Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated knock-in

© 2019, The Author(s). The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR-knock in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits

Platelet glycoprotein VI cluster size is related to thrombus formation and phosphatidylserine exposure in collagen-adherent platelets under arterial shear

Background: Collagen-induced platelet activation is predominantly mediated by glycoprotein (GP) VI through formation of receptor clusters that coincide with the accumulation of signaling molecules and are hypothesized to drive strong and sustained platelet activation. Objectives: To determine the importance of GPVI clusters for thrombus formation in whole blood under shear. Methods: We utilized whole blood microfluidics and an anti-GPVI nanobody (Nb), Nb28, labeled with AlexaFluor 488, to assess the distribution of GPVI on the surface of platelets adhering to a range of collagen-like substrates with different platelet activation potentials. Results: Automated analysis of GPVI surface distribution on platelets supported the hypothesis that there is a relationship between GPVI cluster formation, thrombus size, and phosphatidylserine (PS) exposure. Substrates that supported the formation of macroclusters also induced significantly bigger aggregates, with increased amounts of PS-exposing platelets in comparison to substrates where no GPVI clusters were detected. Furthermore, we demonstrate that only direct inhibition of GPVI binding, but not of downstream signaling, is able to disrupt cluster formation. Conclusion: Labeled anti-GPVI Nb28 permits visualization of GPVI clustering under flow conditions. Furthermore, whilst inhibition of downstream signaling does not affect clustering, it does prevent thrombus formation. Therefore, GPVI macroclustering is a prerequisite for thrombus formation and platelet activation, namely, PS exposure, on highly GPVI-dependent collagen surfaces

MAXI and NuSTAR observations of the faint X-ray transient MAXI J1848-015 in the GLIMPSE-C01 Cluster

We present the results of MAXI monitoring and two NuSTAR observations of the recently discovered faint X-ray transient MAXI J1848-015. Analysis of the MAXI light-curve shows that the source underwent a rapid flux increase beginning on 2020 December 20, followed by a rapid decrease in flux after only $\sim5$ days. NuSTAR observations reveal that the source transitioned from a bright soft state with unabsorbed, bolometric ($0.1$-$100$ keV) flux $F=6.9 \pm 0.1 \times 10^{-10}\,\mathrm{erg\,cm^{-2}\,s^{-1}}$, to a low hard state with flux $F=2.85 \pm 0.04 \times 10^{-10}\,\mathrm{erg\,cm^{-2}\,s^{-1}}$. Given a distance of $3.3$ kpc, inferred via association of the source with the GLIMPSE-C01 cluster, these fluxes correspond to an Eddington fraction of order $10^{-3}$ for an accreting neutron star of mass $M=1.4M_\odot$, or even lower for a more massive accretor. However, the source spectra exhibit strong relativistic reflection features, indicating the presence of an accretion disk which extends close to the accretor, for which we measure a high spin, $a=0.967\pm0.013$. In addition to a change in flux and spectral shape, we find evidence for other changes between the soft and hard states, including moderate disk truncation with the inner disk radius increasing from $R_\mathrm{in}\approx3\,R_\mathrm{g}$ to $R_\mathrm{in}\approx8\,R_\mathrm{g}$, narrow Fe emission whose centroid decreases from $6.8\pm0.1$ keV to $6.3 \pm 0.1$ keV, and an increase in low-frequency ($10^{-3}$-$10^{-1}$ Hz) variability. Due to the high spin we conclude that the source is likely to be a black hole rather than a neutron star, and we discuss physical interpretations of the low apparent luminosity as well as the narrow Fe emission.Comment: 19 pages, 9 figures, 3 tables. Accepted for publication in Ap

Interspecies differences in protein expression do not impact the spatiotemporal regulation of glycoprotein VI mediated activation

Background Accurate protein quantification is a vital prerequisite for generating meaningful predictions when using systems biology approaches, a method that is increasingly being used to unravel the complexities of sub cellular interactions and as part of the drug discovery process. Quantitative proteomics, flow cytometry and western blotting have been extensively used to define human platelet protein copy numbers, yet for mouse platelets, a model widely used for platelet research, evidence is largely limited to a single proteomic dataset in which the total amount of proteins were generally comparatively higher than those found in human platelets. Objectives To investigate the functional implications of discrepancies between levels of mouse and human proteins in the GPVI signalling pathway using a systems pharmacology model of GPVI Methods The protein copy number of mouse platelet receptors was determined using flow cytometry. The Virtual Platelet, a mathematical model of Glycoprotein VI (GPVI) signalling, was used to determine the consequences of protein copy number differences observed between human and mouse platelets. Results and conclusion Despite the small size of mouse platelets compared to human platelets they possessed a greater density of surface receptors alongside a higher concentration of intracellular signalling proteins. Surprisingly the predicted temporal profile of Syk activity was similar in both species with predictions supported experimentally. Super resolution microscopy demonstrates that the spatial distribution of Syk is similar between species, suggesting that the spatial distribution of receptors and signalling molecules in activated platelets, rather than their copy number, is important for signalling pathway regulation

Experimental validation of computerised models of clustering of platelet glycoprotein receptors that signal via tandem SH2 domain proteins

The clustering of platelet glycoprotein receptors with cytosolic YxxL and YxxM motifs, including GPVI, CLEC-2 and PEAR1, triggers activation via phosphorylation of the conserved tyrosine residues and recruitment of the tandem SH2 (Src homology 2) domain effector proteins, Syk and PI 3-kinase. We have modelled the clustering of these receptors with monovalent, divalent and tetravalent soluble ligands and with transmembrane ligands based on the law of mass action using ordinary differential equations and agent-based modelling. The models were experimentally evaluated in platelets and transfected cell lines using monovalent and multivalent ligands, including novel nanobody-based divalent and tetravalent ligands, by fluorescence correlation spectroscopy. Ligand valency, receptor number, receptor dimerisation, receptor phosphorylation and a cytosolic tandem SH2 domain protein act in synergy to drive receptor clustering. Threshold concentrations of a CLEC-2-blocking antibody and Syk inhibitor act in synergy to block platelet aggregation. This offers a strategy for countering the effect of avidity of multivalent ligands and in limiting off-target effects

Comparison of the GPVI inhibitors losartan and honokiol

<p>Losartan and honokiol are small molecules which have been described to inhibit aggregation of platelets by collagen. Losartan has been proposed to block clustering of GPVI but not to affect binding of collagen. Honokiol has been reported to bind directly to GPVI but only at a concentration that is three orders of magnitude higher than that needed for inhibition of aggregation. The mechanism of action of both inhibitors is so far unclear. In the present study, we confirm the inhibitory effects of both agents on platelet aggregation by collagen and show that both also block the aggregation induced by the activation of CLEC-2 or the low affinity immune receptor FcγRIIa at similar concentrations. For GPVI and CLEC-2, this inhibition is associated with a reduction in protein tyrosine phosphorylation of multiple proteins including Syk. In contrast, on a collagen surface, spreading of platelets and clustering of GPVI (measured by single molecule localisation microscopy) was not altered by losartan or honokiol. Furthermore, in flow whole-blood, both inhibitors suppressed the formation of multi-layered platelet thrombi at arteriolar shear rates at concentrations that hardly affect collagen-induced platelet aggregation in platelet rich plasma. Together, these results demonstrate that losartan and honokiol have multiple effects on platelets which should be considered in the use of these compounds as anti-platelet agents.</p

FrenchFISH: Poisson Models for Quantifying DNA Copy Number From Fluorescence In Situ Hybridization of Tissue Sections

Purpose: Chromosomal aberration and DNA copy number change are robust hallmarks of cancer. The gold standard for detecting copy number changes in tumor cells is fluorescence in situ hybridization (FISH) using locus-specific probes that are imaged as fluorescent spots. However, spot counting often does not perform well on solid tumor tissue sections due to partially represented or overlapping nuclei. Materials and Methods: To overcome these challenges, we have developed a computational approach called FrenchFISH, which comprises a nuclear volume correction method coupled with two types of Poisson models: either a Poisson model for improved manual spot counting without the need for control probes or a homogeneous Poisson point process model for automated spot counting. Results: We benchmarked the performance of FrenchFISH against previous approaches using a controlled simulation scenario and tested it experimentally in 12 ovarian carcinoma FFPE-tissue sections for copy number alterations at three loci (c-Myc, hTERC, and SE7). FrenchFISH outperformed standard spot counting with 74% of the automated counts having &lt; 1 copy number difference from the manual counts and 17% having &lt; 2 copy number differences, while taking less than one third of the time of manual counting. Conclusion: FrenchFISH is a general approach that can be used to enhance clinical diagnosis on sections of any tissue by both speeding up and improving the accuracy of spot count estimates

Low dose Btk inhibitors selectively block platelet activation by CLEC-2

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 agammaglobulinaemia (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

Spectral and Timing Analysis of NuSTAR and Swift/XRT Observations of the X-Ray Transient MAXI J0637-430

We present results for the first observed outburst from the transient X-ray binary source MAXI J0637-430. This study is based on eight observations from the Nuclear Spectroscopic Telescope Array (NuSTAR) and six observations from the Neil Gehrels Swift Observatory X-Ray Telescope (Swift/XRT) collected from 2019 November 19 to 2020 April 26 as the 3-79 keV source flux declined from 8.2 × 10-10 to 1.4 × 10-12 erg cm-2 s-1. We see the source transition from a soft state with a strong disk-blackbody component to a hard state dominated by a power-law or thermal Comptonization component. NuSTAR provides the first reported coverage of MAXI J0637-430 above 10 keV, and these broadband spectra show that a two-component model does not provide an adequate description of the soft-state spectrum. As such, we test whether blackbody emission from the plunging region could explain the excess emission. As an alternative, we test a reflection model that includes a physical Comptonization continuum. Finally, we also test a spectral component based on reflection of a blackbody illumination spectrum, which can be interpreted as a simple approximation to the reflection produced by returning disk radiation due to the bending of light by the strong gravity of the black hole. We discuss the physical implications of each scenario and demonstrate the value of constraining the source distance