of assessing EV yields and purity with respect to albumin by various SEC parameters (Sepharose size, column length, fractions collected). This is the first demonstration of the use of Simoa with three commonly used tetraspanins to measure EVs from small volumes of CSF, of great relevance to human CSF biomarker studies, but these methods could also be applied to compare EV isolation methods from other fluids such as cell culture media. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

Evaluation Summary: 

Kaplan and colleagues build upon their earlier work using genetic phenotypes to find and analyze mutations that determine how mRNA start sites are chosen. Here they provide convincing genetic evidence supporting a model in which the Transcription Factor II H (TFIIH) protein complex pushes downstream DNA back into the RNA polymerase active site, creating a window within which the polymerase can choose particular start sites. This will primarily interest those in the transcription field who are thinking about initiation mechanisms. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary: 

In this manuscript the authors use a combination of transcriptomics, metabolomics, and quantitative measurements of growth to characterize the temporal and spatial distribution of cells with different metabolic states within colony of biofilms of the model bacterium Escherichia coli. They show that within the biofilm cells performing different metabolic functions are distributed in different regions of the colonies, and propose a model where nutrient cross-feeding through the amino acid alanine explains the phenotypic heterogeneity observed within the biofilm. The findings reported have potential broad implications for thinking about the spatial structure of communities of all bacterial species. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary:

The manuscript introduces a new molecular screen, MassTitr, to screen for long (36-mer) peptides derived from the human proteome that can bind a specific target. The method is demonstrated using the EVH1 domain of the actin-associated ENAH protein as target. About 100 peptides were isolated, and further analysis identified sequence features that contribute to the binding of the EVH1 domain by these peptides. The human proteome contains many short linear motifs of 4-6 residues that are critical for protein-protein interactions. The work here helps to better understand how the sequence surrounding such motifs contributes to protein-protein interactions.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

Evaluation Summary: 

Local recycling of synaptic vesicles is required to maintain neurotransmission via clathrin-mediated endocytosis and clathrin-independent ultrafast endocytosis. Clathrin also plays a role in ultrafast endocytosis to regenerate vesicles from a recycling endosome. Here the authors have further tested the role of clathrin and clathrin adaptors in synaptic vesicle endocytosis. This paper raises the interesting possibility that adaptor protein AP-2 but not clathrin contributes to the endocytosis of synaptic vesicle proteins. There are some concerns about differential knockdown of clathrin and AP-2 but if the authors can resolve these, this would be an important result. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary:

In this work, Minh Nguyen and colleagues performed single nuclear RNAseq of human dorsal root ganglion (DRG) neurons and classified them into 15 clusters. A bioinformatic comparison to mouse lumbar DRG single nucleus sequencing results is also described. The importance of reporting the single nucleus or single cell molecular profiles of human DRG cannot be overstated. Proper molecular targeting of therapeutics requires knowing this information. Given that the field is just starting to understand the human specific molecular signature of primary somatosensory neurons using single cell/nuclear RNAseq, this study is important and timely, providing one of the first gene expression databases of individual human DRG neurons.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors)

Evaluation Summary:

This manuscript describes follow-up studies on a hit from a proteome-wide screen for peptides that can bind to the EVH1 domain of the ENAH protein, one of three highly similar Ena/VASP actin regulators. The hit investigated is from a protein called PCARE, which selectively binds to ENAH but not the other two members of the Ena/VASP family, EVL and VASP. The authors provide a good explanation for how this selectivity is achieved and develop a peptide, PCARE-Dual, that specifically binds ENAH more tightly, setting out the stage for developing potent and selective inhibitors of ENAH activity. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

Evaluation Summary:

This manuscript is of primary interest to readers in the field of infectious diseases especially the ones involved in COVID-19 research. The identification of immunological signatures caused by SARS-CoV-2 in HIV-infected individuals is important not only to better predict disease outcomes but also to predict vaccine efficacy and to potentially identify sources of viral variants. In here, the authors leverage a combination of clinical parameters, limited virologic information and extensive flow cytometry data to reach descriptive conclusions.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary: 

This manuscript reveals new molecular details about the human calcium sensing receptor (CaSR), a G protein-coupled receptor that maintains calcium homeostasis and is involved in pathological states. This study, together with other recent structural work on this molecule, will have important implications for the design of molecules that control CaSR activity and treat human disease. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

Evaluation Summary:

These are pressing times for nature, standing alone the impact of multiple (human-based) ecological stressors. Wildlife trade is one of these stressors. And, although it is an acute one, it is the easiest solvable global ecological problem. The authors increase dramatically our understanding of legal and illegal trade of amphibians, and offer a wider methodology (however, and importantly, not necessarily a more complex one) to gain a deeper understanding of the causes and consequences) of amphibians' trade. The work will inspire in conservation biologists similar approaches to learn about the trade of other taxa.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary: 

The authors demonstrate deficits in perceptual tests related to fine-time perception in non-speech and speech sounds in a group of patients with stroke aphasia compared to a control group without a lesion. An area in left auditory cortex is defined that is essential for fine-time perception that is shown in a separate group of normal subjects to other areas including the cerebellum. The work in interesting in suggesting an anatomical basis for interaction between cortical and cerebellar system for perceptual timing. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary:

The pleiotropic effects of beneficial mutations have been characterized in various settings, but it is less clear whether and how these pleiotropic patterns change over the course of evolution. Using a technically innovative and intensive experimental design with evolving yeast populations, the authors show that patterns of pleiotropy depend on the evolution environment and can change and vary substantially over relatively short timescales. They also find a surprising amount of variation among replicate populations that increases over time, so generalism or specialism is not deterministic. These technical and conceptual strengths were diminished by insufficient focus on the details of certain treatments that are demonstrative of these broader findings, making the take-home message somewhat unclear.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

Evaluation Summary: 

The manuscript by Lee and co-workers describes the development of 21 unique transgenic mouse lines that express optogenetic sensors and effectors in a cell lineage-specific fashion. The knock-in approach allows the sensors and effectors to be rapidly combined or moved to different backgrounds, such as genetic disease models. Such manipulations are often impractical when using a Cre-based system. This constitutes a vital advantage for many studies. The new mice described here will be very powerful tools to study physiology and alteration in disease models. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary: 

This paper claims to identify a long-sought ligand for the receptor protein tyrosine phosphatase Lar that mediates its functions in neuromuscular junction development, mushroom body development, and photoreceptor axon targeting. If correct, this would be of interest to many developmental neurobiologists, However, further evidence is needed to strongly support this claim. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary: 

This study shows how astrocytic MCL1 can contribute to postnatal maturation of the brain vascular system. Since the development and physiological roles of perivascular astrocyte coverage are not well understood, this manuscript provides potentially important frame works and should be of interest to the broad fields of neuroscientists. 

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

Evaluation Summary:

This study describes an immunopeptidomic-based pipeline to discover new tumor antigens for the development of cancer vaccines. The pipeline is relatively straightforward and exploits molecular mimicry and tumor pathogen cross-reactive T-cells and would be interesting for cancer immunologists. If the utility of the pipeline were demonstrated in more diverse systems, including carcinogen-induced tumors and human settings, this work would provide an immediate impact to the immuno-oncology field and personalized cancer vaccine development.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)