Evolution of resistance to a last-resort antibiotic in Staphylococcus aureus via bacterial competition.

Antibiotic resistance is a key medical concern, with antibiotic use likely being an important cause. However, here we describe an alternative route to clinically relevant antibiotic resistance that occurs solely due to competitive interactions among bacterial cells. We consistently observe that isolates of Methicillin-resistant Staphylococcus aureus diversify spontaneously into two distinct, sequentially arising strains. The first evolved strain outgrows the parent strain via secretion of surfactants and a toxic bacteriocin. The second is resistant to the bacteriocin. Importantly, this second strain is also resistant to intermediate levels of vancomycin. This so-called VISA (vancomycin-intermediate S. aureus) phenotype is seen in many hard-to-treat clinical isolates. This strain diversification also occurs during in vivo infection in a mouse model, which is consistent with the fact that both coevolved phenotypes resemble strains commonly found in clinic. Our study shows how competition between coevolving bacterial strains can generate antibiotic resistance and recapitulate key clinical phenotypes.

Parkinson's Disease Phenotypes in Patient Neuronal Cultures and Brain Organoids Improved by 2-Hydroxypropyl-ß-Cyclodextrin Treatment.

BACKGROUND: The etiology of Parkinson's disease (PD) is only partially understood despite the fact that environmental causes, risk factors, and specific gene mutations are contributors to the disease. Biallelic mutations in the phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) gene involved in mitochondrial homeostasis, vesicle trafficking, and autophagy are sufficient to cause PD. OBJECTIVES: We sought to evaluate the difference between controls' and PINK1 patients' derived neurons in their transition from neuroepithelial stem cells to neurons, allowing us to identify potential pathways to target with repurposed compounds. METHODS: Using two-dimensional and three-dimensional models of patients' derived neurons we recapitulated PD-related phenotypes. We introduced the usage of midbrain organoids for testing compounds. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), we corrected the point mutations of three patients' derived cells. We evaluated the effect of the selected compound in a mouse model. RESULTS: PD patient-derived cells presented differences in their energetic profile, imbalanced proliferation, apoptosis, mitophagy, and a reduced differentiation efficiency to tyrosine hydroxylase positive (TH+) neurons compared to controls' cells. Correction of a patient's point mutation ameliorated the metabolic properties and neuronal firing rates as well as reversing the differentiation phenotype, and reducing the increased astrocytic levels. Treatment with 2-hydroxypropyl-ß-cyclodextrin increased the autophagy and mitophagy capacity of neurons concomitant with an improved dopaminergic differentiation of patient-specific neurons in midbrain organoids and ameliorated neurotoxicity in a mouse model. CONCLUSION: We show that treatment with a repurposed compound is sufficient for restoring the impaired dopaminergic differentiation of PD patient-derived cells. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The Virtual Metabolic Human database: integrating human and gut microbiome metabolism with nutrition and disease.

A multitude of factors contribute to complex diseases and can be measured with 'omics' methods. Databases facilitate data interpretation for underlying mechanisms. Here, we describe the Virtual Metabolic Human (VMH, www.vmh.life) database encapsulating current knowledge of human metabolism within five interlinked resources 'Human metabolism', 'Gut microbiome', 'Disease', 'Nutrition', and 'ReconMaps'. The VMH captures 5180 unique metabolites, 17 730 unique reactions, 3695 human genes, 255 Mendelian diseases, 818 microbes, 632 685 microbial genes and 8790 food items. The VMH's unique features are (i) the hosting of the metabolic reconstructions of human and gut microbes amenable for metabolic modeling; (ii) seven human metabolic maps for data visualization; (iii) a nutrition designer; (iv) a user-friendly webpage and application-programming interface to access its content; (v) user feedback option for community engagement and (vi) the connection of its entities to 57 other web resources. The VMH represents a novel, interdisciplinary database for data interpretation and hypothesis generation to the biomedical community.

Metabolic and signalling network maps integration: application to cross-talk studies and omics data analysis in cancer.

BACKGROUND: The interplay between metabolic processes and signalling pathways remains poorly understood. Global, detailed and comprehensive reconstructions of human metabolism and signalling pathways exist in the form of molecular maps, but they have never been integrated together. We aim at filling in this gap by integrating of both signalling and metabolic pathways allowing a visual exploration of multi-level omics data and study of cross-regulatory circuits between these processes in health and in disease. RESULTS: We combined two comprehensive manually curated network maps. Atlas of Cancer Signalling Network (ACSN), containing mechanisms frequently implicated in cancer; and ReconMap 2.0, a comprehensive reconstruction of human metabolic network. We linked ACSN and ReconMap 2.0 maps via common players and represented the two maps as interconnected layers using the NaviCell platform for maps exploration ( https://navicell.curie.fr/pages/maps_ReconMap%202.html ). In addition, proteins catalysing metabolic reactions in ReconMap 2.0 were not previously visually represented on the map canvas. This precluded visualisation of omics data in the context of ReconMap 2.0. We suggested a solution for displaying protein nodes on the ReconMap 2.0 map in the vicinity of the corresponding reaction or process nodes. This permits multi-omics data visualisation in the context of both map layers. Exploration and shuttling between the two map layers is possible using Google Maps-like features of NaviCell. The integrated networks ACSN-ReconMap 2.0 are accessible online and allows data visualisation through various modes such as markers, heat maps, bar-plots, glyphs and map staining. The integrated networks were applied for comparison of immunoreactive and proliferative ovarian cancer subtypes using transcriptomic, copy number and mutation multi-omics data. A certain number of metabolic and signalling processes specifically deregulated in each of the ovarian cancer sub-types were identified. CONCLUSIONS: As knowledge evolves and new omics data becomes more heterogeneous, gathering together existing domains of biology under common platforms is essential. We believe that an integrated ACSN-ReconMap 2.0 networks will help in understanding various disease mechanisms and discovery of new interactions at the intersection of cell signalling and metabolism. In addition, the successful integration of metabolic and signalling networks allows broader systems biology approach application for data interpretation and retrieval of intervention points to tackle simultaneously the key players coordinating signalling and metabolism in human diseases.

Whole-body cellular mapping in mouse using standard IgG antibodies.

Whole-body imaging techniques play a vital role in exploring the interplay of physiological systems in maintaining health and driving disease. We introduce wildDISCO, a new approach for whole-body immunolabeling, optical clearing and imaging in mice, circumventing the need for transgenic reporter animals or nanobody labeling and so overcoming existing technical limitations. We identified heptakis(2,6-di-O-methyl)-ß-cyclodextrin as a potent enhancer of cholesterol extraction and membrane permeabilization, enabling deep, homogeneous penetration of standard antibodies without aggregation. WildDISCO facilitates imaging of peripheral nervous systems, lymphatic vessels and immune cells in whole mice at cellular resolution by labeling diverse endogenous proteins. Additionally, we examined rare proliferating cells and the effects of biological perturbations, as demonstrated in germ-free mice. We applied wildDISCO to map tertiary lymphoid structures in the context of breast cancer, considering both primary tumor and metastases throughout the mouse body. An atlas of high-resolution images showcasing mouse nervous, lymphatic and vascular systems is accessible at http://discotechnologies.org/wildDISCO/atlas/index.php .

Generalising from conventional pipelines using deep learning in high-throughput screening workflows.

The study of complex diseases relies on large amounts of data to build models toward precision medicine. Such data acquisition is feasible in the context of high-throughput screening, in which the quality of the results relies on the accuracy of the image analysis. Although state-of-the-art solutions for image segmentation employ deep learning approaches, the high cost of manually generating ground truth labels for model training hampers the day-to-day application in experimental laboratories. Alternatively, traditional computer vision-based solutions do not need expensive labels for their implementation. Our work combines both approaches by training a deep learning network using weak training labels automatically generated with conventional computer vision methods. Our network surpasses the conventional segmentation quality by generalising beyond noisy labels, providing a 25% increase of mean intersection over union, and simultaneously reducing the development and inference times. Our solution was embedded into an easy-to-use graphical user interface that allows researchers to assess the predictions and correct potential inaccuracies with minimal human input. To demonstrate the feasibility of training a deep learning solution on a large dataset of noisy labels automatically generated by a conventional pipeline, we compared our solution against the common approach of training a model from a small manually curated dataset by several experts. Our work suggests that humans perform better in context interpretation, such as error assessment, while computers outperform in pixel-by-pixel fine segmentation. Such pipelines are illustrated with a case study on image segmentation for autophagy events. This work aims for better translation of new technologies to real-world settings in microscopy-image analysis.

Reproducible generation of human midbrain organoids for in vitro modeling of Parkinson's disease.

The study of human midbrain development and midbrain related diseases, like Parkinson's disease (PD), is limited by deficiencies in the currently available and validated laboratory models. Three dimensional midbrain organoids represent an innovative strategy to recapitulate some aspects of the complexity and physiology of the human midbrain. Nevertheless, also these novel organoid models exhibit some inherent weaknesses, including the presence of a necrotic core and batch-to-batch variability. Here we describe an optimized approach for the standardized generation of midbrain organoids that addresses these limitations, while maintaining key features of midbrain development like dopaminergic neuron and astrocyte differentiation. Moreover, we have established a novel time-efficient, fit for purpose analysis pipeline and provided proof of concept for its usage by investigating toxin induced PD.

Creation and analysis of biochemical constraint-based models using the COBRA Toolbox v.3.0.

Constraint-based reconstruction and analysis (COBRA) provides a molecular mechanistic framework for integrative analysis of experimental molecular systems biology data and quantitative prediction of physicochemically and biochemically feasible phenotypic states. The COBRA Toolbox is a comprehensive desktop software suite of interoperable COBRA methods. It has found widespread application in biology, biomedicine, and biotechnology because its functions can be flexibly combined to implement tailored COBRA protocols for any biochemical network. This protocol is an update to the COBRA Toolbox v.1.0 and v.2.0. Version 3.0 includes new methods for quality-controlled reconstruction, modeling, topological analysis, strain and experimental design, and network visualization, as well as network integration of chemoinformatic, metabolomic, transcriptomic, proteomic, and thermochemical data. New multi-lingual code integration also enables an expansion in COBRA application scope via high-precision, high-performance, and nonlinear numerical optimization solvers for multi-scale, multi-cellular, and reaction kinetic modeling, respectively. This protocol provides an overview of all these new features and can be adapted to generate and analyze constraint-based models in a wide variety of scenarios. The COBRA Toolbox v.3.0 provides an unparalleled depth of COBRA methods.