Nuclei determine the spatial origin of mitotic waves

Traveling waves play an essential role in coordinating mitosis over large distances, but what determines the spatial origin of mitotic waves remains unclear. Here, we show that such waves initiate at pacemakers, regions that oscillate faster than their surroundings. In cell-free extracts of Xenopus laevis eggs, we find that nuclei define such pacemakers by concentrating cell cycle regulators. In computational models of diffusively coupled oscillators that account for nuclear import, nuclear positioning determines the pacemaker location. Furthermore, we find that the spatial dimensions of the oscillatory medium change the nuclear positioning and strongly influence whether a pacemaker is more likely to be at a boundary or an internal region. Finally, we confirm experimentally that increasing the system width increases the proportion of pacemakers at the boundary. Our work provides insight into how nuclei and spatial system dimensions can control local concentrations of regulators and influence the emergent behavior of mitotic waves.status: publishe

Impact of storage conditions on the human stool metabolome and lipidome : preserving the most accurate fingerprint

Faecal metabolomics markedly emerged in clinical as well as analytical chemistry through the unveiling of aberrations in metabolic signatures as reflection of variance in gut (patho)physiology and beyond. Logistic hurdles, however, hinder the analysis of stool samples immediately following collection, inferring the need of biobanking. Yet, the optimum way of storing stool material remains to be determined, in order to conserve an accurate snapshot of the metabolome and circumvent artifacts regarding the disease and parameter(s) under observation. To address this problem, this study scrutinised the impact of freeze-thaw cycling, storage duration, temperature and aerobicity, thereby using ultra-high performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS)-based polar metabolomics and lipidomics methodologies for faecal metabolomics. Both targeted (n > 400) and untargeted approaches were implemented to assess storage effects on individual chemical classes of metabolites as well as the faecal fingerprint. In general, recommendations are that intact stool samples should be divided into aliquots, lyophilised and stored at -80 degrees C for a period no longer than 18 weeks, and avoiding any freeze-thawing. The first preservation week exerted the most decisive impact regarding storage temperature, i.e. 12.1% and 6.4% of the polar metabolome experienced a shift at -20 degrees C and at -80 degrees C, respectively, whereas 8.6% and 7.9% was observed to be changed significantly for the lipidome. In addition, aside from the negligible impact of aerobicity, the polar metabolome appeared to be more dependent on the storage conditions applied compared to the lipidome, which emerged as the more stable fraction when assessing the storage duration for 25 weeks. If the interest would greatly align with particular chemical classes, such as branched-chain amino acids or short-chain fatty acids, specific storage duration recommendations are reported. The provided insights on the stability of the faecal metabolome may contribute to a more reasoned design of experiments in biomarker detection or pathway elucidation within the field of faecal metabolomics. (C) 2020 Elsevier B.V. All rights reserved

Reaction injection molding of hydrophilic-in-hydrophobic femtolitre-well arrays

Patterning of micro- and nanoscale topologies and surface properties of polymer devices is of particular importance for a broad range of life science applications, including cell-adhesion assays and highly sensitive bioassays. The manufacturing of such devices necessitates cumbersome multiple-step fabrication procedures and results in surface properties which degrade over time. This critically hinders their wide-spread dissemination. Here, we simultaneously mold and surface energy pattern microstructures in off-stoichiometric thiol-ene by area-selective monomer self-assembly in a rapid micro-reaction injection molding cycle. We replicated arrays of 1,843,650 hydrophilic-in-hydrophobic femtolitre-wells with long-term stable surface properties and magnetically trapped beads with 75% and 87.2% efficiency in single- and multiple-seeding events, respectively. These results form the basis for ultrasensitive digital biosensors, specifically, and for the fabrication of medical devices and life science research tools, generally.status: publishe

Lung epithelial and myeloid innate immunity in influenzaassociated or COVID-19-associated pulmonary aspergillosis: an observational study

International audienceBACKGROUND: Influenza-associated pulmonary aspergillosis (IAPA) and COVID-19-associated pulmonary aspergillosis (CAPA) affect about 15% of critically ill patients with influenza or COVID-19, respectively. These viral-fungal coinfections are difficult to diagnose and are associated with increased mortality, but data on their pathophysiology are scarce. We aimed to explore the role of lung epithelial and myeloid innate immunity in patients with IAPA or CAPA. METHODS: In this observational study, we retrospectively recruited patients who had been admitted to the intensive care unit (ICU) of University Hospitals Leuven, Belgium, requiring non-invasive or invasive ventilation because of severe influenza or COVID-19, with or without aspergillosis, between Jan 1, 2011, and March 31, 2021, whose bronchoalveolar lavage samples were available at the hospital biobank. Additionally, biobanked in vivo tracheobronchial biopsy samples from patients with IAPA or CAPA and invasive Aspergillus tracheobronchitis admitted to ICUs requiring invasive ventilation between the same dates were collected from University Hospitals Leuven, Hospital Network Antwerp (Belgium), and Amiens-Picardie University Hospital (France). We did nCounter gene expression analysis of 755 genes linked to myeloid innate immunity and protein analysis of 47 cytokines, chemokines, and growth factors on the bronchoalveolar lavage samples. Gene expression data were used to infer cell fractions by use of CIBERSORTx, to perform hypergeometric enrichment pathway analysis and gene set enrichment analysis, and to calculate pathway module scores for the IL-1β, TNF-α, type I IFN, and type II IFN (IFNγ) pathways. We did RNAScope targeting influenza virus or SARS-CoV-2 RNA and GeoMx spatial transcriptomics on the tracheobronchial biopsy samples. FINDINGS: Biobanked bronchoalveolar lavage samples were retrieved from 166 eligible patients, of whom 40 had IAPA, 52 had influenza without aspergillosis, 33 had CAPA, and 41 had COVID-19 without aspergillosis. We did nCounter gene expression analysis on bronchoalveolar lavage samples from 134 patients, protein analysis on samples from 162 patients, and both types of analysis on samples from 130 patients. We performed RNAScope and spatial transcriptomics on the tracheobronchial biopsy samples from two patients with IAPA plus invasive Aspergillus tracheobronchitis and two patients with CAPA plus invasive Aspergillus tracheobronchitis. We observed a downregulation of genes associated with antifungal effector functions in patients with IAPA and, to a lesser extent, in patients with CAPA. We found a downregulated expression of several genes encoding proteins with functions in the opsonisation, recognition, and killing of conidia in patients with IAPA versus influenza only and in patients with CAPA versus COVID-19 only. Several genes related to LC3-associated phagocytosis, autophagy, or both were differentially expressed. Patients with CAPA had significantly lower neutrophil cell fractions than did patients with COVID-19 only. Patients with IAPA or CAPA had downregulated IFNγ signalling compared with patients with influenza only or COVID-19 only, respectively. The concentrations of several fibrosis-related growth factors were significantly elevated in the bronchoalveolar lavage fluid from patients with IAPA versus influenza only and from patients with CAPA versus COVID-19 only. In one patient with CAPA, we visualised an active or very recent SARS-CoV-2 infection disrupting the epithelial barrier, facilitating tissue-invasive aspergillosis. INTERPRETATION: Our results reveal a three-level breach in antifungal immunity in IAPA and CAPA, affecting the integrity of the epithelial barrier, the capacity to phagocytise and kill Aspergillus spores, and the ability to destroy Aspergillus hyphae, which is mainly mediated by neutrophils. The potential of adjuvant IFNγ in the treatment of IAPA and CAPA should be investigated. FUNDING: Research Foundation Flanders, Coronafonds, the Max Planck Society, the Fundação para a Ciência e a Tecnologia, the European Regional Development Fund, "la Caixa" Foundation, and Horizon 2020