Search

S100A8/A9, also known as “calprotectin” or “MRP8/14,” is an alarmin primarily secreted by activated myeloid cells with antimicrobial, proinflammatory, and prothrombotic properties. Increased plasma levels of S100A8/A9 in thrombo-inflammatory diseases are associated with thrombotic complications. We assessed the presence of S100A8/A9 in the plasma and lung autopsies from patients with COVID-19 and investigated the molecular mechanism by which S100A8/A9 affects platelet function and thrombosis. S100A8/A9 plasma levels were increased in patients with COVID-19 and sustained high levels during hospitalization correlated with poor outcomes. Heterodimeric S100A8/A9 was mainly detected in neutrophils and deposited on the vessel wall in COVID-19 lung autopsies. Immobilization of S100A8/A9 with collagen accelerated the formation of a fibrin-rich network after perfusion of recalcified blood at venous shear. In vitro, platelets adhered and partially spread on S100A8/A9, leading to the formation of distinct populations of either P-selectin or phosphatidylserine (PS)-positive platelets. By using washed platelets, soluble S100A8/A9 induced PS exposure but failed to induce platelet aggregation, despite GPIIb/IIIa activation and alpha-granule secretion. We identified GPIbα as the receptor for S100A8/A9 on platelets inducing the formation of procoagulant platelets with a supporting role for CD36. The effect of S100A8/A9 on platelets was abolished by recombinant GPIbα ectodomain, platelets from a patient with Bernard-Soulier syndrome with GPIb-IX-V deficiency, and platelets from mice deficient in the extracellular domain of GPIbα. We identified the S100A8/A9-GPIbα axis as a novel targetable prothrombotic pathway inducing procoagulant platelets and fibrin formation, in particular in diseases associated with high levels of S100A8/A9, such as COVID-19.

Supplementary Materials and Methods, Supplementary Figures S1-S12

Supplementary Table 3 HD and MPN samples.

Supplementary Table 1 VEGFAC Top Differentially Expressed Genes by Cluster

MM ALL Donor Details

Supplementary Table 2 Gene sets for GSEA

Supplementary Table 6 qRT PCR Probes

Supplementary Table 5 Antibodies

Supplementary Table 7 NGS Panel

A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe a step-wise, directed-differentiation protocol in which organoids are generated from induced pluripotent stem cells committed to mesenchymal, endothelial, and hematopoietic lineages. These 3D structures capture key features of human bone marrow—stroma, lumen-forming sinusoids, and myeloid cells including proplatelet-forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of the organoid occurred following TGFβ stimulation and engraftment with myelofibrosis but not healthy donor–derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow–like milieu. This enabling technology is likely to accelerate the discovery and prioritization of novel targets for bone marrow disorders and blood cancers.Significance:We present a human bone marrow organoid that supports the growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment and provides a much-needed ex vivo tool for the prioritization of new therapeutics.See related commentary by Derecka and Crispino, p. 263.This article is highlighted in the In This Issue feature, p. 247

The growing number of manufactured nanomaterials (MNMs) used in consumer products and industrial applications is leading to increased exposures whose consequences are not yet fully understood in terms of potential impacts on human and ecosystem health. Thus, there is an urgent need to further develop high throughput testing for hazard prediction in nanotoxicology. The zebrafish (Danio rerio) embryo has emerged as a vertebrate model organism for nanotoxicity studies, as it provides superior characteristics for microscopy-based screening and can describe the complex interactions of MNMs with a living organism. Automated microscopy coupled with image acquisition has facilitated hazard assessment of chemicals by quantification of various endpoints (e.g., mortality, malformations, hatching), an approach which we employed here to address adverse effects of a selected library of MNMs of different compositions and surface functionalities. We investigated metal and metal oxide MNMs with mean primary particle sizes of 5 to 50 nm, different chemical compositions (silica, ceria, titania, zinc oxide and silver) and various surface coatings / functionalisation, including OECD reference materials. In addition, we tested as proxies representatives for of nanoplastics polystyrene and polymethylmethacrylate nanoparticles which were surface modified by either amino- or carboxyl-groups. Our systematic analysis to establish quantitative property-activity relationships revealed a pronounced toxicity of silver and amino-modified polystyrene nanoparticles (NPs), evidenced by reduced survival and malformations. Interference with hatching, however, was the most sensitive endpoint, as zinc oxide as well as silver NPs reduced hatching already at lower concentrations (i.e., 0.5 and 1 µg/mL, respectively) and early time points (3 days post fertilization, dpf). Interestingly, carboxylated polystyrene NPs and carboxylated or amino-modified polymethylmethacrylate NPs were non-toxic, highlighting both surface modification and core chemistry of nanoplastics as key determinants of biocompatibility.

Lipids contribute to hematopoiesis and membrane properties and dynamics, however, little is known about the role of lipids in megakaryopoiesis. Here, a lipidomic analysis of megakaryocyte progenitors, megakaryocytes, and platelets revealed a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake and de novo lipogenesis impaired megakaryocyte differentiation and proplatelet production. In vivo, mice on a high saturated fatty acid diet had significantly lower platelet counts, which was prevented by eating a PUFA-enriched diet. Fatty acid uptake was largely dependent on CD36, and its deletion in mice resulted in thrombocytopenia. Moreover, patients with a CD36 loss-of-function mutation exhibited thrombocytopenia and increased bleeding. Our results suggest that fatty acid uptake and regulation is essential for megakaryocyte maturation and platelet production, and that changes in dietary fatty acids may be a novel and viable target to modulate platelet counts.

Aims Thrombotic complications and vasculopathy have been extensively associated with severe COVID-19 infection; however, the mechanisms inducing endotheliitis and the disruption of endothelial integrity in the microcirculation are poorly understood. We hypothesized that within the vessel wall, pericytes preferentially take up viral particles and mediate the subsequent loss of vascular integrity. Methods and results Immunofluorescence of post-mortem patient sections was used to assess pathophysiological aspects of COVID-19 infection. The effects of COVID-19 on the microvasculature were assessed using a vascular organoid model exposed to live viral particles or recombinant viral antigens. We find increased expression of the viral entry receptor angiotensin-converting enzyme 2 on pericytes when compared to vascular endothelium and a reduction in the expression of the junctional protein CD144, as well as increased cell death, upon treatment with both live virus and/or viral antigens. We observe a dysregulation of genes implicated in vascular permeability, including Notch receptor 3, angiopoietin-2, and TEK. Activation of vascular organoids with interleukin-1β did not have an additive effect on vascular permeability. Spike antigen was detected in some patients’ lung pericytes, which was associated with a decrease in CD144 expression and increased platelet recruitment and von Willebrand factor (VWF) deposition in the capillaries of these patients, with thrombi in large vessels rich in VWF and fibrin. Conclusion Together, our data indicate that direct viral exposure to the microvasculature modelled by organoid infection and viral antigen treatment results in pericyte infection, detachment, damage, and cell death, disrupting pericyte-endothelial cell crosstalk and increasing microvascular endothelial permeability, which can promote thrombotic and bleeding complications in the microcirculation.

In specialized cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviors. The mechanisms by which β1-tubulin, the platelet and megakaryocyte (MK) lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational tubulin polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell derived MK, and healthy human donor platelets. We find distinct patterns of polymodification in MK and platelets, mediated by the antagonistic activities of the cell specific expression of tubulin tyrosine ligase like enzymes and cytosolic carboxypeptidase enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganization required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved.

A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe a step-wise, directed-differentiation protocol in which organoids are generated from iPSCs committed to mesenchymal, endothelial and hematopoietic lineages. These 3-dimensional structures capture key features of human bone marrow - stroma, lumen-forming sinusoidal vessels and myeloid cells including pro-platelet forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of the organoid occurred following TGFβ stimulation and engraftment with myelofibrosis but not healthy donor-derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow-like milieu. This enabling technology is likely to accelerate discovery and prioritization of novel targets for bone marrow disorders and blood cancers.Significance StatementWe present a 3D, vascularised human bone marrow organoid that supports growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment, and provides a much-needed, ex vivo tool for prioritization of new therapeutics.

Inherited bleeding disorders (IBDs) comprise an extremely heterogeneous group of diseases that reflect abnormalities of blood vessels, coagulation proteins, and platelets. Previously the UK-GAPP study has used whole-exome sequencing in combination with deep platelet phenotyping to identify pathogenic genetic variants in both known and novel genes in approximately 40% of the patients. To interrogate the remaining "unknown" cohort and improve this detection rate, we employed an IBD-specific gene panel of 119 genes using the Congenica Clinical Interpretation Platform to detect both single-nucleotide variants and copy number variants in 126 patients. In total, 135 different heterozygous variants in genes implicated in bleeding disorders were identified. Of which, 22 were classified pathogenic, 26 likely pathogenic, and the remaining were of uncertain significance. There were marked differences in the number of reported variants in individuals between the four patient groups: platelet count (35), platelet function (43), combined platelet count and function (59), and normal count (17). Additionally, we report three novel copy number variations (CNVs) not previously detected. We show that a combined single-nucleotide variation (SNV)/CNV analysis using the Congenica platform not only improves detection rates for IBDs, suggesting that such an approach can be applied to other genetic disorders where there is a high degree of heterogeneity.

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.

Germline defects affecting the DNA-binding domain of the transcription factor FLI1 are associated with a bleeding disorder that is characterized by the presence of large, fused α-granules in platelets. We investigated whether the genes showing abnormal expression in FLI1-deficient platelets could be involved in platelet α-granule biogenesis by undertaking transcriptome analysis of control platelets and platelets harboring a DNA-binding variant of FLI1. Our analysis identified 2,276 transcripts that were differentially expressed in FLI1-deficient platelets. Functional annotation clustering of the coding transcripts revealed significant enrichment for gene annotations relating to protein transport, and identified Sorting nexin 24 (SNX24) as a candidate for further investigation. Using an induced pluripotent stem cell-derived megakaryocyte model, SNX24 expression was found to be increased during the early stages of megakaryocyte differentiation and downregulated during proplatelet formation, indicating tight regulatory control during megakaryopoiesis. CRISPR-Cas9 mediated knockout (KO) of SNX24 led to decreased expression of immature megakaryocyte markers, CD41 and CD61, and increased expression of the mature megakaryocyte marker CD42b (P=0.0001), without affecting megakaryocyte polyploidisation, or proplatelet formation. Electron microscopic analysis revealed an increase in empty membrane-bound organelles in SNX24 KO megakaryocytes, a reduction in α-granules and an absence of immature and mature multivesicular bodies, consistent with a defect in the intermediate stage of α-granule maturation. Co-localization studies showed that SNX24 associates with each compartment of α-granule maturation. Reduced expression of CD62P and VWF was observed in SNX24 KO megakaryocytes. We conclude that SNX24 is required for α-granule biogenesis and intracellular trafficking of α-granule cargo within megakaryocytes.

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.

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.

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.

ObjectiveThrombotic complications and vasculopathy have been extensively associated with severe COVID-19 infection, however the mechanisms by which endotheliitis is induced remain poorly understood. Here we investigate vascular permeability in the context of SARS-CoV-2-mediated endotheliitis in patient samples and a vascular organoid model.Methods and ResultsWe report the presence of the Spike glycoprotein in pericytes associated with pericyte activation and increased endothelial permeability in post-mortem COVID-19 lung autopsies. A pronounced decrease in the expression of the adhesion molecule VE-cadherin is observed in patients with thrombotic complications. Interestingly, fibrin-rich thrombi did not contain platelets, did not colocalize with tissue factor and have heterogenous levels of Von Willebrand factor, suggesting a biomarker-guided therapy might be required to target thrombosis in severe patients. Using a 3D vascular organoid model, we observe that ACE2 is primarily expressed in pericytes adjacent to vascular networks, consistent with patient data, indicating a preferential uptake of the S glycoprotein by these cells. Exposure of vascular organoids to SARS-CoV-2 or its antigens, recombinant trimeric Spike glycoprotein and Nucleocapsid protein, reduced endothelial cell and pericyte viability as well as CD144 expression with no additive effect upon endothelial activation via IL-1β.ConclusionsOur data suggest that pericyte uptake of SARS-CoV-2 or Spike glycoprotein contributes to vasculopathy by altering endothelial permeability increasing the risk of thrombotic complications.

Schlafen 14 (SLFN14) has recently been identified as an endoribonuclease responsible for cleaving RNA to regulate and inhibit protein synthesis. Early studies revealed that members of the SLFN family are capable of altering lineage commitment during T-cell differentiation by using cell-cycle arrest as a means of translational control by RNase activity. SLFN14 has been reported as a novel gene causing an inherited macrothrombocytopenia and bleeding in human patients; however, the role of this endoribonuclease in megakaryopoiesis and thrombopoiesis remains unknown. To investigate this, we report a CRISPR knock-in mouse model of SLFN14 K208N homologous to the K219N mutation observed in our previous patient studies. We used hematological analysis, in vitro and in vivo studies of platelet and erythrocyte function, and analysis of spleen and bone marrow progenitors. Mice homozygous for this mutation do not survive to weaning age, whereas heterozygotes exhibit microcytic erythrocytosis, hemolytic anemia, splenomegaly, and abnormal thrombus formation, as revealed by intravital microscopy, although platelet function and morphology remain unchanged. We also show that there are differences in erythroid progenitors in the spleens and bone marrow of these mice, indicative of an upregulation of erythropoiesis. This SLFN14 mutation presents distinct species-specific phenotypes, with a platelet defect reported in humans and a severe microcytic erythrocytosis in mice. Thus, we conclude that SLFN14 is a key regulator in mammalian hematopoiesis and a species-specific mediator of platelet and erythroid lineage commitment.

Essentials Identifying genetic variants in platelet disorders is challenging due to its heterogenous nature. We combine WES, RNAseq, and python-based bioinformatics to identify novel gene variants. We find novel candidates in patient data by cross-referencing against a murine RNAseq model of thrombopoiesis. This innovative combined bioinformatic approach provides novel data for future research in the field. ABSTRACT: Background The UK Genotyping and Phenotyping of Platelets study has recruited and analyzed 129 patients with suspected heritable bleeding. Previously, 55 individuals had a definitive genetic diagnosis based on whole exome sequencing (WES) and platelet morphological and functional testing. A significant challenge in this field is defining filtering criteria to identify the most likely candidate mutations for diagnosis and further study. Objective Identify candidate gene mutations for the remaining 74 patients with platelet-based bleeding with unknown genetic cause, forming the basis of future re-recruitment and further functional testing and assessment. Methods Using python-based data frame indexing, we first identify and filter all novel and rare variants using a panel of 116 genes known to cause bleeding across the full cohort of WES data. This identified new variants not previously reported in this cohort. We then index the remaining patients, with rare or novel variants in known bleeding genes against a murine RNA sequencing dataset that models proplatelet-forming megakaryocytes. Results Filtering against known genes identified candidate variants in 59 individuals, including novel variants in several known genes. In the remaining cohort of "unknown" patients, indexing against differentially expressed genes revealed candidate gene variants in several novel unreported genes, focusing on 14 patients with a severe clinical presentation. Conclusions We identified candidate mutations in a cohort of patients with no previous genetic diagnosis. This work involves innovative coupling of RNA sequencing and WES to identify candidate variants forming the basis of future study in a significant number of undiagnosed patients.

The assessment of platelet spreading through light microscopy, and the subsequent quantification of parameters such as surface area and circularity, is a key assay for many platelet biologists. Here we present an analysis workflow which robustly segments individual platelets to facilitate the analysis of large numbers of cells while minimising user bias. Image segmentation is performed by interactive learning and touching platelets are separated with an efficient semi-automated protocol. We also use machine learning methods to robustly automate the classification of platelets into different subtypes. These adaptable and reproducible workflows are made freely available and are implemented using the open source software KNIME and ilastik.

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 CeO2, Ag, TiO2, ZnO and SiO2 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. TiO2 and SiO2 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. CeO2 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.

Purpose The purpose of this paper is to find the applications, advantages and challenges of human resource information system (HRIS) from different sectors of Pakistan. It also finds the demographic perspective of HRIS. Design/methodology/approach Questionnaire was adopted from previous studies. Responses were gathered through social websites and physically. The questionnaire was sent to 491 respondents from which only 99 responded. Reliability, t-testing and chi-square were used for the analysis. Findings Result shows that HRIS is widely used in Pakistani organizations for the purpose of accessing employees’ information, absence monitoring and performance appraisal to get quick responses to information, reduction of error and paperwork. It helps standardizing the program and manpower requirement, but lack of commitment by the lower management is the most significant barrier for its implementation because of fear of change by them. It is also found that there is no association between gender and adoption of HRIS but different employment position perceives differently the adoption of HRIS. Research limitations/implications The research is limited to the Pakistani organizations. The research paper is useful for the Pakistani human resources professionals, as it gives the local perspective of HRIS. Originality/value The previous results are from the developed countries and there is merely any reliable paper found on the several aspects of HRIS in Pakistani context. The research has also focused on the demographic variables that are employment position and gender with respect to their perception on the adoption of HRIS.

Microtubules are ubiquitously expressed cytoskeletal structures responsible for a host of cellular processes from division to cargo transport. In specialised cells, the expression of specific isoforms of tubulin and their subsequent post-translational modifications are thought to drive and co-ordinate unique morphologies and behaviours. The mechanisms by which {beta}-1 tubulin (encoded by TUBB1), the platelet and megakaryocytic specific {beta}-tubulin isoform required for platelet production and function, drives these processes remains poorly understood. We investigate the effects of two key tubulin post-translational polymodifications (polyglutamylation and polyglycylation) on the glutamate rich C-terminus of {beta}-1 tubulin using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived megakaryocytes, and healthy human donor platelets. We find that while megakaryocytes (MKs) are positive for both polymodifications, polyglycylation is substantially reduced on platelets. On platelet activation, the marginal band becomes heavily polyglutamylated, which drives the mobilisation of motor proteins, including axonemal dynein, to achieve the shape change required for the haemostatic role of platelets. Finally, we show that a number of modifying enzymes (Tubulin Tyrosine Like Ligases (TTLLs) and Cytosolic Carboxypeptidases (CCPs)) are up-regulated through MK maturation. In platelets, a single polyglutamylase (TTLL7) is expressed to mediate the polyglutamylation of the marginal band required for shape change on activation. Finally, we report a novel disease causing gene in multiple families (TTLL10) resulting in bleeding despite normal platelet production and function. This work highlights the importance of a complex regulatory mechanism driven by both cell specific tubulin isoform expression and differential post-translational modification to drive specialist function, the loss of which results in disease states.nnnnO_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/595868_fig1.gif" ALT="Figure 1">nView larger version (20K):norg.highwire.dtl.DTLVardef@2ea097org.highwire.dtl.DTLVardef@131582corg.highwire.dtl.DTLVardef@93b671org.highwire.dtl.DTLVardef@1aee018_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1.C_FLOATNO Graphical AbstractIn this work we report a system of reversible polymodification (polyglutamylation and polyglycylation) of the MK and platelet specific -1 tubulin isoform required for platelet production and function. These polymodifications are driven by an up-regulation of key Tubulin Tyrosine Like Ligases (TTLLs) which mediate the addition of single glutamate or glycine residues (initiases which trigger monogluytamylation or monoglycylation), and elongases which extend a glutamate or glycine tail (polyglutamylation and polyglycylation respectively). These processes are reversed by Cytosolic Carboxypeptidase (CCP) enzymes). MKs express a range of TTLLs and CCPs, while platelets only express a single polyglutamylase, TTLL7.nnC_FIG

Nanomaterial (NM) surface chemistry has an established and significant effect on interactions at the nano-bio interface, with important toxicological consequences for manufactured NMs, as well as potent effects on the pharmacokinetics and efficacy of nano-therapies. In this work, the effects of different surface modifications (PVP, Dispex AA4040, and Pluronic F127) on the uptake, cellular distribution, and degradation of titanium dioxide NMs (TiO2 NMs, ~10 nm core size) are assessed and correlated with the localization of fluorescently-labeled serum proteins forming their coronas. Imaging approaches with an increasing spatial resolution, including automated high throughput live cell imaging, correlative confocal fluorescence and reflectance microscopy, and dSTORM super-resolution microscopy, are used to explore the cellular fate of these NMs and their associated serum proteins. Uncoated TiO2 NMs demonstrate a rapid loss of corona proteins, while surface coating results in the retention of the corona signal after internalization for at least 24 h (varying with coating composition). Imaging with two-color super-resolution dSTORM revealed that the apparent TiO2 NM single agglomerates observed in diffraction-limited confocal microscopy are actually adjacent smaller agglomerates, and provides novel insights into the spatial arrangement of the initial and exchanged coronas adsorbed at the NM surfaces.

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.

Superresolution microscopy has become increasingly widespread over the past 5 years and allows users to image biological processes below the diffraction limit of traditional fluorescence microscopy where resolution is restricted to approximately 250 nm. Superresolution refers to a wide range of techniques which employ different approaches to circumvent the diffraction limit. Two of these approaches, structured illumination microscopy (SIM) and single-molecule localization microscopy (SMLM), which provide a doubling and tenfold increase in resolution respectively, are dominating the field. This is partly because of the insights into biology they offer and partly because of their commercialization by the main microscope manufacturers. This chapter provides background to the two techniques, practical considerations for their use, and protocols for their application to platelet biology.

Key Points Platelet activation in vitro results in a more rapid and greater upregulation of TLT-1 surface expression compared with P-selectin. TLT-1 is more rapidly translocated to the surface of activated platelets than P-selectin during thrombus formation in vivo.

Platelets are anucleate and mostly ribosome-free cells within the bloodstream, derived from megakaryocytes within bone marrow and crucial for cessation of bleeding at sites of injury. Inherited thrombocytopenias are a group of disorders characterized by a low platelet count and are frequently associated with excessive bleeding. SLFN14 is one of the most recently discovered genes linked to inherited thrombocytopenia where several heterozygous missense mutations in SLFN14 were identified to cause defective megakaryocyte maturation and platelet dysfunction. Yet, SLFN14 was recently described as a ribosome-associated protein resulting in rRNA and ribosome-bound mRNA degradation in rabbit reticulocytes. To unveil the cellular function of SLFN14 and the link between SLFN14 and thrombocytopenia, we examined SLFN14 (WT/mutants) in in vitro models. Here, we show that all SLFN14 variants colocalize with ribosomes and mediate rRNA endonucleolytic degradation. Compared to SLFN14 WT, expression of mutants is dramatically reduced as a result of post-translational degradation due to partial misfolding of the protein. Moreover, all SLFN14 variants tend to form oligomers. These findings could explain the dominant negative effect of heterozygous mutation on SLFN14 expression in patients’ platelets. Overall, we suggest that SLFN14 could be involved in ribosome degradation during platelet formation and maturation.

Platelets are anucleate and mostly ribosome-free cells within the bloodstream, derived from megakaryocytes within bone marrow and crucial for cessation of bleeding at sites of injury. Inherited thrombocytopenias are a group of disorders characterized by alow platelet count and are frequently associated with excessive bleeding. SLFN14 is one of the most recently discovered genes linked to inherited thrombocytopenia where several heterozygous missense mutations in SLFN14 were identified to cause defective megakaryocyte maturation and platelet dysfunction. Yet, SLFN14 was recently described as a ribosome-associated protein resulting in rRNA and ribosome-bound mRNA degradation in rabbit reticulocytes. To unveil the cellular function of SLFN14 and the link between SLFN14 and thrombocytopenia, we examined SLFN14 (WT/mutants) in in vitro models. Here, we show that all SLFN14 variants co-localize with ribosomes and mediate rRNA endonucleolytic degradation and ribosome clearance. Compare dto SLFN14 WT, expression of mutants is dramatically reduced as a result of post-translational degradation due to partial misfolding of the protein. Moreover, all SLFN14 variants tend to form oligomers. These findings could explain the dominant negative effect of heterozygous mutation on SLFN14 expression in patients’ platelets. Overall we suggest that SLFN14 could be involved in ribosome degradation during platelet formation and maturation.

The British Heart Foundation (BHF) annual image competition, Reflections of Research, provides a glimpse into the cutting-edge research that the BHF funds

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.