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.

(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 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.

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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.

(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:

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.

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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.

(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 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.

(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 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.

(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 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.

(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:

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.

(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 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.

(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 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.

(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 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.

(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 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.

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Evaluation Summary:

This paper provides evidence on public opinion from six European countries on key attributes according to which they believe COVID-19 vaccines should be prioritized. The paper presents significant and valuable findings supported by solid evidence.

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Evaluation Summary:

This article is of potential interest to researchers working on primate social behaviour, as it presents a novel mechanism for how an association with non-relatives can be favoured under kin selection. In a wild mandrill population, mothers are observed to preferentially lead offspring to associate with paternal half-sibs, a potential mechanism for encouraging nepotistic interactions between their offspring and other members of their group. The authors' explanation for their results was considered to be only partially supported by the data and a more measured and nuanced presentation would be appropriate.

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Evaluation Summary:

In this manuscript, Li et al directly compare different editing strategies for human pluripotent stem cells. They demonstrate that prime editing is more efficient and precise, compared with double-strand break-based methods. They also confirm the suitability of prime editing for the introduction of different mutations related to Parkinson's disease as a model.

(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. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary:

This manuscript will be of interest to researchers working in chronobiology and metabolism. The authors have found evidence that starvation decreases the abundance of the fungal circadian clock protein white collar complex (WCC), even though WC-1 is required for responses to starvation. This observation is interesting, but the authors should consider that WCC has several other functions (as a light receptor, in transcriptional regulation) that are not necessarily clock connected. As such the most interesting result from this paper is that the standard model for the molecular mechanism of the fungal circadian clock does not explain the persistence of normal rhythms under extreme starvation conditions, where the levels of clock proteins are drastically altered.

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Evaluation Summary:

This paper challenges a fundamental view concerning why males of most animals have a higher germline mutation rate than females. Evidence is provided to show that it is not simply the fact that males have more cell divisions in the germline, but instead, most of the mutations arise from a different balance of DNA damage vs. DNA repair. The case is supported by data from multiple species, from de novo mutation rate estimates from pedigrees, and from fits to a simple heuristic model. This work will be of interest to the broad field of DNA mutations and DNA repair, as well as evolutionary and phylogenomics researchers.

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Evaluation Summary:

Interactions between transposons and the Drosophila host genome are governed by dedicated H3K9me3-enriched loci that are selected for producing anti-transposon piRNAs through binding by the HP1 variant Rhino in Drosophila. The authors identify Kipferl, a ZAD zinc-finger protein, as helping to guide Rhino to G-rich motifs found at piRNA-producing loci in the female germline. The work thus reveals the involvement of a factor binding specific DNA sequences in piRNA biogenesis. The findings are of broad interest to the fields of heterochromatin and transposon biology.

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Evaluation Summary:

This manuscript reports the cryo-EM structure of HOPS, a heterohexameric tether that participates in the fusion of late endosomes, autophagosomes, and AP-3 vesicles with lysosomes. The structure will be of interest to a wide range of cell biologists and structural biologists who study membrane traffic. However, while the structural data are elegant, the functional interpretations need further support.

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Evaluation Summary:

Xing and colleagues present a cryoEM structure of the protein phosphatase 2A (PP2A)-B56 holoenzyme in complex with protein phosphatase methyltransferase-1 (PME-1). The structure reveals that PME-1 blocks the substrate binding site of PP2A by inserting an unstructured loop. This unexpected inhibitory mechanism is also coupled to a large conformation change in the PP2A-B56 holoenzyme and PME-1. Combined with biochemical and cellular assays, the authors suggest how PME-1 can regulate p53-mediated DNA damage responses via inhibiting PP2A. This manuscript will be of importance for structural biologists as well as colleagues in the p53 field.

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Evaluation Summary:

This manuscript will be of interest to researchers in the phage-microbial host interaction field. Notably, the interplay between bacteria and their viral predators has regained broad interest in recent years given the discovery of numerous innate immunity-like phage defense systems. The identification of phage-mediated counter-defense strategies is therefore not only of prime importance for our basic understanding of predator-prey arms races but also for medical applications such as phage therapy.

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Evaluation Summary:

This paper is of interest for a wide range of readers who study the biology of lipid modifying enzymes, especially as it relates to interfacial reaction kinetics in biological membranes. This study aimed to obtain detailed biochemical insights into the mutual relationship between PI(4,5)P2 lipids and their kinase PIP5K, which engage in an exciting pattern-forming reaction on membranes. The authors find cooperative recruitment of PIP5K to the membrane, oligomerization-enhanced catalytic efficiency and indications of allosteric regulation. Although of very high interest and featuring mostly convincing data, there are concerns about the interpretation of whether the observed phenomenon is dimer specific or related to higher-order oligomerization. In addition, there are inconsistencies in the data presentation.

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Evaluation Summary:

This reported crystal structure of nearly-full-length cGMP-dependent protein kinase β (PGK1β) provides new insights into how the activity of the PKG catalytic domain is held in check by intramolecular interactions between both the upstream regulatory cGMP-binding domains and the autoinhibitory segment and the catalytic domain, and how cGMP binding to the cGMP-binding domains can relieve these inhibitory constraints leading to an increase in catalytic activity. The structure of the activating PKGIα R177Q CNB-A domain mutant, which resembles a cGMP-bound wild type CNB-A domain, provides a nice explanation for how this point mutation activates PKG Iα and leads to the development of the TAAD (Thoracic Aortic Aneurysms and Dissections) syndrome. The work will be of specific interest to the cyclic nucleotide community, and to the broader signaling community in general.

(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 article applies a systems biology approach to understand the mechanism of action of 3-chloropiperidines (a class of anticancer drugs) in cancer cells and evaluate their sensitivity to drugs. It integrates transcriptomic and open-chromatin data and utilizes sound statistical frameworks for building a sensitivity model. The author's methodology can be applied to early-stage drug discovery. This paper will be of interest to the large class of people who tried to understand how omics data will help drug discovery. It sets a new framework to integrate transcriptome and chromatin accessibility data to identify the key mechanisms of action and provide potential disease targets, which will help speed up the early phases of drug discovery.

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Evaluation Summary:

This study is of interest to the readership interested in the different cell types present in the mouse adult ovary and shows how cellular states change during the four phases of the estrous cycle. This is a valuable resource for the community.

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Evaluation Summary:

The authors first develop a new flexible and robust method to detect deviations from Mendelian inheritance in genomic data from gametes. The authors then apply this method to study deviations from Mendelian inheritance in human sperm data, but find no evidence for it. Even though this is a negative result, and overall the results are expected based on previous studies. the reviewers agreed that the research is rigorous and valuable.

(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:

Bae et al. examine the regulatory role of the PH domain of the AKT Ser/ Thr kinase, finding a key set of interactions that mediate autoinhibitory interactions between PH and kinase domains. The work provides additional mechanistic understanding of the E17K mutation, a common variant in human cancers. This manuscript will be of great interest to scientists focused on protein kinase regulation and molecular mechanisms that control signal transduction.

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Evaluation Summary:

Using data from a tetraplegic individual, the authors show that the neural representations for attempted single finger movements after multiple years after the injury is still organized in a way that is typical for healthy participants. They also show that the representational structure does not change during task training on a simple finger classification task, and that the representational structure - even without active motor outflow or sensory inflow - switches from a motor representation to a sensory representation during the trial. The results have important implications for the use and training of BCI devices 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. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary:

The manuscript shows that bisphosphonate-related osteonecrosis of the jaw, a rare complication of osteoporosis treatment, was prevented in mice using a novel treatment which works by reversing the associated oral inflammation. The work in this manuscript has the potential to be impactful if limitations are addressed. It will be of interest to investigators in the bone and dental fields who conduct pre-clinical research.

(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:

Chaudhary and colleagues follow up their preliminary study on mitochondrial genome copy number in AML with this current study by looking if the expression of specific genes encoding mitochondrial components could provide further insight into AML prognosis. Multivariate analysis was used to identify those genes whose expression was prognostic of patient outcome, which led to the identification of three mitochondrial genes whose expression was used to build a multivariate risk model for childhood AML patients. Altogether, the work by Chaudhary and colleagues interestingly builds on their previous work and suggests that mitochondria may influence AML outcomes, and measuring mitochondrial parameters may help assess patient risk. However, the authors will need to identify the novelty of their findings over the previous reports from their own group.

(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 models the evolution of simple multicellular life cycles using evolutionary game theory. The authors discuss natural selection between different life cycles by modeling growth, fragmentation, and interactions between propagules, discovering conditions for selection of a single life cycle or coexistence of multiple ones. Overall, the model is biologically intuitive, the results are rigorous, and the implications for the evolution of multicellularity are interesting.

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Evaluation Summary:

This manuscript is of broad interest to readers in the fields of vitamin D and obesity. It utilises a Mendelian randomization framework to separate the genetically predicted effects of adiposity at two timepoints in the lifecourse, childhood and adulthood. The key claims of the manuscript are well supported by the data. Higher childhood body size had a direct effect on lower vitamin D levels in early life, while in midlife, childhood body size impacted on adult obesity to result in lower vitamin D levels.

(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 focuses on identifying how metabolism can influence the response of cartilage cells to inflammation. This has relevance to the painful disease known as osteoarthritis. Modulation of cell metabolism in the right direction can serve to protect joint cartilage from the negative effects of inflammation which causes onset and disease progression.

(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:

Bouvette et al. describe a new software for fully automated cryo-EM sample screening and data acquisition, making use of deep-learning-based algorithms for the detection of regions and objects of interest. This is the first example of software for fully automated grid screening, which is of great interest to the cryo-EM community, to free skilled researchers and engineers from a serious of tedious tasks, so that they can devote more time to method development or finding answers to interesting biological and medical questions.

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Evaluation Summary:

We all have had days where there were multiple distinct memorable experiences that we successfully remember as distinct. This paper for the first time focuses on the important question of whether the resting/sleeping hippocampus maintains a clear distinction between replays of different environments and finds that in fact, replays of different tracks are distinct in the sense that both the right sets of neurons are coactive AND their firing rates in replay reflect their firing rates during experiences.

(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 by Yue et al describes studies investigating the role of sensory neuron versus arteriole expression of Trpv1 in body temperature control. This is a detail about the contribution of different cells which has significance because of the reported on-target side-effect of hyperthermia by Trpv1-antagonists. The study shows that the effects on body temperature are predominantly produced through sensory neurons. From these studies it is speculated that the actions of Trpv1 might be pharmacologically modified to permit dissociation of the effects on neurogenic inflammation and the undesirable effects on body temperature.

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Evaluation Summary:

This experiment investigated the link between the role of B lymphocytes to neutrophils for the achievement of LPS tolerance. The authors found that B cells can modulate the tissue-damaging properties of neutrophil leukocytes by influencing neutrophil Cxcr4 signaling in a mouse model of bacterial sepsis.

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Evaluation Summary:

This study shows a direct link between inflammation and cholesterol metabolism in endothelial cells. Specifically, the authors show a pathway by which the major inflammatory factor, NF kappa B, activates a gene called STARD10, which, in turn, leads to the activation of the cholesterol biosynthetic pathway. The study, therefore, provides important insights into the inter-relationship between cholesterol metabolism and inflammation at the molecular level.

(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 identifies pH as a common factor that underlies eco-evolutionary dynamics related to priority effects, which play an important role in community assembly. Using multiple lines of evidence, the data support the overall conclusions of the manuscript that pH-mediated priority effects in the nectar microbiome are the drivers of alternative community states. This manuscript will be of broad interest to readers in ecology and evolutionary biology.

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Evaluation Summary:

The authors present an analysis of SARS-CoV-2 variant of concern movements into and within the Netherlands. The primary finding is that flight bans (in conjunction with other NPIs) were grossly insufficient at stopping the invasion of new variants into The Netherlands over a one-year period. Although consistent with similar analyses of other regions early in the pandemic, this manuscript provides additional evidence of the inadequacy of flight bans at stopping the spread of variants that are already widespread globally, especially (but not only) when importations continue via ground travel. The reviewers have questioned the rigor of the statistical models and the presentation of the main result, including analyses that were included but do not appear to contribute to the main argument.

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Evaluation Summary:

This paper will be of interest to scientists working to understand Chlamydia trachomatis persistence, and host pathogen interaction in general. The authors report the surprising observation that the mechanism of restriction of bacterial growth is through the inhibition of c-Myc signaling by IFNg as opposed to IDO-dependent depletion of tryptophan levels, as had been previously suggested.

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Evaluation Summary:

Although immunotherapy has revolutionized the cancer field, most tumors do not respond, and in those that do respond, acquired resistance is often inevitable. Several mechanisms have been proposed to be involved in acquired resistance to immunotherapy. In the present study, the authors show that tumor cells from multi-cellular structures protect the inner core of tumor cells via the prevention of penetration by lytic molecules. The formation of these structures is mediated by anti-tumor T cells even with tumors that have retained their immunogenic neoantigens. This work identifies a novel possible resistance mechanism to immune-mediated tumor killing.

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Evaluation Summary:

This work sets out to develop a better machine learning-based predictor of survival/prognosis for patients diagnosed with pancreatic cancer, by developing a large combinatorial family of machine learning methods based on a high-dimensional set of -omics and other patient data features; using ten publicly available data sets. A reduced set of features (giving rise to a signature called AIDPS that involves 9 genes) was identified. Unfortunately, the authors used all ten data sets both in the discover stage and in the validation stage of their study. There was also a large mismatch between the initial number of covariates (15,288 genes) and the number of samples (n=1280). The combinatorial ensemble of ML models makes for an unwieldy methodology that is difficult to interpret or duplicate.

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Evaluation Summary:

This study by Zander et al provides a valuable transcriptomic resource of murine CD4 T cell subsets in chronic viral infection. This study will be of broad interest to a wide range of researchers focused on studying CD4 T cell biology.

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Evaluation Summary:

Women are a mosaic of two population of cells, one with the paternal X-chromosome and the other with the maternal one in the active state due to random X-chromosome inactivation (XCI) that occurs during embryogenesis. During aging, one of the two populations dominates the other in a significant proportion of women. This skewing of XCI is of unknown etiology and its impact on health remains enigmatic. In this study, Amy L. Robert et al, demonstrate that skewing may not be benign and it is associated with a modest but significant increased risk of cardiovascular disease and cancer.

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Evaluation Summary

This study aims to characterise the brain dynamics of different disorders of consciousness by studying patients in a minimally conscious state and those with unresponsive wakefulness syndrome, along with healthy controls. The authors apply elegant analyses to the dynamics of brain functional connectivity to successfully discriminate between healthy controls and patients, revealing reduced metastability and a contracted network repertoire in disorders of consciousness. Overall, the study provides important new information on the mechanisms of disorders of consciousness and the functional brain networks involved.

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 is of interest within the fields of DNA replication, developmental biology and oncology. Focusing on the YAP protein, a major regulator of tissue growth and repair, it identifies an interesting new role in DNA replication dynamics, beyond its known role in gene transcription regulation. A series of experimental manipulations support the key claims of the paper. Additional control experiments, as well as mechanistic insight into how RIF1 and YAP interact, and insight into how that interaction influences replication timing would make the paper stronger.

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Evaluation Summary:

The authors present a manuscript addressing an important unmet need, specifically focused on understanding the effects of high protein on hematopoiesis. This information can be of interest to basic biologists and clinicians who specialize in the areas of various diseases associated with elevated protein concentration (e.g. infections, inflammation, multiple myeloma, renal failure, etc). This is in part what makes for the complexity in studying this entity as the consequences of such disparate diseases are difficult to parcel out as causes of which specific disease manifestations. Furthermore, the presented work is done in an invertebrate model without additional confirmation in other model systems. Taken together, the work, which is plentiful in experiments, provides an incomplete understanding of cause and effect, leading to overinterpretation of results and overstating of derived 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:

The authors make an important contribution to our understanding of the universal mechanism of unloading of sugars from the phloem (the vascular tissue dedicated to long-distance sugar transport in plants) into root tip cells. Specifically, the authors investigate the pores (called plasmodesmata) present in the cell wall separating phloem cells from those cells into which sugars get unloaded in roots, which they found to have the same characteristic structure in all plant species investigated. The physical properties of these particular plasmodesmata suggest that they are especially suited for efficient and selective phloem unloading. The paper is relevant for audiences studying plant physiology and development. There are a few criticisms of the modelling work.

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Evaluation Summary:

Zaforas et al. conducted a high-quality study on a very complex topic, using advanced layer-specific neuronal recording techniques. Their findings might be especially interesting for pre-clinical and clinical researchers as well as clinicians in the field of SCI-related sensory pathologies such as neuropathic pain. However, methodological limitations prevent clear mechanistic insight into the underlying causes of their effects.

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Evaluation Summary:

The study claims to demonstrate an interplay between awareness and bottom-up attention and explains their joint effects within an established normalization framework. How awareness fits into current computational theory is an important and timely undertaking that has a far-reaching impact on our understanding of visual and cognitive function. Although the study uses control experiments and analyses to reinforce their claims, shortcomings in their experimental approach require further clarification and data to adequately support the study's conclusions.

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Evaluation Summary:

This work investigates how prestimulus alpha neural oscillations differentially modulate sensory signal and noise during visual detection and demonstrates that alpha power correlates with the subject's perceptual discriminability but not with decision criterion, supporting that alpha power modulates sensory signals more strongly than noise. The key conceptual claim is directly related to existing claims in the literature, although this is an unusually elegant experimental demonstration of the phenomenon.

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Evaluation Summary:

In this study, the authors investigated how predictions modulate performance using a combination of pharmacological experiments, high-density EEG, Bayesian modeling, and machine learning. This is an interesting study with a complex set of analyses. The detailed assessment and interpretation of all the findings could be strengthened by providing a more unified and hypotheses-driven 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:

This study identifies a novel Shank3 mutation from individuals with ADHD-like syndrome and tests the impacts of this mutation together with other known Shank3 mutations on inter- and intramolecular protein-protein interactions of Shank3 involving the N-terminal SPN and Ank repeats. The results indicate that Shank3 mutations have diverse impacts on the intramolecular SPN-Ank domains and the interaction of Shank3 with other proteins including delta-catenin, fodrin, and CaMKIIa. Overall, the results of the study are novel and of high quality. Considering the lack of detailed biochemical understanding on various Shank3 mutations associated with PMS, ASD, and schizophrenia, this study is a meaningful step forward in the basic understanding of Shank3 functions and related pathophysiology.

(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:

Park and colleagues examined the activity and function of different projection neuron types, pyramidal tract (PT) and intratelencephalic (IT) neurons, in the primary motor cortex using a joystick manipulation task in mice. During forelimb movements, the activity of IT neurons was more correlated with movement kinematics than that of PT neurons was, and inactivation of IT neurons caused larger effects on movement kinematics (amplitude and velocity). The results highlight different activity patterns and functions between PT and IT neurons. Discussion among reviewers focused on two main issues. One centered on the interpretation of the PT neural activity; the second on the evidence underlying the claim of a dissociation between the PT and IT 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. Reviewer #1 agreed to share their name with the authors.)”

Evaluation Summary:

This article will be of interest to evolutionary biologists and behavioural ecologists. It aims to quantify the fitness benefits of helping with the breeding attempts of others vs. seeking own breeding attempts via dispersal. It is generally considered that helping is less profitable than breeding, but occurs when superior reproductive options are constrained. Using a long-term dataset of birds, the authors call into question this assumption, and propose that both reproductive tactics can in fact have similar fitness returns, resulting in mixed-kin societies.

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Evaluation Summary:

All reviewers think the study will be really valuable for the field, especially after re-writing to include a detailed comparison with results that were previously published. We all appreciate the clear identification of a gamete sub-population, and also thought that the discovery of low activation of all VSG expression sites was intriguing and will be of considerable interest to those in the field.

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Evaluation Summary:

This work builds on previous work by the same team, demonstrating that the bacterial protein CspA, which inactivates host complement by binding to the host complement inhibitor FH, is a determinant of host range for the Lyme disease bacterium. Additionally, the authors present phylogenetic analysis of CspA and related protein sequences, which supports the hypothesis that inactivation of host complement has evolved independently in three bacterial genospecies.

(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 paper describes the development and validation of an automatic approach that leverages machine vision and learning techniques to quantify dynamic facial expressions of emotion. The potential clinical and translational significance of this automated approach is then examined in a "proof-of-concept" follow-on study, which leveraged video recordings of depressed individuals watching humorous and sad video clips.

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Evaluation Summary:

This study provides a compelling and significant advance on the understanding of how gene regulation by the histone methyltransferase MES-4 underlies germ cell survival in C. elegans, with the major claims being nicely substantiated. The critical and surprising finding is that the degeneration of mes-4 mutant primordial germ cells is due to inappropriate upregulation of genes on the silenced X chromosome, and not failure to activate germline-expressed genes, though reduced levels of germline gene expression were observed. An X-linked target of mes-4, lin-15b, is necessary for the degeneration phenotype. The work could be improved by clarification of the relationship between X and autosomal gene expression, especially in consideration with the action of the other histone methyltransferase MET-1, but otherwise it is excellent.

(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:

Dongre et al. build on previous social learning research on wild vervet monkeys to investigate the role of a particular social behaviour, muzzle-muzzle contact, in aiding the acceptance of a novel food and provide interesting observations on the potential for male monkeys immigrating from one social group to another in spreading this novel behaviour. With a more robust and transparent analysis, this manuscript has the potential to provide significant insights into innovation and social learning in animals.

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 will be of interest to scientists studying neuroinflammation and searching for potential therapeutic targets. The findings here have revealed the effects of an anti-inflammatory cytokine, human IL-37 (hIL-37), in the central nervous system of mice. The data support the conclusions within the current mouse models. Since hIL-37 is not naturally expressed in mice, more evidence related to human cells or tissues would strengthen the physiological significance.

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:

Rabies-mediated monosynaptic retrograde tracing is a powerful method to characterize the connectivity of neural circuits. The CVS-N2c strain of rabies virus shows significantly higher efficiency of transsynaptic spread and less toxicity than the more commonly used SAD B19 strain but has been limited in use by an arduous and lengthy packaging process and low resultant titers. Here, Sumser et al. present a method that significantly speeds up the production process while reducing off-target expression. They also introduce a suite of novel reagents (34 novel plasmids) for monosynaptic tracing with the CVS-N2c strain that they commendably, have already deposited with Addgene. The work is an important advance that will reinvigorate rabies-mediated circuit tracing.

(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:

Cohesin is an evolutionarily conserved protein complex that plays essential roles in mitotic chromosome structure and function. Previous studies suggest that multiple activities of cohesin are required only prior to the onset of chromosome segregation. Using a Mcd1-AID and a Mcd1-TEV to either degrade or cleave cohesin's kleisin subunit Mcd1 of the yeast Saccharomyces cerevisiae, this study shows that cohesion plays also a role in anaphase organizing the centromeric regions, providing new evidence that cohesin function is critical for chromosome structure and segregation during and after the onset of chromosome segregation. The work is of relevance for students of chromosome biology and cell division.

(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:

Results from observational studies examining an association between the antihypertensive drugs and the risk of breast cancer reported inconsistent findings. This study uses a two-sample Mendelian randomization approach (MR), which overcomes the limitation of observational studies by using genetic variants as a proxy for modifiable exposures, to investigate the relationship between the use of antihypertensive medication and breast cancer risk. Using publicly available data and including a comprehensive assessment of antihypertensive drugs, the authors identified two SNPs that were associated with breast cancer risk. While the findings suggest that antihypertensive medication use may be associated with breast cancer risk, there are some methodological issues that need to 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. The reviewers remained anonymous to the authors.)

Evaluation Summary:

FLT3 (Fms Related Receptor Tyrosine Kinase 3) activation occurs in a subset of acute myeloid leukemia (AML) cases and is associated with poor prognosis. This work is focused on the mechanisms of resistance to FLT3 inhibitors in AML. The authors show that the combination of the FLT3 inhibitor and an mTORC1 inhibitor reduces tumor burden and prevents relapse in FLT3 mutant AML. This paper is of interest in scientists and physicians investigating AML as well as scientists studying signaling pathways.

(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:

Overall, this study will make significant contributions to developmental neuroscience and vision science as they attempt to study how prenatal and postnatal maturation influence structural-functional measurements in the early and high-level visual cortex. These results will be of broad interest as it is a novel attempt to study processes that might be innate or genetically wired and those that emerge due to worldly experiences within the sensory systems. The authors are addressing an important and timely question based on a large and impressive infant database.

(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 paper demonstrates that artificial neural networks can be used to accurately predict the responses of biologically-detailed neuron models to synaptic inputs, and hence to approximate the behaviour of networks of such neurons. This study potentially opens the door to massively reduced simulation times for biologically-detailed neuronal network simulations without recourse to supercomputers and hence will be of broad interest to computational 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 #3 agreed to share their name with the authors.)

Evaluation Summary:

This paper describes novel insights into the potential function of innexin proteins, which are electrical synapse-forming proteins with often quite enigmatic in vivo functions. The authors describe here potential functions in synapse tiling. The paper should be of interest to researchers with interests in molecular mechanisms governing nervous system 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 #3 agreed to share their name with the authors.)

Evaluation Summary:

In this paper the authors report an updated theoretical model describing in mathematical terms how the Hsf1 transcription factor is activated in yeast in response to heat shock, and demonstrate that rather than denatured mature proteins, Hsf1 activation involves newly synthesized proteins that sequester the Hsp70 chaperone away from the inactive Hsp70/Hsf1 complex, releasing active Hsf1. They also describe a general role for the Sis1 co-chaperone in maintaining the fitness of yeast cells under stress conditions, such as heat shock, that is independent of regulation of Hsf1.

(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 name with the authors.)

Evaluation Summary:

The authors bioinformatically analyze previous scRNA-seq datasets of the developing mouse soft palate to identify differential signaling pathway activities in the heterogeneous palatal mesenchyme. Identifying TGF-beta signaling pathway activity with the perimysial cells, they hypothesize and test whether TGF-beta signaling in the perimysial cells might regulate palatal muscle formation. This paper will be of high interest to developmental biologists interested in the molecular regulation of tissue interactions that occur during mammalian palate morphogenesis.

(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 will be of interest to readers curious about collective behavior, biological rhythms, and models of synchronized oscillations. The authors study a remarkable species of fireflies in which individual males flash non-rhythmically and erratically, but sufficiently large groups flash rhythmically and synchronously. Unlike nearly all previous models of biological oscillations, the authors' model accounts for their data quantitatively and with no adjustable parameters, at least in the experimental set-up 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. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary:

This study provides insight into the identity of the sodium channel controlling excitability in proprioceptors. Using pharmacology, gene KO, behavior, and histology, the authors show quite convincingly that NaV1.1 in sensory neurons is essential for normal motor behavior and contributes to proprioceptor excitability. The work has interesting implications for human subjects with inherited variants of Nav1.1.

(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:

Some children conceived by assisted reproductive technologies (ART) exhibit metabolic differences compared to those conceived naturally, and the causes are unknown. This work reveals possible explanations for the metabolic differences and provides opportunities to improve ART and prevent the differences. This is a valuable contribution and will be of special interest to practitioners of ART, as well as to developmental and reproductive biologists.

(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.)