Exploiting Mass Spectrometry to Unlock the Mechanism of Nanoparticle-Induced Inflammasome Activation

Nanoparticles (NPs) elicit sterile inflammation, but the underlying signaling pathways are poorly understood. Here, we report that human monocytes are particularly vulnerable to amorphous silica NPs, as evidenced by single-cell-based analysis of peripheral blood mononuclear cells using cytometry by time-of-flight (CyToF), while silane modification of the NPs mitigated their toxicity. Using human THP-1 cells as a model, we observed cellular internalization of silica NPs by nanoscale secondary ion mass spectrometry (nanoSIMS) and this was confirmed by transmission electron microscopy. Lipid droplet accumulation was also noted in the exposed cells. Furthermore, time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealed specific changes in plasma membrane lipids, including phosphatidylcholine (PC) in silica NP-exposed cells, and subsequent studies suggested that lysophosphatidylcholine (LPC) acts as a cell autonomous signal for inflammasome activation in the absence of priming with a microbial ligand. Moreover, we found that silica NPs elicited NLRP3 inflammasome activation in monocytes, whereas cell death transpired through a non-apoptotic, lipid peroxidation-dependent mechanism. Together, these data further our understanding of the mechanism of sterile inflammation

Dendritic Core-Multishell Nanocarriers in Murine Models of Healthy and Atopic Skin

Dendritic hPG-amid-C18-mPEG core-multishell nanocarriers (CMS) represent a novel class of unimolecular micelles that hold great potential as drug transporters, e.g., to facilitate topical therapy in skin diseases. Atopic dermatitis is among the most common inflammatory skin disorders with complex barrier alterations which may affect the efficacy of topical treatment. Here, we tested the penetration behavior and identified target structures of unloaded CMS after topical administration in healthy mice and in mice with oxazolone-induced atopic dermatitis. We further examined whole body distribution and possible systemic side effects after simulating high dosage dermal penetration by subcutaneous injection. Following topical administration, CMS accumulated in the stratum corneum without penetration into deeper viable epidermal layers. The same was observed in atopic dermatitis mice, indicating that barrier alterations in atopic dermatitis had no influence on the penetration of CMS. Following subcutaneous injection, CMS were deposited in the regional lymph nodes as well as in liver, spleen, lung, and kidney. However, in vitro toxicity tests, clinical data, and morphometry- assisted histopathological analyses yielded no evidence of any toxic or otherwise adverse local or systemic effects of CMS, nor did they affect the severity or course of atopic dermatitis. Taken together, CMS accumulate in the stratum corneum in both healthy and inflammatory skin and appear to be highly biocompatible in the mouse even under conditions of atopic dermatitis and thus could potentially serve to create a depot for anti-inflammatory drugs in the skin

Enabling planetary science across light-years. Ariel Definition Study Report

Ariel, the Atmospheric Remote-sensing Infrared Exoplanet Large-survey, was adopted as the fourth medium-class mission in ESA's Cosmic Vision programme to be launched in 2029. During its 4-year mission, Ariel will study what exoplanets are made of, how they formed and how they evolve, by surveying a diverse sample of about 1000 extrasolar planets, simultaneously in visible and infrared wavelengths. It is the first mission dedicated to measuring the chemical composition and thermal structures of hundreds of transiting exoplanets, enabling planetary science far beyond the boundaries of the Solar System. The payload consists of an off-axis Cassegrain telescope (primary mirror 1100 mm x 730 mm ellipse) and two separate instruments (FGS and AIRS) covering simultaneously 0.5-7.8 micron spectral range. The satellite is best placed into an L2 orbit to maximise the thermal stability and the field of regard. The payload module is passively cooled via a series of V-Groove radiators; the detectors for the AIRS are the only items that require active cooling via an active Ne JT cooler. The Ariel payload is developed by a consortium of more than 50 institutes from 16 ESA countries, which include the UK, France, Italy, Belgium, Poland, Spain, Austria, Denmark, Ireland, Portugal, Czech Republic, Hungary, the Netherlands, Sweden, Norway, Estonia, and a NASA contribution

The dynamic envelope of a fusion class II virus : Molecular reorganizations during prefusion stages of semliki forest virus

The aim of this study was to explore a membrane fusion mechanism, prevailing in alphaviruses and known as virus class 11 fusion. The model virus for this mechanism is Semliki Forest virus (SFV) of the alphavirus family. Contrary to class 1 fusion, for which the influenza virus is die prototype along with HIV, class II fusion mechanism involves membrane proteins mainly folded in beta-sheet structures, not alpha-helices as in the class I case. The fusion class II viruses enter the cell by fusion with the endosomal membrane. Acidification is a prerequisite for the fusion step to occur and to fulfill the infection in vivo. Virus fusion can be triggered experimentally by acidification in the presence of a target membrane. The acidification would transform the virus into a fusogenic state, after which it is prone to interact with the membrane. Thus, by mimicking the environment in the endosome, stages in the fusion process can be studied under well-defined conditions. In the present work I have focused on the dynamic transformation of SFV at stages preceding membrane fusion. To do so, the accessibility of functional domains in the virus envelope was explored with the aid of antibodies and various biochemical methods. Electron cryo-microscopy (cryo-EM) was used to capture intermediate forms of the virus. This provides data for threedimensional (3D) structure determination to reveal details and fusion related variations. Pseudo-atomic resolution structures of the virus particle from combined x-ray and cryo-EM data enabled protein assignment to densities within the cryoEM map. In addition, experiments with formaldehyde (FA) cross-linking of virus particles were performed to gain understanding of its morphological effects. The aim was to develop a method to safely prepare virus specimens for structural studies and, possibly, enhance structural details. In the structures solved, details of acid-induced rearrangements were conclusively identified. The major changes occur as an expansion of the external domain. In the raised shell layer the expanded area show widened shell openings and dissolved protein contacts. In the stalks of the protruding spikes the two glycoproteins separate, but keep together in the spike head lobes, while in the sub-membrane domain the envelope contacts with the nucleocapsid (NC) is released. In spite of the essentially retained virus morphology, several antibody epitopes, including the receptor binding domain and the fusion loop, become exposed in a pH dependent manner. This implies that subtle local rearrangements might represent essential functional stages. In summery, by exploring prefusion stages as they occur in a native virion, this thesis tries to fill a gap of knowledge in viral membrane fusion. It shows details of the virus class II fusion mechanism not earlier discussed. It presents new observations and demonstrates sequential stages of rearrangements in the virus structural domains related to the adaptation of a fusogenic state. The results suggest a cooperative action between the two envelope proteins at stages beyond the control of fusion loop exposure. Furthermore, it also presents a procedure to safely prepare virus specimens for structural studies under close to native conditions

Divorce in the family business : unfolding the legal problems by learning from practice

Purpose – The purpose of this paper is to explore the case of divorce in family business from a legal perspective and highlight the problems of applying family law in the family business context. Design/methodology/approach – The authors rely on legal analysis and interviews with estate distribution executors to discuss problems with the legal rules and how they are practiced. Findings – The findings show that the law is ill fitted to the situation where there is a family business included in the division of marital property. In divorce, family law dictates the division of marital property and the family business is reduced to an asset to be divided like any other. Critical issues are identified and elaborated. Research limitations/implications – Divorce and other disruptions to the family system should be considered in family business consultancy among other threats to the business. The legal perspective on divorce in the family business offered here primarily concerns ownership issues. The impact of divorce on management is equally in need of exploration, which is the suggestion for further studies. Practical implications – The paper illuminates in which ways the business is hampered from divorcing owners and discuss critical issues with applying family law in a family business context. Social implications – Policymakers should establish rules in which shares in an unlisted business are by default assigned to separate property until something else is contracted. Originality/value – New light is shed on the practical problems of interpreting family law in a family business context advancing the understanding of family aspects in family business management

The effects of lipophilic substances on the shape of erythrocytes demonstrated by a new in vitro-method

Abstract Low aqueous solubility of lipophilic agents, such as free fatty acids, hampers proper in vitro demonstration of biological effects, yielding an ambiguous in vitro-in vivo correlation. We have therefore developed a method for evaluating the acute effects of lipophilic substances on the shape of erythrocytes and estimated EC50 and Hill coefficient according to the sigmoidal Emax model. The test substance dissolved in medium-chain triglyceride is coated on a polycarbonate slide which serves as a cover sheet of a Bürker chamber. Freshly collected finger-tip blood is diluted with autologous EDTA-plasma and introduced into the chamber. After ten min at 37 C, the cells are photographed under microscope and the fractions of normal and defect cells are evaluated. No staining is needed and the cells are kept viable during the test period. With increasing chain length, fatty acids, aliphatic amines and alcohols all increased the fraction of defect erythrocytes in a concentration-dependent manner. The results indicate that several fatty acids are very potent in their acute actions on erythrocytes, and that this effect is due to chain length rather than conformation. Conclusion: The technique offers a screening method for testing the harmful effects of small amounts of lipophilic substances on erythrocytes

Acid-induced movements in the glycoprotein shell of an alphavirus turn the spikes into membrane fusion mode

In the icosahedral (T = 4) Semliki Forest virus, the envelope protomers, i.e. E1–E2 heterodimers, make one-to-one interactions with capsid proteins below the viral lipid bilayer, transverse the membrane and form an external glycoprotein shell with projections. The shell is organized by protomer domains interacting as hexamers and pentamers around shell openings at icosahedral 2- and 5-fold axes, respectively, and the projections by other domains associating as trimers at 3- and quasi 3-fold axes. We show here, using cryo- electron microscopy, that low pH, as occurs in the endosomes during virus uptake, results in the relaxation of protomer interactions around the 2- and the 5-fold axes in the shell, and movement of protomers towards 3- and quasi 3-fold axes in a way that reciprocally relocates their putative E1 and E2 domains. This seemed to be facilitated by a trimerization of transmembrane segments at the same axes. The alterations observed help to explain several key features of the spike-mediated membrane fusion reaction, including shell dissolution, heterodimer dissociation, fusion peptide exposure and E1 homotrimerization