Accurate and Versatile 3D Segmentation of Plant Tissues at Cellular Resolution, bioRxiv, 2020-01-19
ABSTRACTQuantitative analysis of plant and animal morphogenesis requires accurate segmentation of individual cells in volumetric images of growing organs. In the last years, deep learning has provided robust automated algorithms that approach human performance, with applications to bio-image analysis now starting to emerge. Here, we present PlantSeg, a pipeline for volumetric segmentation of plant tissues into cells. PlantSeg employs a convolutional neural network to predict cell boundaries and graph partitioning to segment cells based on the neural network predictions. PlantSeg was trained on fixed and live plant organs imaged with confocal and light sheet microscopes. PlantSeg delivers accurate results and generalizes well across different tissues, scales, and acquisition settings. We present results of PlantSeg applications in diverse developmental contexts. PlantSeg is free and open-source, with both a command line and a user-friendly graphical interface.
biorxiv plant-biology 100-200-users 2020Primordial emergence of a nucleic acid binding protein via phase separation and statistical ornithine to arginine conversion, bioRxiv, 2020-01-19
AbstractDe novo emergence, and emergence of the earliest proteins specifically, demands a transition from disordered polypeptides into structured proteins with well-defined functions. However, can peptides confer evolutionary relevant functions, let alone with minimal abiotic amino acid alphabets? How can such polypeptides evolve into mature proteins? Specifically, while nucleic acids binding is presumed a primordial function, it demands basic amino acids that do not readily form abiotically. To address these questions, we describe an experimentally-validated trajectory from a phase-separating polypeptide to a dsDNA-binding protein. The intermediates comprise sequence-duplicated, functional proteins made of only 10 amino acid types, with ornithine, which can form abiotically, as the only basic amino acid. Statistical, chemical modification of ornithine sidechains to arginine promoted structure and function. The function concomitantly evolved – from phase separation with RNA (coacervates) to avid and specific dsDNA binding – thereby demonstrating a smooth, gradual peptide-to-protein transition with respect to sequence, structure, and function.
biorxiv biochemistry 0-100-users 2020Rigor and Transparency Index, a new metric of quality for assessing biological and medical science methods, bioRxiv, 2020-01-19
AbstractThe reproducibility crisis in science is a multifaceted problem involving practices and incentives, both in the laboratory and in publication. Fortunately, some of the root causes are known and can be addressed by scientists and authors alike. After careful consideration of the available literature, the National Institutes of Health identified several key problems with the way that scientists conduct and report their research and introduced guidelines to improve the rigor and reproducibility of pre-clinical studies. Many journals have implemented policies addressing these same criteria. We currently have, however, no comprehensive data on how these guidelines are impacting the reporting of research. Using SciScore, an automated tool developed to review the methods sections of manuscripts for the presence of criteria associated with the NIH and other reporting guidelines, e.g., ARRIVE, RRIDs, we have analyzed ∼1.6 million PubMed Central papers to determine the degree to which articles were addressing these criteria. The tool scores each paper on a ten point scale identifying sentences that are associated with compliance with criteria associated with increased rigor (5 pts) and those associated with key resource identification and authentication (5 pts). From these data, we have built the Rigor and Transparency Index, which is the average score for analyzed papers in a particular journal. Our analyses show that the average score over all journals has increased since 1997, but remains below five, indicating that less than half of the rigor and reproducibility criteria are routinely addressed by authors. To analyze the data further, we examined the prevalence of individual criteria across the literature, e.g., the reporting of a subject’s sex (21-37% of studies between 1997 and 2019), the inclusion of sample size calculations (2-10%), whether the study addressed blinding (3-9%), or the identifiability of key biological resources such as antibodies (11-43%), transgenic organisms (14-22%), and cell lines (33-39%). The greatest increase in prevalence for rigor criteria was seen in the use of randomization of subjects (10-30%), while software tool identifiability improved the most among key resource types (42-87%). We further analyzed individual journals over time that had implemented specific author guidelines covering rigor criteria, and found that in some journals, they had a big impact, whereas in others they did not. We speculate that unless they are enforced, author guidelines alone do little to improve the number of criteria addressed by authors. Our Rigor and Transparency Index did not correlate with the impact factors of journals.
biorxiv scientific-communication-and-education 0-100-users 2020Skd3 (human CLPB) is a potent mitochondrial protein disaggregase that is inactivated by 3-methylglutaconic aciduria-linked mutations, bioRxiv, 2020-01-19
ABSTRACTCells have evolved specialized protein disaggregases to reverse toxic protein aggregation and restore protein functionality. In nonmetazoan eukaryotes, the AAA+ disaggregase Hsp78 resolubilizes and reactivates proteins in mitochondria. Curiously, metazoa lack Hsp78. Hence, whether metazoan mitochondria reactivate aggregated proteins is unknown. Here, we establish that a mitochondrial AAA+ protein, Skd3 (human CLPB), couples ATP hydrolysis to protein disaggregation and reactivation. The Skd3 ankyrin-repeat domain combines with conserved AAA+ elements to enable stand-alone disaggregase activity. A mitochondrial inner-membrane protease, PARL, removes an autoinhibitory peptide from Skd3 to greatly enhance disaggregase activity. Indeed, PARL-activated Skd3 dissolves α-synuclein fibrils connected to Parkinson’s disease. Human cells lacking Skd3 exhibit reduced solubility of various mitochondrial proteins, including anti-apoptotic Hax1. Importantly, Skd3 variants linked to 3-methylglutaconic aciduria, a severe mitochondrial disorder, display diminished disaggregase activity (but not always reduced ATPase activity), which predicts disease severity. Thus, Skd3 is a potent protein disaggregase critical for human health.
biorxiv biochemistry 0-100-users 2020Trans-ethnic and ancestry-specific blood-cell genetics in 746,667 individuals from 5 global populations, bioRxiv, 2020-01-19
SUMMARYMost loci identified by GWAS have been found in populations of European ancestry (EA). In trans-ethnic meta-analyses for 15 hematological traits in 746,667 participants, including 184,535 non-EA individuals, we identified 5,552 trait-variant associations at P<5×10−9, including 71 novel loci not found in EA populations. We also identified novel ancestry-specific variants not found in EA, including an IL7 missense variant in South Asians associated with lymphocyte count in vivo and IL7 secretion levels in vitro. Fine-mapping prioritized variants annotated as functional, and generated 95% credible sets that were 30% smaller when using the trans-ethnic as opposed to the EA-only results. We explored the clinical significance and predictive value of trans-ethnic variants in multiple populations, and compared genetic architecture and the impact of natural selection on these blood phenotypes between populations. Altogether, our results for hematological traits highlight the value of a more global representation of populations in genetic studies.
biorxiv genetics 100-200-users 2020A standardized and reproducible method to measure decision-making in mice, bioRxiv, 2020-01-18
AbstractProgress in neuroscience is hindered by poor reproducibility of mouse behavior. Here we show that in a visual decision making task, reproducibility can be achieved by automating the training protocol and by standardizing experimental hardware, software, and procedures. We trained 101 mice in this task across seven laboratories at six different research institutions in three countries, and obtained 3 million mouse choices. In trained mice, variability in behavior between labs was indistinguishable from variability within labs. Psychometric curves showed no significant differences in visual threshold, bias, or lapse rates across labs. Moreover, mice across laboratories adopted similar strategies when stimulus location had asymmetrical probability that changed over time. We provide detailed instructions and open-source tools to set up and implement our method in other laboratories. These results establish a new standard for reproducibility of rodent behavior and provide accessible tools for the study of decision making in mice.
biorxiv neuroscience 200-500-users 2020