Inhibitors of the tyrosine kinase Btk have been proposed as novel antiplatelet agents. In this study we show that low concentrations of the Btk inhibitor ibrutinib block CLEC-2-mediated activation and tyrosine phosphorylation including Syk and PLCγ2 in human platelets. Activation is also blocked in patients with X-linked agammaglobulinemia (XLA) caused by a deficiency or absence of Btk. In contrast, the response to GPVI is delayed in the presence of low concentrations of ibrutinib or in patients with XLA, and tyrosine phosphorylation of Syk is preserved. A similar set of results is seen with the second-generation inhibitor, acalabrutinib. The differential effect of Btk inhibition in CLEC-2 relative to GPVI signalling is explained by the positive feedback role involving Btk itself, as well as ADP and thromboxane A2 mediated activation of P2Y12 and TP receptors, respectively. This feedback role is not seen in mouse platelets and, consistent with this, CLEC-2-mediated activation is blocked by high but not by low concentrations of ibrutinib. Nevertheless, thrombosis was absent in 8 out of 13 mice treated with ibrutinib. These results show that Btk inhibitors selectively block activation of human platelets by CLEC-2 relative to GPVI suggesting that they can be used at 'low dose' in patients to target CLEC-2 in thrombo-inflammatory disease.

Motivation Localization microscopy data is represented by a set of spatial coordinates, each corresponding to a single detection, that form a point cloud. This can be analyzed either by rendering an image from these coordinates, or by analyzing the point cloud directly. Analysis of this type has focused on clustering detections into distinct groups which produces measurements such as cluster area, but has limited capacity to quantify complex molecular organisation and nano-structure. Results We present a segmentation protocol which, through the application of persistence based clustering, is capable of probing densely packed structures which vary in scale. An increase in segmentation performance over state-of-the-art methods is demonstrated. Moreover we employ persistent homology to move beyond clustering, and quantify the topological structure within data. This provides new information about the preserved shapes formed by molecular architecture. Our methods are flexible and we demonstrate this by applying them to receptor clustering in platelets, nuclear pore components, endocytic proteins and microtubule networks. Both 2D and 3D implementations are provided within RSMLM, an R package for pointillist based analysis and batch processing of localization microscopy data. Availability RSMLM has been released under the GNU General Public License v3.0 and is available at https://github.com/JeremyPike/RSMLM. Tutorials for this library implemented as Binder ready Jupyter notebooks are available at https://github.com/JeremyPike/RSMLM-tutorials. Supplementary information Supplementary data are available at Bioinformatics online.

Particles present in diesel exhaust have been proposed as a significant contributor to the development of acute and chronic lung diseases, including respiratory infection and allergic asthma. Nanoceria (CeO2 nanoparticles) are used to increase fuel efficiency in internal combustion engines, are present in exhaust fumes, and could affect cells of the airway. Components from the environment such as biologically derived proteins, carbohydrates, and lipids can form a dynamic layer, commonly referred to as the "protein corona" which alters cellular nanoparticle interactions and internalization. Using confocal reflectance microscopy, we quantified nanoceria uptake by lung-derived cells in the presence and absence of a serum-derived protein corona. Employing mass spectrometry, we identified components of the protein corona, and demonstrated that the interaction between transferrin in the protein corona and the transferrin receptor is involved in mediating the cellular entry of nanoceria via clathrin-mediated endocytosis. Furthermore, under these conditions nanoceria does not affect cell growth, viability, or metabolism, even at high concentration. Alternatively, despite the antioxidant capacity of nanoceria, in serum-free conditions these nanoparticles induce plasma membrane disruption and cause changes in cellular metabolism. Thus, our results identify a specific receptor-mediated mechanism for nanoceria entry, and provide significant insight into the potential for nanoparticle-dependent toxicity.

Manufactured nanomaterials (MNMs) selected from a library of over 120 different MNMs with varied compositions, sizes, and surface coatings were tested by four different laboratories for toxicity by high-throughput/-content (HT/C) techniques. The selected particles comprise 14 MNMs composed of CeO, Ag, TiO, ZnO and SiO with different coatings and surface characteristics at varying concentrations. The MNMs were tested in different mammalian cell lines at concentrations between 0.5 and 250 µg/mL to link physical-chemical properties to multiple adverse effects. The cell lines are derived from relevant organs such as liver, lung, colon and the immune system. Endpoints such as viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied. Soluble MNMs, Ag and ZnO, were toxic in all cell types. TiO and SiO MNMs also triggered toxicity in some, but not all, cell types and the cell type-specific effects were influenced by the specific coating and surface modification. CeO MNMs were nearly ineffective in our test systems. Differentiated liver cells appear to be most sensitive to MNMs, Whereas most of the investigated MNMs showed no acute toxicity, it became clear that some show adverse effects dependent on the assay and cell line. Hence, it is advised that future nanosafety studies utilise a multi-parametric approach such as HT/C screening to avoid missing signs of toxicity. Furthermore, some of the cell type-specific effects should be followed up in more detail and might also provide an incentive to address potential adverse effects in vivo in the relevant organ.

Deep vein thrombosis (DVT) is linked to local inflammation. A role for both neutrophil extracellular traps (NETs) and the assembly of inflammasomes (leading to caspase-1–dependent interleukin-1β activation) in the development of DVT was recently suggested. However, no link between these 2 processes in the setting of thrombosis has been investigated. Here, we demonstrate that stimulation of neutrophils induced simultaneous formation of NETs and active caspase-1. Caspase-1 was largely associated with NETs, suggesting that secreted active caspase-1 requires NETs as an adhesive surface. NETs and their components, histones, promoted robust caspase-1 activation in platelets with the strongest effect exerted by histones 3/4. Murine DVT thrombi contained active caspase-1, which peaked at 6 hours when compared with 48-hour thrombi. Platelets constituted more than one-half of cells containing active caspase-1 in dissociated thrombi. Using intravital microscopy, we identified colocalized NETs and caspase-1 as well as platelet recruitment at the site of thrombosis. Pharmacological inhibition of caspase-1 strongly reduced DVT in mice, and thrombi that still formed contained no citrullinated histone 3, a marker of NETs. Taken together, these data demonstrate a cross-talk between NETs and inflammasomes both in vitro and in the DVT setting. This may be an important mechanism supporting thrombosis in veins.