Evaluation Summary:

The authors describe a newly developed software, ProteInfer, that analyses protein sequences to predict their functions. It is based on a single convolutional neural network scan for all known domains in parallel. This software provides a convincing approach for all computational scientists as well as experimentalists working near the interface of machine learning and molecular biology.

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Evaluation Summary:

The authors aim to tackle a fundamental question with their study: whether there is a direct age-associated increase of transcriptional noise. To investigate this question, they develop tools to analyze single-cell sequencing data from mouse and human aging datasets. Ultimately, application of their novel tool (Scallop) suggests that transcriptional noise does not change with age, changes in transcriptional noise can be attributed to other sources such as subtle shifts in cell identity. This study is in principle of broad interest, but it currently lacks a definitive demonstration of the robustness of Scallop. Systematic testing of this new package would ultimately strengthen the key conclusion of the work and give additional users more confidence when using the tool to estimate expression noise.

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Evaluation Summary:

In addition to providing extensive proteomics profiling datasets. this manuscript is fundamental that sheds light on the importance of appropriate experimental design for mouse disease model which have been overlooked so far. The results look quite solid based on the proper methodology. This type of work is extremely valuable to many biomedical scientists in the field for conducting reproducible research especially in the preclinical studies and properly interpreting the results.

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Evaluation Summary:

This paper will be of interest to scientists within the field of neuromuscular disorders and has potential clinical relevance. It reveals a novel targeted strategy to improve the pathophysiology of children with neonatal brachial plexus injury. 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 is a paper that will be of particular interest to neuroscientists with a focus on food intake and neural responses to food ingestion. This paper provides new insights into how the body responds to weight loss and helps identify those that may not be successful.

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Evaluation Summary:

The authors use theoretical models to examine the joint evolution of different cheating strategies: selfish cheating (not contributing to a common good), and manipulative cheating (inducing a competitor to preferentially provide benefits to the cheat). The models seem well formulated and the results robust. That said, improvements could be made to the presentation to clarify the assumptions and wider applicability of the model. An improved article would provide a better understanding of the mechanisms behind cheating, which would be of interest to readers working on the evolution of cooperation, potentially opening up new directions for theoretical and empirical work.

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Evaluation Summary:

This work has potential value for researchers in several areas of cognitive and systems neuroscience. Range adaptation is a widespread property in neuronal circuits, and a network mechanism that relates neuronal adaptation to behavioral outputs is a valuable addition to the literature. However, limitations in the current framing and analyses leave some uncertainty about the interpretation of the results and their broader applicability.

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Evaluation Summary:

This manuscript tackles the timely and interesting research question of whether meningeal lymphatic drainage is required for the control of brain infection with Toxoplasma gondii. It contains a sophisticated experimental approach using cutting-edge methods, it has an easy-to-follow narrative, and comes up with an interesting albeit negative finding which the authors even tried to explain by an additional set of experiments. Although there are some limitations and weaknesses of the paper in its present form it will certainly contribute to the growing body of literature on how the once "immune-privileged" CNS is protected against environmental challenges.

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Evaluation Summary:

This manuscript shows that mutations in the gene encoding an enhancer chromatin-modifying enzyme MLL3 cooperate with Myc overexpression to drive hepatocellular carcinoma in mouse models. The authors identify Cyclin Dependent Kinase Inhibitor 2A (Cdkn2a) as a critical direct target gene of MLL3. Overall, the manuscript makes a compelling case that MLL3 is a hepatocellular carcinoma tumor suppressor that directly binds and activates the Cdkn2a locus. This study provides important insights for cancer biologists and those interested in specific epigenetic mechanisms that regulate liver cancer development. Editorial and some experimental suggestions were made to strengthen the work.

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Evaluation Summary:

By performing homeostatic longitudinal IgH repertoire analysis of human memory B cells and plasma cells, authors draw two major unique conclusions; first, a high degree of clonal persistence in individual memory B cell subsets with inter individual convergence in memory B and plasma cells; second, reactivation of persisting memory B cells with new rounds of affinity maturation during proliferation and differentiation into plasma cells. These conclusions provide a significant insight into how human memory B and plasma cells are generated in a homeostatic condition.

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Evaluation Summary:

Keine et al study the role of the RhoGTPase Rac1 in neurotransmitter release by ablating this protein at an age when synapses are in an almost mature stage. They describe an increase in synaptic strength, which they interpret as an increase in release probability or fusogenicity of synaptic vesicles. They also describe subtle effects in the timing of release, which point towards a mild defect in positional priming. The study delivers important information on the role of Rac1.

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Evaluation Summary:

This study develops a resource allocation model for E. coli growing under steady-state conditions. The model describes both growth rate and yield and has been subjected to validation by comparison with a compiled data set. The manuscript addresses an important problem of interest to a wide range of investigators. At the same time, the authors would need to explore different assumptions for the housekeeping proteome fraction (phi_q) to ensure the model is robust.

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Evaluation Summary:

The current study examined in detail the role of oxytocin neurons in the hypothalamus in regulating food intake. The current study extends our understanding of the role of this peptide in regulating complex behaviors.

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Evaluation Summary:

Hu and colleagues describe the discovery and characterization of a new class of reversible palmitoylation (PLM) binding site TEAD inhibitors. X-ray co-crystallographic analysis reveals that the ligand class, identified from a screen of 30,000 small molecules, binds to a new site within the auto-PLM site. The TM2 lead compound inhibits the growth of NF2-deficient cell lines. The discovery has the potential to significantly impact the design and development of new effective TEAD inhibitors. Some clarification or additional data are required to support and justify some of the authors' claims regarding the molecular significance of this new class of inhibitors.

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Evaluation Summary:

Recent studies of the brainstem locus coeruleus (LC) noradrenaline system have demonstrated a partially modular organization in which specific classes of neurons can serve distinct functions or exhibit module-specific co-activity. However, how noradrenaline cell classes function in a modular way is not clear. The authors have accomplished a technical feat by recording up to eight LC neurons at once using ex-vivo, multi-patch recordings. In doing so, two empirically-derived classes of LC neurons were identified and the analysis of electrical coupling between these neurons established some principles of local circuit communication occurring preferentially within the defined cell classes.

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Evaluation Summary:

This manuscript is of interest to individuals working on genome stability and B lymphocyte development. Using knockouts for the genes encoding the structure-selective endonucleases GEN1 and MUS81 in mice, the authors show that the absence of both proteins is incompatible with embryonic development, with selective loss in mature B-cells inhibiting germinal center formation. This is the first study of these enzymes in an organismic context and in primary cells, revealing insight into the in vivo consequences of loss of MUS81 and GEN1 functions not previously accessible through studies in cultured cells.

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Evaluation Summary:

This paper explores the use of 2D high-resolution template-matching (2DTM) to locate and discriminate highly similar macromolecules within cryo-EM images of focused ion beam-milled cells. It demonstrates that differences in the 2DTM signal-to-noise ratios for located targets against multiple search templates can effectively segregate a mixed population of similar structures, as well as present a formal analysis strategy for probabilistic assignment of species within the mixed population. Because the identification of distinct structural states of macromolecular complexes inside the cell is a fundamental problem in 3D visual proteomics, this paper will be of broad interest to both structural and cell biologists.

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Evaluation Summary:

The notion of transcription factors as composed of interchangeable parts where DNA binding activity can be separated from activation activity has been a dominant paradigm in molecular biology for decades. However, recent evidence suggests that activation domains may contribute to binding specificity as well. This paper describes the use of single-molecule imaging of endogenously tagged transcription factors to dissect how transcription factors move in the nucleus and how these dynamics are related to functional protein domains. These results will be of interest to the transcription and gene regulation fields, but the conclusions require additional experimental support.

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Evaluation Summary:

The authors describe their work on an atlas of associations between polygenic scores for 129 different traits representing a variety of quantitative phenotypes and diseases, and a large set of metabolites measured in up to 83,000 participants in the UK Biobank. These associations are all available via a public browser, and may be used to identify candidate intermediate phenotypes, as well as potential biomarkers of disease.

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Evaluation Summary:

This study investigates the potential role of kin selection in driving social behaviours among siblings in wild mountain gorillas. Using an impressive dataset of 14 years for 157 individuals the authors find some evidence for kin recognition in guiding biases for affiliative and aggressive behaviours. However, the results of the current study will be more convincing if a number of major concerns with the analysis can be addressed.

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Evaluation Summary:

This paper will be of broad interest to clinicians and scientists in the area, providing clinical trial data on how the efficacy of monoclonal antibodies targeting SARS-CoV-2 varies according to the variant of concern. The clinical outcome data were consistent with previously reported in vitro data, which are being used to inform the clinical use of monoclonal antibodies. However, as the trial was stopped early, conclusions regarding the efficacy and safety of the individual monoclonal antibodies are limited.

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Evaluation Summary:

The origin of virulence in pathogenic microbes is not understood for many microbial species. The concept of 'accidental virulence' was proposed as a mechanism by which a microbe could acquire the capacity for virulence through interaction with other microbial species, such as amoeba. This paper adds an important new dimension to that concept by showing that the capacity for virulence can emerge from abiotic interactions, such as adherence to plastic.

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Evaluation Summary:

The work by Volante et al. studied a new plasmid partition system, in which the authors discovered that four or more contiguous ParS sequence repeats are required to assemble a stable partitioning ParAB complex and to activate the ParA ATPase. The work reveals a new plasmid partitioning mechanism in which the mechanic property of DNA and its interaction with the partition complex may drive the directional movement of the plasmid.

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Evaluation Summary:

In this interesting study the authors combined innovative object-based conflict assays with optogenetic silencing to probe the role of the perirhinal cortex in motivational conflict. The manuscript is well-written and the approach was adequate. The findings provide new insight into how conflicting motivation is processed and would benefit from additional analysis and experimental investigation to more conclusively support the interpretations.

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Evaluation Summary:

In this manuscript, the authors identify enhancer-associated (e)RNAs that are specifically associated with esophageal adenocarcinoma. Based on combining the data with analyses of patient gene expression data and epigenetic data from cell lines, they conclude that eRNAs are markers of enhancers relevant to the transition from Barrett's esophagus to cancer. This work provides new insights into the epigenetic alterations that occur in cancer progression, and it will be of interest to the cancer and epigenetics fields.

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Evaluation Summary:

This paper describes results obtained from multi-cellular imaging of CA1 cells using large-field-of-view miniscopes in rats performing a shock avoidance task. By exploiting behavioral (barriers) and pharmacological (scopolamine) manipulations the authors explore cell remapping dynamics during aversive learning. This work will be of interest to the neuroscience community by setting new methodological standards and providing data for across-species comparisons.

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Evaluation Summary:

The current paper is of interest to cell biologists studying ciliogenesis and specifically vertebrate photoreceptors, which are specialized cilia. The study shows that mutations in the small GTP binding protein ARL3 known to cause dominant inherited retinal dystrophies in humans result in ARL3 hyperactivity, disrupting the normal ciliary gradient of ARL3 activity and leading to altered retinal development. The authors demonstrate restored normal nuclear distribution by overexpression of ARL3 effectors, suggesting that the active mutants disrupt nuclear migration, at least in part, by sequestering ARL3 effectors. Overall, the experiments are properly controlled, executed, and analyzed and involve a series of extensive biochemical analyses complemented with in vivo phenotypic assessment. The development of a method to analyze snapshots of the interaction between ARL3 and its interactors is also a strength of the paper, however, significant concerns remain regarding links between nuclear migration failure and ciliogenesis in the outer segment, and alternative possibilities that could explain the phenotype of the ARL3 Y90C mutant with respect to its sequestration of the GEF ARL13B. Addressing these major concerns would improve the manuscript and could have considerable impact on the field.

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Evaluation Summary:

The authors show the utility of an AAV-based approach in non-human primates to develop an improved model of Huntington's disease. They have presented a very thorough, carefully executed, body of work that will be of benefit to a range of researchers studying HD or developing therapies for HD. While this extends the work from an earlier paper (that presented the tools used to induce phenotypes) the results presented are new, relevant, and important to the community.

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Evaluation Summary:

In this work, the authors test a multivalent vaccine design they term Mos-tri-RBD, consisting of three linked spike receptor binding domains, one based on Omicron sub-lineage BA.1 and the others with different SARS-CoV-2 variant mutations. Immunization with this construct either as a prime or booster vaccine resulted in better neutralization of the Omicron and Beta variants relative to the same design, but with the ancestral receptor binding domain, and supports the notion that vaccination with variant sequences may broaden the neutralization capacity of vaccines against divergent variants.

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Evaluation Summary

This study investigates the question of whether distinct brain areas differentially encode time during the learning of a simple motor timing task. The key novel result is that early in training the dynamics of the medial prefrontal cortex (mPFC) provides the best code for time, but later in training, the basal ganglia and in particular, the striatum provides a better code. In addition, the study shows that inactivation of mPFC produces a delayed learning effect, while inactivation of the striatum after learning led to impaired performance. The observation that temporal coding and the necessity of brain area for task performance transfers from medial prefrontal cortex to the striatum during learning is an intriguing observation for our understanding of the neural mechanisms underlying temporal processing in sensorimotor control.

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Evaluation Summary:

In this manuscript, the authors explore the circuit mechanism underlying mating-induced change of odor preference in Drosophila. Olfactory cues during mating induce a long-lasting increase in attraction to polyamines in female flies. The authors use a combination of neurogenetics, imaging, and behaviour to identify elements of the mushroom body and lateral horn circuitry involved in this behaviour. The importance of mushroom body plasticity in female postmating changes highlights a novel pathway for these changes and reveals the variety of mechanisms by which the brain can encode experience and adapt behavior. This paper will be of interest to scientists within the field of reproductive behaviors and neuroscience of internal states.

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Evaluation Summary:

This study uses media-based conversion of stem cell cultures towards to investigate how cell cycle regulation affects the transition of cell populations between pluripotent and differentiated states. Through a detailed analysis of cell cycle properties in different primed subpopulations, under a range of growth conditions, the authors propose that both the maintenance of pluripotency as well as the conversion towards a more differentiated state is influenced by selective shortening of the cell cycle in different primed subpopulations. By using new reporter systems and long-term imaging, this study thus sheds new light on the old question of whether extracellular signals control differentiation in cell populations through selection or induction.

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Evaluation Summary:

Prior work has linked posterior urethral valves (PUV), a common cause of end stage renal disease in children, with chromosomal abnormalities and rare copy number variants, but the genetic causes of PUV remain incompletely defined. In this study, the authors have used diverse ancestry whole-genome sequencing association studies to identify two novel genes and an enrichment of rare duplications and inversions affecting candidate cis-regulatory elements as possible causes of this rare condition, illustrating the potential for this approach to other rare conditions.

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Evaluation Summary:

This manuscript reports a new method for high-throughput analysis of C. elegans feeding behaviour that overcomes shortcomings of existing methods. It is a useful technique that will be interesting for scientists studying feeding dynamics in worms.

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Evaluation Summary:

This manuscript describes cellular and developmental defects at late embryonic stages during Wolbachia-induced cytoplasmic incompatibility (CI), which occurs when male insects harboring the endosymbiont bacteria Wolbachia fertilize eggs of uninfected females, triggering embryonic lethality, usually at the first nuclear division. This work presents evidence that the mechanism of late embryonic defects is independent from the ones responsible for early embryonic defects. The experiments are technically superb, and the strength of evidence provided is compelling, including beautiful single-embryo PCR analyses and convincing light microscopy. While the overall significance might be limited, the knowledge will be useful to those in the fields of cytoplasmic incompatibilities and insect embryo development.

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Evaluation Summary:

This manuscript, contains fundamental findings that substantially advance understanding of an important research question, mostly uses appropriate and validated methodology in line with the current state-of-the-art, with good support for the claims, and the message of the manuscript will have a profound and lasting influence on neuroscience. In essence, the manuscript reports that dopamine converts spike-timing-dependent synaptic depression into potentiation that requires cAMP/PKA second messenger cascade and protein synthesis. The mechanism enables a separate synaptic input to induce heterosynaptic potentiation in previously primed synapses, which is shown in a network model to have desirable computational properties. The significance of the findings is threefold: First, it is the longest-lasting synaptic eligibility trace identified so far; second, the mechanism enables memory linking between temporally separate events; and third, it indicates a novel function of postsynaptic reactivation events. In addition, the finding may inspire new reinforcement learning algorithms in machine learning.

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Evaluation Summary:

This work will be of interest to the field of researchers that generate human stem cell-derived kidney organoids to model genetic kidney diseases. It describes a novel and crucial role of the protein CEP83 in mesoderm patterning, which further determines whether kidney tissues can form correctly. Using cutting-edge technologies the authors provide strong data, which support the key claims of this manuscript. This work is of high impact due to the relevance of CEP83 mutations in human kidney disease.

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Evaluation Summary:

This manuscript describes de novo dominant toxic mutations in CRMP1 in 3 probands with a shared neurodevelopmental phenotype. The authors show that the mutations lead to reduced protein production from recombinant expression and that the mutations correlate with shorter neurites in cultured cells. This is the first report of mutations in CRMP1 in humans, encoding a cytoskeletal regulator protein. The results could have implications for physicians, geneticists, neurodevelopmental scientists, and cell biologists.

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Evaluation Summary:

The study is of interest to researchers in the field of cell biology, especially mechanosensing. The work identifies a new context to evaluate the activity of MSL proteins in mechanosensing by identifying two novel suppressors of MSL10 as components of the ER-PM contact sites (EPCS). The work has significance for both the plant and the animal science community providing the basics for various avenues of further research in the area of mechanobiology.

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Evaluation Summary:

This manuscript tackles the important question of what proteins regulate asymmetrical cell division in Mycobacteria. This will be of interest to all individuals interested in bacterial physiology. The data are sound, but some of the conclusions need to be tempered or bolstered, in relation to the models proposed.

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Evaluation Summary:

This manuscript is an important body of work that addresses the role of the integrated stress response (ISR) and the role of the GCN2 protein kinase in prostate cancer. The studies comprehensively elucidate how GCN2 and amino acid transporters and uptake promote prostate cancer proliferation, as well as the therapeutic potential of inhibiting this pathway. This work, therefore, provides insights for both identification of new mechanisms and experimental therapeutics in prostate cancer.

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Evaluation Summary:

Fluid flows in networks are ubiquitous, and in many living systems the networks are not static but instead can rearrange over time. Using vascular networks formed by the slime mold Physarum polycephalum, Marcbach et al. demonstrate that there is a time delay between the change in the flow and the change in the network geometry. They present a mechanical model of vein-radius adaptation leveraging the negative normal stress response of the actin cytoskeletal network lining the vein walls. More generally, the authors make use of the unique advantage of this simple model vascular system to connect the local shear rate to the network reorganisation and how it depends on its architecture. There are features to their work that are new to the literature and that can be impactful in advancing the field.

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Evaluation Summary:

This manuscript examines the role of transposable element (TE) expression and the transcription factor FOXO on aging of Drosophila melanogaster. Increased TE expression in aged organisms compared to their younger counterparts has been observed in several animals, including Drosophila. Here, the authors show that artificially inducing transcription of a specific TE can reduce fly lifespan and exacerbate some aging phenotypes-paraquat resistance and rhythmicity. The authors also argue that the detrimental effects of increased TE expression can be rescued by FOXO expression, although this is less convincing.

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Evaluation Summary:

This paper establishes a visually mediated gap-jumping behavioral task in freely moving mice, and shows that mice can perform the task using only monocular cues with little performance deficit, perhaps at the cost of additional active sensing movements before executing the jumping maneuver. Further, using acute optogenetic inhibition, the authors establish that the primary visual cortex is used to perform this task. Using vision to judge distance - such as the width of a gap to be crossed - is crucial for survival across taxa, and this new paradigm could be informative to those interested in using mice to study such vision-based estimation under naturalistic conditions.

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Evaluation Summary:

Yang et al. provide a scientifically sound and compelling manuscript characterizing mid-to-late gestation trophoblast and decidual organoids as ex vivo models to study vertically transmitted microbial infections, using human cytomegalovirus as a model pathogen. They demonstrate organoids have tissue-specific immunological responses and susceptibilities to viral infection.

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Evaluation Summary:

This paper will be of interest to researchers in the fields of motor control, visual perception, learning and brain plasticity, sight loss and rehabilitation. The paper shows the contributions of sensory-motor experience to the development of visuo-motor recalibration abilities using careful experimental methods and analyses, comparing a rare population of late-operated cataract patients with control groups.

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Evaluation summary

Ribonucleotide reductases (RNRs) have fascinated biologists and chemists, as these enzymes catalyze the conversion of ribonucleotides (NDPs or NTPs) to deoxynucleotides (dNDP or dNTPs), which are essential for DNA biosynthesis in all organisms. Given this role, they have been postulated to be the link in the transition from an RNA/protein to a DNA world. In addition, RNRs use an array of protein, metal-based, and nucleotide radicals for the reaction they catalyze. This paper creatively combines two methods of analysis to propose a new evolutionary model for the diversification observed for the RNR family into the three classes: I, II and III. The work is of interest to students of molecular evolution, RNRs and colleagues interested in the origin of life.

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Evaluation Summary:

The role of satellite glial cells in the sympathetic nervous system has not been extensively investigated. Using targeted ablation of SGCs, the authors demonstrate that satellite glia has a profound effect on neuronal activity and the survival of sympathetic neurons. The peripheral sympathetic system is responsible for a wide spectrum of activities, including blood flow, heart rate, respiration, and digestion.

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Evaluation Summary:

Kikumoto and colleagues explore the question of how stimulus- and response-related mental representations are stored and selected in working memory. The authors use a combination of decoding and representational similarity analysis on EEG data to provide evidence for conjunctive representations of action plans. This work would potentially be of great interest to readers in the field of working memory, motor preparation, and selective attention.

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Evaluation Summary:

This paper presents an exploitation/exploration paradigm using a model-based approach in 18 patients treated with GPi DBS for Tourette's syndrome. Their main observation is that despite DBS (used as a proxy of GPi inhibition) doesn't have any effect on the overall performance of the subjects, it has a significant effect on the probability of exploration. This work will be interesting for scientists working in fundamental and clinical neurosciences.

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Evaluation Summary:

In this highly innovative study, the authors use combinatorial gene expression analysis to study the development of the gill arch of the little skate. This process depends on Shh and Fgf ligand-derived endodermal cells at the endoderm-ectoderm junction, providing insight into not only the fundamental developmental mechanisms regulating brachial arch formation in cartilaginous fishes, but also highlighting a unique relationship between inhibition of Wnt and Hedgehog signaling pathways in the context of early appendage development. The work will be of interest to developmental biologists and colleagues studying Wnt and Hedgehog signaling pathways.

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Evaluation Summary:

This work is valuable for those who study how diet and metabolism impact neurological function, specifically learning and memory since it investigates the impact of high-fat diet intake during the preadolescent period on memory performances. The data convincingly showed the possibility to reverse memory deficits related to obesity by manipulating selected hippocampal circuits. The claims would benefit from additional controls and analyses.

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Evaluation Summary:

This paper, of interest to both basic scientists and clinicians, addresses the clinically important condition of reduced muscle mass in human chronic liver disease. It seeks causative mechanisms under these conditions. It uses in vivo and in vitro techniques to draw associations between bile acid concentrations and muscle disease. They implicate a specific bile acid for the first time.

(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 derivation of cardiomyocytes from the first and second heart fields is a well-studied phenomenon in animal models, however, due to ethical concerns, has not been studied in human heart development. The authors utilize hiPSC technology to demonstrate that it is the FHF and SHF that give rise to cardiomyocytes which is an important step in furthering our understanding of early human heart development.

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Evaluation Summary:

Detecting and quantifying balancing selection is a notoriously difficult challenge. In this study, the authors use both empirical analyses and simulations to characterize the amount of balancing selection in the human genome, focusing specifically on the contribution of polymorphic deletions. These results will be of interest to population and human geneticists. Although the presented evidence supports some degree of balancing selection among shared ancient polymorphisms, these findings primarily rely on the elimination of alternative explanations rather than a direct estimation of the extent of balancing selection. The conclusions are also based on simulations of a single demographic model without testing the robustness to other plausible model parameters.

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Evaluation Summary:

This manuscript is of broad interest to readers in the field of Alzheimer's disease, neurodegeneration, and single-cell omics. The identification of shared pathways across different cell types and ages is an important contribution to our understanding of APOE4 gene regulation in a cell type-specific manner. A combination of snRNAseq in APOE mouse models and human iPSC cells supports the key claims in the paper.

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Evaluation Summary:

This study will interest basic and clinical scientists and, potentially, device manufacturers interested in the regulation of heart rate in health and disease. A major control of the heart is from the nervous system originating in a neuronal cluster sitting outside the heart called the stellate ganglia. This study has identified the neural code associated with a healthy heart and describes how it changes in disease. Whether the change in code is cause or effect remains equivocal although normalising the code may have valued therapeutic benefit. The study opens the way for sophisticated mimicking of healthy neural code applied to a diseased heart as a potential electroceutical approach.

(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:

DNA double-strand breaks are a major threat to genome stability. In this study, the roles of two DNA repair proteins, Brc-1 and Smc-5, are investigated in C. elegans meiotic cells, to investigate the DSB repair pathways using the homolog or the sister chromatid as template . The results highlight a regulatory role of Brc-1 and Smc-5 as repressors of repair with the sister chromatid. The experiments are generally well executed, and the findings will be of interest to the DNA repair and C. elegans meiosis communities.

(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:

The study presented in this manuscript is of interest to cartilage biologists studying the mechanisms of chondrocyte differentiation. The authors investigated transcriptomic profiles of hESC-derived articular and growth plate chondrocytes. To characterize the regulatory landscapes with respective transcriptomes, they mapped chromatin accessibility in hESC derived chondrocyte lineages and mouse embryonic chondrocytes using ATAC-sequencing and revealed lineage-specific gene regulatory networks. They further validated functional interactions of two transcription factors, Runx2 and RELA, with their predicted genomic targets. This study could help us understand chondrocyte differentiation.

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Evaluation Summary:

This study presents novel experimental data from a mutant mouse model lacking microglia (Pu.1-/- mouse line), indicating that these cells have an important role in the embryonic establishment of critical neural circuits in the brainstem generating breathing motor behavior in mice. This paper is of interest to scientists within the field of microglia as well as respiratory neurobiology as it provides original key information about a new role of microglia in the embryonic development of respiratory circuits. Overall, the data are clearly presented and rigorous. Some of the conclusions should be toned down as the data in another microglia depletion model do not support some claims of the paper.

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Evaluation Summary:

Apoptotic regulators have long been known to often be expressed in pairs of pro- and anti-apoptotic isoforms. This demonstration of how a program of these splicing changes contributes to immune responses adds an important new understanding of both apoptosis and T cell biology.

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Evaluation Summary:

This paper's central findings - that wtf genes are old, rapidly evolving, and often meiotic drivers - are important and of broad interest to evolutionary biologists and geneticists. The study's main claims are supported by convincing evidence from comparative genomic data, phylogenetic analyses, and functional experiments. However, support for the verbal model of wtf persistence is currently incomplete.

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Evaluation Summary:

This work applies hybrid-capture sequencing for coronavirus (CoV) surveillance in bats. Given that bats are a major reservoir for animal-to-human virus spillover events, which have caused several major epidemics/pandemics, this is a very important field of research. The reported hybrid-capture method shows some clear advantages over amplicon-based viral sequencing, which is the established standard in the field. This new approach has clear merits that are well supported by the data presented and is likely to become an important tool in viral surveillance programs that ultimately aim to predict/prevent/prepare for future pandemics. The work will be of interest to microbiologists, particularly those studying viruses or interested in genomics surveillance.

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Evaluation Summary:

The current manuscript presents a computational model of numerosity estimation. The model relies on center-surround contrast filters at different spatial scales with divisive normalization between their responses. Using dot arrays as visual stimuli, it is shown that the summed normalized responses of the filters are sensitive to numerosity and insensitive to the low-level visual features of dot size and spacing. Importantly, the model provides an explanation of various spatial and temporal illusions in visual numerosity perception.

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Evaluation Summary:

The human small heat shock protein (sHSP) HSPB5 is an ATP-independent molecular chaperone involved in maintaining protein homeostasis. This manuscript reports on dynamic interactions between the disordered N-terminal region (NTR) and the structured alpha-crystallin domain (ACD) in HSPB5 oligomers. The authors show that two mutations, associated with early cataract and myopathy development, disrupt the interaction of the ACD core with the unfolded NTRs and generate a much more dynamic and hyperactive version of the chaperone. These findings will be of interest to colleagues studying molecular chaperones and their implications for disease 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 manuscript is of broad interest to researchers working in the area of lower urinary tract dysfunction. It describes a novel method to reliably study bladder function; the approach allows for monitoring bladder filling and emptying in freely moving, non-anaesthetized animals without the need for catheter implantation. This work has optimized a machine learning algorithm for defining the outline of the urinary bladder border from fluoroscopic images of mice that received subcutaneous injections of iodinated radiocontrast media. The advantage is that with images taken at 30 images/second and with monitoring bladder dynamics requiring hours-long observation periods, this very large number of generated images no longer requires manual analysis.

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Evaluation Summary:

The identification of an autoinhibitory mechanism of the dynein intermediate chain provides an important contribution to our understanding of dynein assembly and illustrates the plethora of regulatory mechanisms attainable by intrinsically disordered proteins. This paper provides insight into the autoinhibited inactive state of dynein as well as the activation mechanism. A wide range of biophysical approaches is used, providing a very nice example of how these diverse technologies can be applied in concert and in a synergistic manner to study an important question in the realm of "unstructured biology".

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Evaluation Summary:

The paper describes an interesting, but very abstract extension of normative choice theories. By linking economic and foraging theory, the paper would potentially be of interest to a broad audience in behavioral economics and neuroscience. However, the results in their current form have several important limitations: the lack of a significant validation, such as an account for well-known behavioral or neural effects that would not be explained by alternative theories, a quantitative performance comparison between the proposed EDM and other models in realistic behavioral situations, and a specific link between the actual processes and limitations of real brains and the EDM.

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Evaluation Summary:

This study reports candidate causal genes in genome-wide association studies that exhibit a discordant pattern of association, namely a higher waist-hip ratio simultaneously with a lowered risk of type 2 diabetes. Identification of such genes could provide insights into why some individuals with obesity are not developing type 2 diabetes, knowledge that ultimately could shed light on the complex interplay between fat distribution and type 2 diabetes. The work is of relevance to the fields of genetics of diabetes and obesity.

(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:

Advances in the discovery of novel anti-platelet therapeutics remains an unmet need. This manuscript by Chaurasia et al. describes a novel signaling pathway involving Notch1 and its ligand, Delta-like ligand-4 (DLL4) in driving platelet activation and thrombus formation. The authors provide convincing mechanistic studies to show that blockade of this pathway may serve as a new therapeutic approach to prevent/treat thrombosis. The work will be of great interest to individuals in the hematology and thrombosis 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. The reviewers remained anonymous to the authors.)

Evaluation Summary:

The authors describe new approaches to improve the analysis of single-molecule tracking data to uncover multiple diffusive states of proteins in living cells. This paper will be of interest to researches from the fields of experimental biology, who are interested in tracking of proteins using microscopy, as well as computational scientists who are interested in devising novel methodologies for analysis of multiple-particle tracking data. The paper presents two advanced techniques for estimation of motion parameters (such as diffusion coefficients) and contains rigorous evaluation using simulated and real biological 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 #3 agreed to share their name with the authors.)

Evaluation Summary:

The shrimp market is growing globally, with 8.12 tons produced in 2020. The market size is thought to reach $55 billion by 2027. Most of the market comes from farms, the white shrimp, Penaeus vannamei, being one of the most commonly farmed species worldwide. The present study provides a single cell transcriptional atlas of the white shrimp, P. vannamei, immune cells in the hemolymph, known as hemocytes. White shrimp single cell RNA sequencing studies uncovered two macrophage-like populations, one of them with markers similar to mammalian macrophages. These findings redefine the current classification of shrimp immune cells which has been done using morphological approaches and via targeted qPCR studies but never using single cell transcriptomics.

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Evaluation Summary:

PLK4 is the master regulator of centriole biogenesis, but whether it is also key for centriole amplification during differentiation of multiciliated cells (MCCs) has been questioned based on PLK4 chemical inhibition. Here, using mouse models engineered to lack PLK4 or PLK4 activity, LoMastro et al provide very compelling evidence that PLK4 and its activity are essential for centriole amplification in MCCs. Moreover, they show that centriole amplification in MCCs drives expansion of their apical surface. The findings will be of interest to cell biologists and experts interested in multi-ciliogenesis-related pathologies.

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Evaluation Summary:

To precisely diagnose DM2 caused by CCTG repetition in CNBP, the authors established a Cas9-mediated target enrichment system followed by Nanopore sequencing and analysis. The authors are fully aware of the limitations of the current diagnostic tests of DM2 and efficiently presented what novel findings have been revealed by the Cas9 nanopore sequencing. The findings of the current study suggest that Cas9 nanopore sequencing can be very useful for accurate genetic diagnosis of DM2 and understanding the genotype-phenotype correlation of this disease.

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Evaluation Summary:

This manuscript will be of broad interest to the field of muscle biology, muscle physiology, exercise physiology, metabolism and circadian rhythms. This manuscript identifies a new molecular pathway that connects circadian rhythms to muscle structure and function through titin isoform switching.

(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 identifies an important role for a lytic polysaccharide monooxygenase in allorecognition in the filamentous fungus Neurospora crassa, which is independent of the catalytic activity of this remarkable class of proteins. The study's findings are strongly supported through an interdisciplinary approach, combining microscopy with genetics and biochemistry. The study will be of great interest to fungal biologists and microbiologists, as well as biochemists studying carbohydrate-active enzymes.

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Evaluation Summary:

This manuscript reports that Epac2, a downstream effector of cAMP, positively regulates cocaine reward by altering dopamine release properties in the striatum. These results provide insight into Epac2 as a potential presynaptic molecular target through which dopamine signaling and drug taking might be manipulated and is of interest to scientists studying dopamine transmission and substance use disorders.

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Evaluation Summary:

This paper will be of broad interest to those working on the regulation of gene expression and mycobacteria as it deals with the collaboration of two important transcription regulators. A combination of experiments indicates how a complex of two regulators selectively turns on gene expression of a few genes in intracellular pathogen Mycobacterium tuberculosis.

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Evaluation Summary:

This manuscript addresses the important question of how cell types acquire regional identity during embryonic development. The authors study the role of TBXT in the establishment of posterior identity and show unexpected temporally restricted and cell-specific modes of acquisition of posterior identities in neural crest and spinal cord cells. They conclude that Wnt signaling influences posterior identity acquisition in neural crest cells whereas FGF is the main driver for spinal cord axial patterning.

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Evaluation Summary:

This paper addresses an interesting an important topic bearing on the initiation of labor at the end of pregnancy, invoking interleukin-33 in an alteration of Ca2+ homeostasis in uterine smooth muscle. The study implicated altered IL-33 expression in the third trimester of pregnancy in the endoplasmic reticular stress that might be involved in initiating labor.

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Evaluation Summary:

This manuscript provides new insights into one of the most enigmatic brain regions; the posterior cingulate cortex. Using electrophysiological recordings from dorsal and ventral PCC subregions, the authors provide evidence for a dorsal-executive and ventral-episodic functional subdivision. This paper will be of high interest to a broad range of neuroscientists.

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Evaluation Summary:

This paper is of interest to neuroscientists studying the organization of neural activity and of behavior. The authors link the apparently scale-free distributions of behavioral metrics with scale-free distributions of neural activity, and then explore computationally mechanistic models that could account for these observations. While the alternative view set up in the introduction - that scale-free neural activity is "'background activity', not linked to behavior" - is perhaps overly simplistic, the analysis is thorough, and the mechanistic insights garnered from the computational modeling are intriguing.

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Evaluation Summary:

The authors use a mouse stroke model to address a potential cellular source of functional recovery. Using multiple lineage tracing paradigms, they show that undifferentiated progenitor cells that migrate from the subventricular zone produce trophic factors including VEGF that promote functional and cellular recovery. These findings will be of interest to the neuroscience community, and those who study neural repair.

(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:

In this study, the authors assess the decline of retinal function in a mouse model of slow photoreceptor degeneration. The authors use a linear-nonlinear receptive field model to characterize functional changes across some RGC populations and information theory to assess the fidelity of RGC signaling. They show remarkable preservation of cone-driven ganglion cell light responses in advanced stages of a retinitis pigmentosa model when most rods have died, and cone morphologies are dramatically altered. The results are presented clearly in the text and figures and are scholarly discussed. However, there are several technical and conceptual concerns with the conclusions that can be drawn.

(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:

Meechan et al. present a systematic approach to semi-automate an ultramicrotome operation for targeting a specific plane aided by x-ray tomography measurements. It is a fundamental work of great interest to any users of using electron microscopy (EM), particularly when targeting the imaging of thin sections in a select region of interest by ultramicrotomy, or when targeting volume EM of select sample regions. The manuscript documents with exceptional detail a workflow including both microtome modifications and software adaptations for semi-automated targeting of structures with micrometer precision, resulting in a faster and more accurate orientation of the image acquisition planes for volume electron microscopy, a task that has traditionally been difficult and time-consuming. Therefore, this work will reduce sample preparation labor and, critically, facilitate the comparison of the ultrastructure of multiple samples. The method is based on X-ray imaging acquisition prior to any sectioning and proposes a solution for the two instruments commercially available in the field, and by transparently sharing all the data, hardware, and software, and by describing every detail of the workflow, this fundamental method can be readily adopted by any practitioner, enabling its wide application - it is a key step in the field regarding speed-up, accuracy, and reproducibility in electron microscopy.

(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:

In this manuscript, the authors developed a sensitive single particle tracking method for endogenous AMPA receptors. They found that AMPAR-containing vesicles showed reduced mobility near stimulation sites, likely due to increased F-actin bundling in dendritic shafts. The study found a novel mechanism of AMPAR trafficking using state-of-the-art labeling and analysis techniques, and thus will be of great interest for broad audience. However, their conclusion requires additional experimental support.

(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:

Roux and colleagues measured spiking activity and local field potentials predominantly from the hippocampus and also a few surrounding structures in the medial temporal lobe from patients with pharmacologically intractable epilepsy while the patients performed a cued-recall task. They report differences in local spike-field coherence measurements between hits and misses in the gamma frequency band and differences in both local and distal spike-field coherence measurements between hits and misses in the theta frequency band. The authors further report differences in the timing of spikes between pairs of neurons, with hits correlated with putative downstream neurons firing about 30 ms after putative upstream neurons and misses correlated with delays of about 60 ms. Overall, these are interesting observations that provide intriguing data to further think about how neurons in the medial temporal lobe correlate with recognition memory.

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Evaluation Summary:

In this important paper, Chen and colleagues identify a species of fungus, Ophiocordyceps sp. BRM1, that is able to grow on Aglaia sp. plants despite their production of rocaglate inhibitors of the eIF4A translation initiation factor. Through a series of solid experiments, the authors identify an amino acid substitution encoded in the fungal eIF4A gene that preserves eIF4A activity in the presence of these compounds. The authors conclude the substitution evolved to bypass this defense mechanism, similar to the way in which the plant itself bypasses it. The work will be of interest to fungal biologists and colleagues studying plant-microbe interactions.

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Evaluation Summary:

This article may be of interest to researchers working on predator-prey interactions in the fields of biomechanics and neurosensory biology. It presents a mathematical model that outputs possible escape trajectories given parameters relevant to the predator-prey system of interest. The premise of the modeling is attractive, as it includes the time required for prey to turn, but the methods as presently reported raise questions about the validity of some of the conclusions.

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Evaluation Summary:

Embryonic behavior is a widespread phenomenon that remains poorly understood in any system. Ardiel et al. describe new quantitative methods for imaging late embryo behavior in C. elegans, which will be of great interest as a technical innovation. They identify a novel rhythmic behavior (which they call slow wave twitch) in very late embryogenesis that includes repeated periods of quiescence, and show that this behavior depends on a known pro-sleep neuron and neuropeptide. Although the biological function of the rhythmic sleep behavior is unclear, it has the potential to serve as a model for understanding the mechanisms and purposes of sleep in other model organisms.

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Evaluation Summary:

This valuable paper addresses an important question about the neuroanatomical markers of individual and sex differences in sleep spindle frequency. The authors report associations between an anatomical marker - the length of the white matter fibre bundles underlying the thalamocortical loop and sleep spindle frequency, and highlight that the length of the white matter projections from the thalamus to the frontal cortex mediates sex differences in the sleep spindle frequency. This work advances the field of sleep and brain research by showing for the first time the association between the anatomy of a specific brain network and specific functional characteristics.

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Evaluation Summary:

The authors study the requirement for Lis1, a dynein binding protein, in T cells, and present solid evidence that the requirement differs between different T cell lineages, suggesting cell division mechanisms differ across these cell lineages. This work is valuable for cell biologists and immunologists interested in mechanisms that contribute to cell proliferation and differentiation.

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Evaluation Summary:

This methodological manuscript is of potential interest to the audience in the fields of neural development, tissue morphogenesis, and image analysis technologies. The authors developed an image registration tool and created a digital atlas to reflect the anatomical distribution of neuronal birthdates in the developing zebrafish hindbrain. The manuscript would further benefit from better documentation of the claimed temporal dynamics, the methods, and the validity of biological inference.

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Evaluation Summary:

This highly interesting manuscript will be relevant to colleagues studying cancer and those developing cancer therapies. The work describes the use of a large-scale CRISPR screen to identify mechanisms underlying resistance to the hypomethylating anti-cancer agent decitabine, which acts by inhibiting the DNA methyltransferase DNMT1. A specific focus is given to allosteric mechanisms of resistance that emerge, including those that appear to act as gain-of-function mutations in both DNMT1 and its interacting partner UHRF1. These findings showcase the power of large-scale genomic editing screens for the discovery of novel drug resistance mechanisms, which may guide the development of next-generation cancer therapies.

(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 is of broad interest to readers in the field of animal behavior and the evolution of cooperation. This work experimentally investigates the effect of differences in group size and group composition on reproductive behavior, by using an impressive sample of semi-wild populations of ostriches. While the paper does not address some aspects of groups, such as relatedness and parentage, overall, this paper is a complete analysis of the breeding ecology of this system and can serve as a blueprint for more of such work in the fields of cooperation, group living and breeding ecology.

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Evaluation Summary:

Trichomonas vaginalis is a sexually transmitted protist that colonizes its host by transitioning from flagellar locomotion to an adherent ameboid movement. In this manuscript, Wang and coauthors use a wide range of experimental approaches to investigate the function of a novel actin capping protein in T. vaginalis cytoadherence and cell motility. The work provides an intriguing example of how an unusual capping protein may impact cytoskeletal organization and cell behavior.

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Evaluation Summary:

This simulation study is of interest to geneticists, especially those carrying out Genome-wide Association Studies (GWAS). It compares two major approaches for dealing with "population structure"in GWAS: Principal Component Analysis (PCA) and Linear Mixed-effects Models (LMMs). This is a subject of considerable practical importance and the study nicely reviews the theoretical underpinnings and concludes - based on the review and the extensive simulations - that there is every reason to believe LMMs to be superior (although PCA is more widely used). Although this point has been made before, it is worth making again given the ubiquity of these analyses. There are some concerns about the general validity of the claim given that the simulations fail to address several real-world problems.

(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 presents evidence that the adipocyte-derived protein Ism1, which signals through a typical receptor tyrosine kinase, induces unique phosphoproteome signatures when compared to insulin, and regulates skeletal muscle force production. The manuscript should be of interest to those who study integrated physiology and skeletal muscle physiology. While the data suggest there may be some effects on myofiber size, further study is needed before any conclusions can be made as to what, if any, effects Ism1 has on myofiber size.

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Evaluation Summary:

Herneisen et al provide a comprehensive and thorough exploration of Ca2+ responsive changes in the Toxoplasma proteome and the resulting phosphorylation events during the transition from intracellular residing parasites to egress from the host cell. Furthermore, a novel temperature stability profiling method of all proteins responding to Ca2+ concentration with a change in stability is a novel applicable tool that here is used to map Ca2+-responsive proteins in the parasites. They provide a compelling analysis of the complex data and carefully validate their findings using genetics and cell biology. This work is of the highest quality in the field.

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Evaluation Summary:

The present manuscript provides a valuable single-cell transcriptomic resource to understand normal hematopoiesis in humans and the age-dependent cellular and molecular alterations. It addresses very important questions in hematopoietic stem cell biology, such as the molecular changes underlying their aging and their perturbation in the context of myelodysplastic syndrome, and will be of interest to readers in the field of hematopoiesis and associated diseases, aging, and single-cell RNA sequencing.

(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 uses a mouse model of hyperacusis to further explore the hypothesis that this condition may be mediated by cortical hyperactivity. The authors here provide interesting optogenetic and calcium imaging experiments that reinforce this hypothesis and refine our understanding of the related plastic changes that are involved.

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Evaluation Summary:

This is a carefully designed and analysed fMRI study investigating how neural representations in the hippocampus, entorhinal cortex, and ventromedial prefrontal cortex change as a function of local and global spatial learning. It will be of much interest to researchers studying the differentiation and integration of memories and the formation of cognitive maps. The results provide new insight into how local and global knowledge about our environment is represented, but some of the conclusions and interpretations could be strengthened with additional analyses.

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Evaluation Summary:

The work details a new acquisition method of defocus corrected large area cryo-EM (DeCo-LACE). The data-acquisition approach is highly complementary to the research group's previous work of using high-resolution 2D template-matching (2DTM) to identify macromolecular complexes in dense and heterogeneous cellular specimens. Notably and importantly, the data-acquisition approach minimizes sampling bias. Overall, DeCo-LACE is a very interesting approach to locating large ribosomal subunits in FIB-lamella at scale.

(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:

In this study, Trolle et al aimed to introduce methionine, threonine, isoleucine, and valine biosynthetic pathways into Chinese Hamster Ovary (CHO) cells. While this was unsuccessful for methionine, threonine, and isoleucine, introduction of valine synthesis rendered CHO cells partially independent on exogenous valine. Although introduction of essential amino acid biosynthetic pathways into mammalian cells is of potentially broad interest to the fields of synthetic biology, biotechnology and metabolism, there were concerns regarding incomplete demonstration that the introduction of valine pathway into CHO cells is sufficient to sustain homeostasis in the absence of exogenous valine. Further metabolic/biochemical characterization of valine-producing CHO cells is warranted.

(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:

Remarkably, Katydids, insects related to grasshoppers and crickets, have ears in their left and right forelegs. Pulver and colleagues show convincingly how two specialized chambers lining the hearing organs function as sound resonators that effectively boost the perception of high ultrasonic frequencies. This enables Katydids to detect the echolocating pulses of their bat predators before they home in on them for a meal. This study uses an impressive combination of approaches, but the manuscript would be improved by greater clarity.

(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 idea of individual aging trajectories of single cells is important and the authors provide sufficient evidence that there is some stochasticity that directs individual cells towards certain routes of aging - at least in budding yeast. Investigating the link between rDNA silencing and protein homeostasis, this study thus addresses an interesting and exciting question. The authors show how age-dependent loss of rDNA silencing might contribute to protein aggregation. Importantly, the paper furthers the understanding of distinct aging trajectories and raises important questions about how these processes might be relevant in multicellular organisms.

(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 increased use of gene and exome sequencing of individuals for diagnostic purposes has led to the identification of numerous single nucleotide variants (SNVs). However, annotating the probable clinical significance of every newly identified variant relies on multiple criteria, and in silico predictions can be used by curation experts to classify variants in databases. Since the reliability of such predictions is of paramount importance, this study compares the performance of 31 computational tools in classifying the pathogenicity of SNVs in the human adult globin genes and proposes an improved approach to achieve balanced predictions. The paper will be of interest to scientists and clinicians in the field of hemoglobinopathies.

(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:

By integrating in silico predictions and mass-spectrometry, this manuscript tackles the problem of annotating the currently nameless stretches of genomic sequence that actually code for proteins. The hundreds of protein coding fruit fly genes described here offer new inroads for studying some of the very youngest functional elements in genomes, particularly those that have recently emerged from non-coding DNA sequences. To clarify the biological significance of the present study, the authors should both highlight the genes mostly like to encode functional products and conduct a comparison to published datasets that used different methods to identify such genes in fruit flies.

(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 article constructs a four-step assessment of the informativeness of a clinical trial that measures its feasibility, reporting, importance, and risk of bias. This is a potentially highly relevant methodology for the class of trials for which it is defined, namely 'clinically directed randomized controlled trials'. It could also be translated and validated in other areas, using data from a wider set of sources beyond the trial registry clinicaltrials.gov. However, the extended longitudinal nature of the assessment and its potential subjectivity limit this tool's utility to being a retrospective diagnostic rather than as a prospective diagnostic and/or fix for at-risk designs.

(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. All three Reviewers agreed to share their names with the authors.)

Evaluation Summary:

Goering and colleagues investigate subcellular RNA localization across different cell types and species. The major insight is that there may be general mechanisms and specifically conserved proteins that regulate RNA localization in diverse cell types and morphologies. This manuscript will be of interest to those studying gene expression and how its regulation occurs within the cell.

(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 explores a topic of high interest to cell and cancer biologists - the role of actin polymerization, and here specifically the role of fascin, in the nucleus. The authors show that fascin regulates nuclear actin, chromatin organization, response to DNA damage, and demonstrate the need for control of steady-state nuclear levels to avoid cell death. Studying nuclear actin is technically challenging, and the authors deploy some novel technologies towards this goal. There are some very elegant experiments in this paper that suggest fascin has an important role in regulating nuclear actin and other important aspects of cancer cell behaviour. The work could be enhanced by the authors considering adding some additional experiments and providing clarifications and some further details or discussion.

(This preprint has been reviewed by eLife. We include the joint public review from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. All three Reviewers agreed to share their names with the authors.)

Evaluation Summary:

This is an excellent manuscript that addresses the role of the molecule (RETICULON 1A / RTN1A) in the biology of human Langerhans cells (the epidermal resident dendritic cell). The study shows that RTN1A critically regulates the retention within the epidermis versus the emigration from the epidermis of these cells. Since Langerhans cells are central in the induction of immune responses (e.g. in vaccinations, allergic hypersensitivities) as well as in the maintenance of immunological tolerance (e.g. in autoimmune diseases of the skin) this manuscript will be of wide interest to the scientific community working in the fields of immunology/vaccinology, dermatology, cell biology and beyond.

(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 extends previous work from the same group on the mechanism of 5' splice site recognition using co-localization single-molecule spectroscopy. There are three important conclusions: 1) the association of the U1 snRNP with the 5' splice site is largely determined by the snRNP itself and does not require other splicing factors; 2) sequence features of the 5' splice site determine whether a short-lived complex with U1 dissociates or transitions into a longer-lived, "productive" complex, potentially mediated by stabilized contacts with U1 associated proteins; and 3) the ability to form the longer-lived complex cannot be accurately predicted by base-pairing potential alone, as presumed by many predictive algorithms. Currently, a test for the role of specific protein-RNA contacts is lacking; additionally, a comparison with other nucleic acid recognition events is missing, particularly those also showing a two-step binding mechanism. This work will be of interest to colleagues in the splicing field as well as to others in fields where nucleic acid recognition by snRNPs plays a major role.

(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 work will be of interest to biological and clinical specialists interested in the fields of behavioural neuroscience, biological psychiatry, neuroendocrinology, and developmental psychology for its focus on the origins of adult aggressive behavior in early life stress. The authors used an unbiased transcriptomic analysis and identified the thyroid hormone system as a potential mediator of the enduring impact of early stress and aberrant aggressive behavior in adulthood.

(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.)