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Dynamic replacement (DR) is a ground improvement technique that has been used now for almost 50 years. During the formation of a DR column a crater is created which is then filled with a coarse material and compacted again. The length, diameter and shape of such a column cannot be observed directly, which makes the design and execution more troublesome. In the article presented are the dimensions and shapes of 18 columns from eight different test fields. They were formed by means of pounders of various masses (9 or 11.5 Mg) and dimensions (1.00 or 1.05 m in diameter, 1.8 or 2.0 m in height). Based on the observations and measurements, it was concluded that the shape and diameter of a DR column is influenced by the parameters of the soft soil that is supposed to be improved (its thickness, physical state and location in the profile), as well as by the diameter of the pounder. It was revealed that, as the length of the columns increased, the column shapes changed from: a cylinder, through a truncated cone, a barrel to an asymmetric barrel. The diameters of all of the columns were 1.4–2.8 times larger than the diameters of the used pounders and the largest values were noted along the depth of the weakest layer. The presented results may be useful to the profession. When the thickness of the weak soil, its type and state are known and the technological parameters are similar to the ones presented in this paper, it is possible to predict the shape and diameter of the columns depending on the diameter of the pounder.

Stiffness is one of the most important characteristics of geomaterials, and at the same time one of the most difficult to evaluate. It can be described by means of various stress-strain moduli, whose values strongly depend on the strain range and on the method of determination. The aim of the article is to evaluate and compare selected stiffness parameters (Young’s modulus E and shear modulus G) of an anthropogenic soil on the basis of triaxial tests. The experiments were carried out on samples consisting of loess mixed with sand. Loess is a collapsible aeolian sediment with a high calcium carbonate content and so it is a very challenging material for geotechnical applications. The addition of sand improves its properties and increases its suitability for earthworks. The specimens were compacted with normal Proctor energy at the optimal water content, which ensured repeatability of the results. Standard triaxial tests (drained and undrained) were carried out at the effective confining stresses in the range of 50 – 350 kPa. The specimens’ deformation was measured by means of external and local displacement transducers. Additionally, bender elements were used to assess the initial soil stiffness. The applied research methods allowed determination of the deformation characteristics in the range from very small to large strains. The stiffness moduli were assessed using different definitions and methods. It was confirmed that the stiffness of loess is improved by its proper compaction and addition of sand, when compared to the results available in literature for natural loesses.

The development of measurement technologies allows for acquiring various data. The Terrestrial Laser Scanning (TLS) technology is frequently combined with classic geodetic measurements – tacheometry or levelling. This article presents a process of the diaphragm wall monitoring during excavation supported with a top-down method. The construction technology applied required proper planning and performance of measurements in difficult construction site conditions in the city centre. TLS allowed for limiting works at daytime and performing monitoring during the night break in works at the construction site as well as limiting the impact of the subsoil process vibrations on the values of displacements and deformations determined. The authors present a comparison of the results of displacement and deformation measurements with a terrestrial laser scanning and tacheometric measurement method. The possibilities of using the data acquired, among others, for the indication of filtration areas, spatial surface deformation analyses and assessment of the wall execution compliance with the design are presented. The analyses carried out show that the TLS may be used in the investment process from the very beginning, being a component of the Building Information Modelling (BIM).

The author juxtaposes two extreme approaches to the relationship between consciousness and the physical world: phenomenological-idealistic (represented by Edmund Husserl) and radically naturalistic (represented by Paul Churchland). These two positions are interpreted in terms of opposite if symmetrical types of reduction (on the one hand, the reduction of the world to a sense for consciousness, and on the other hand, the reduction of consciousness to an element of the physical world). They emerge as two ways of abstracting from the ambivalence of ordinary experience, in which consciousness and the physical world are both mutually entangled and non-identical with each other. In conclusion, the author argues that contemporary philosophy, which follows both the idealistic and the naturalistic path, fails to solve the problem of this relationship.

The interest in rural tourism in Poland and Romania is the result of processes originating from economic and social factors reflecting the needs of both rural inhabitants and tourists. A significant growth in the field of rural tourism leads to implementation of innovative products in the development of tourist services. The aim of the study presented in this paper is to show the essence of innovation as a component of the contemporary tourist industry, and to present the type of innovations that occur in rural tourism in Poland and in Romania. The theoretical part analyses innovation in the field of rural tourism. The empirical part, in turn, presents case studies of tourist businesses in rural areas, taking into account the implemented innovations, in both Poland and Romania. The case studies are presented based on interviews with owners of tourist facilities.

Objectives: Reports on an increasing number of hospitalizations in other European countries and the lack of epidemiological data on the prevalence of bronchiectasis in Poland constituted motivation for the authors to investigate temporal changes of the registered incidence and hospitalization due to bronchiectasis in Silesian voivodeship, and to evaluate spatial variability of the disease in the study region.Material and Methods: The study is a descriptive epidemiological project. Temporal and spatial variability of coefficients describing numbers of newly diagnosed cases and first time hospitalizations due to bronchiectasis (code J47 according to International Statistical Classification of Diseases and Related Health Problems, 10th revision (ICD-10)) were evaluated based on the registered data available from the National Health Found (2006-2010) and the data from MZ/Szp-11 reports (2000-2011). The data concerned adults aged ≥ 19 years, inhabitants of Silesian voivodeship. Maps of incidence or hospitalization rates due to bronchiectasis were constructed by the use of a geographical information system ArcGIS.Results: The obtained results show a stable trend of reported new diseases, whereas the number of first time hospitalizations is increasing. Values of the standardized incidence were 19.9-25.1/100 000 inhabitants, and values of the standardized first-time hospitalization were 1.2-2.9/100 000 inhabitants. The reported rates of bronchiectasis indicate significant spatial differences in epidemiological situation in the study region.Conclusions: The findings showed territorial variability of the incidence and hospitalization of bronchiectasis recorded in Silesian voivodeship. The observed variability might result from regional differences in the availability of specialized medical services. [ABSTRACT FROM AUTHOR]

Ethylcellulose polymer was used as a coating material in the preparation of controlled release fertilizers. The materials have been prepared with the use of an immersion method. The mass ratio of polymer to fertilizer was in the range of 0.165–0.285 and the layer thickness was in the range of 204–244 μm. Mechanical properties of the prepared materials were significantly better in comparison with the initial fertilizer. Measurements of time and the degree of release of mineral components from the obtained materials were determined with a standard method. Ethylcellulose-coated materials have met the requirements of controlled release fertilizers.

This paper presents the results of studies on the preparation of cellulosic membranes, from a solution in 1-ethyl-3- methylimidazolium acetate (EMIMAc), using the phase inversion method. Initially, the membranes were obtained by coagulation of the polymer film in water and primary alcohols (methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol), 1-hexanol, 1-octanol) resulting in membranes with significantly differing morphologies. Subsequently, composite membranes were produced, with the support layer being a membrane with the largest pores, and the skin layer a membrane with smaller pores. The resulting membranes were tested for physicochemical and transport properties. The morphology of the membrane surfaces and their cross-sections were investigated by using a scanning electron microscope (SEM). The structure of the membranes, on the other hand, was investigated by FTIR spectroscopy and WAXS structural analysis.

In the era of the development of modern measurement technologies, their interconnectedness is of high importance. This paper presents a review of the most popular, currently utilised measuring marks in tachymetric measurements and in object laser scanning. This paper presents the authors’ own solutions that facilitate the linkage of data acquired through terrestrial laser scanning with tachymetric measurements. The proposed marks used to perform orientation on the scanned surfaces were successfully tested in the field with the use of laser scanners manufactured by the Z+F, Leica and Riegl companies. The document describes the consecutive steps that eliminate individual problems that arise during both tachymetric measurements and laser scanning. As a result of the work, a new kinds of marks were created allowing tachymetric measurements and laser scanning at the level of accuracy that is required for basic engineering measurements. This paper also presents a discussion on how to prepare the marks yourselves and the marks durability on the surface of the surveyed object.

The authors present possible applications of thermal data as an additional source of information on an object’s behaviour during the technical assessment of the condition of a concrete surface. For the study one of the most recent propositions introduced by Zoller + Fröhlich company was used, which is an integration of a thermal camera with a terrestrial laser scanner. This solution enables an acquisition of geometric and spectral data on the surveyed object and also provides information on the surface’s temperature in the selected points. A section of the dam’s downstream concrete wall was selected as the subject of the study for which a number of scans were carried out and a number of thermal images were taken at different times of the day. The obtained thermal data was confronted with the acquired spectral information for the specified points. This made it possible to carry out broader analysis of the surface and an inspection of the revealed fissure. The thermal analysis of said fissure indicated that the temperature changes within it are slower, which may affect the way the concrete works and may require further elaboration by the appropriate experts. Through the integration of a thermal camera with a terrestrial laser scanner one can not only analyse changes of temperature in the discretely selected points but on the whole surface as well. Moreover, it is also possible to accurately determine the range and the area of the change affecting the surface. The authors note the limitations of the presented solution like, inter alia, the resolution of the thermal camera.

The registered xyz coordinates in the form of a point cloud captured by terrestrial laser scanner and the intensity values (I) assigned to them make it possible to perform geometric and spectral analyses. Comparison of point clouds registered in different time periods requires conversion of the data to a common coordinate system and proper data selection is necessary. Factors like point distribution dependant on the distance between the scanner and the surveyed surface, angle of incidence, tasked scan’s density and intensity value have to be taken into consideration. A prerequisite for running a correct analysis of the obtained point clouds registered during periodic measurements using a laser scanner is the ability to determine the quality and accuracy of the analysed data. The article presents a concept of spectral data adjustment based on geometric analysis of a surface as well as examples of geometric analyses integrating geometric and physical data in one cloud of points: cloud point coordinates, recorded intensity values, and thermal images of an object. The experiments described here show multiple possibilities of usage of terrestrial laser scanning data and display the necessity of using multi-aspect and multi-source analyses in anthropogenic object monitoring. The article presents examples of multisource data analyses with regard to Intensity value correction due to the beam’s incidence angle. The measurements were performed using a Leica Nova MS50 scanning total station, Z+F Imager 5010 scanner and the integrated Z+F T-Cam thermal camera.

The aim of the study was to determine the possibility of reducing fat content in fatty sponge-cake products by addition of inulin. 200 g/kg, 440 g/kg, 680 g/kg and 100% of fat was substituted with 20 g/kg, 35 g/kg, 50 g/kg, and 62.5 g/kg of inulin, respectively. The authors used two types of fat: with low and high content of trans isomers – containing 2.1 g/kg and 511.2 g/kg of fatty acid methyl esters (FAME), respectively. An analysis of crumb quality and the evaluation of sensory discriminants were undertaken.

The purpose of this work was evaluation of stability of emulsions containing modified fats obtained by enzymatic interesterification of mutton tallow with walnut oil, determination of the optimal range of thickener content, and preferable processing conditions. Modified fats were produced by enzymatic interesterification of mutton tallow with walnut oil. It has been shown that by intentional forcing of hydrolysis process by addition 13 wt% of water into reaction mixture the highest amount of polar fraction in the final product was observed. The physicochemical parameters of the obtained fats were determined in this study. To reveal the stability of the investigated emulsions the comprehensive tests with Turbiscan enabling detailed investigation of the droplet size distribution, dispersion index, morphology, and microstructure were performed. Additionally, to get a full picture of the emulsions stability the rheological measurements in oscillatory mode were carried out. Taking into consideration the physicochemical properties of the investigated emulsions it was found that emulsions containing carboxymethylcellulose exhibited the highest stability. The emulsifiers formed during enzymatic interesterification allow formation of a stable emulsion system. This offers the unique opportunity of using interesterified fat as the oil phase in preparation of emulsions for food, cosmetic and pharmaceutical industries.

Parameters of soil constitutive models are not constant. This mainly concerns the strain parameters such as K, G or Eoed modules. What influences their values is not only soil type, structure and consistency, but also the history of stress and strain states. So, it is the question of the current state but also of what happened to the subsoil in the past (regarding geological and anthropological activity) and what impact would have the planned soil–structure interaction.

The article presents results of a bibliometric analysis of the scientific output made by researchers from Institute of Information Science and Book Studies at Nicolas Copernicus University in Torun during 1976-2007. The research material includes set of 873 items, especially: books and their editions, articles published in journals and collective works, literature reviews, interviews and translations. Authors of this article make a quantitative analysis of the material, take into account dynamic of publications' growth, organizational and employment status of Institute, during the 32-year period of its activity.

Abstract: Fibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "antithrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (α-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and αIIbβ3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2rγnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ibα) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet α-granules to sites of vascular injury., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Plasma cell-free DNA (cfDNA), a marker of disease severity in sepsis, is a recognized driver of thromboinflammation and a potential therapeutic target. In sepsis, plasma cfDNA is mostly derived from neutrophil extracellular trap (NET) degradation. Proposed NET-directed therapeutic strategies include preventing NET formation or accelerating NET degradation. However, NET digestion liberates pathogens and releases cfDNA that promote thrombosis and endothelial cell injury. We propose an alternative strategy of cfDNA and NET stabilization with chemokine platelet factor 4 (PF4, CXCL4). We previously showed that human PF4 (hPF4) enhances NET-mediated microbial entrapment. We now show that hPF4 interferes with thrombogenicity of cfDNA and NETs by preventing their cleavage to short-fragment and single-stranded cfDNA that more effectively activates the contact pathway of coagulation. In vitro, hPF4 also inhibits cfDNA-induced endothelial tissue factor surface expression and von Willebrand factor release. In vivo, hPF4 expression reduced plasma thrombin-antithrombin (TAT) levels in animals infused with exogenous cfDNA. Following lipopolysaccharide challenge, Cxcl4-/- mice had significant elevation in plasma TAT, cfDNA, and cystatin C levels, effects prevented by hPF4 infusion. These results show that hPF4 interacts with cfDNA and NETs to limit thrombosis and endothelial injury, an observation of potential clinical benefit in the treatment of sepsis.

Background: Metastasis is the main cause of death in patients with colorectal cancer (CRC). Apart from platelets, platelet-derived microparticles (PMPs) are also considered important factors that can modify the activity of cancer cells. PMPs are incorporated by cancer cells and can also serve as intracellular signalling vesicles. PMPs are believed to affect cancer cells by upregulating their invasiveness. To date, there is no evidence that such a mechanism occurs in colorectal cancer. It has been shown that platelets can stimulate metalloproteases (MMPs) expression and activity via the p38MAPK pathway in CRC cells, leading to their elevated migratory potential. This study aimed to investigate the impact of PMPs on the invasive potential of CRC cells of various phenotypes via the MMP-2, MMP-9 and p38MAPK axis., Methods: We used various CRC cell lines, including the epithelial-like HT29 and the mesenchymal-like SW480 and SW620. Confocal imaging was applied to study PMP incorporation into CRC cells. The presence of surface receptors on CRC cells after PMP uptake was evaluated by flow cytometry. Transwell and scratch wound-healing assays were used to evaluate cell migration. The level of C-X-C chemokine receptor type 4 (CXCR4), MMP-2, and MMP-9 and the phosphorylation of ERK1/2 and p38MAPK were measured by western blot. MMP activity was determined using gelatine-degradation assays, while MMP release was evaluated by ELISA., Results: We found that CRC cells could incorporate PMPs in a time-dependent manner. Moreover, PMPs could transfer platelet-specific integrins and stimulate the expression of integrins already present on tested cell lines. While mesenchymal-like cells expressed less CXCR4 than epithelial-like CRC cells, PMP uptake did not increase its intensity. No significant changes in CXCR4 level either on the surface or inside CRC cells were noticed. Levels of cellular and released MMP-2 and MMP-9 were elevated in all tested CRC cell lines after PMP uptake. PMPs increased the phosphorylation of p38MAPK but not that of ERK1/2. Inhibition of p38MAPK phosphorylation reduced the PMP-induced elevated level and release of MMP-2 and MMP-9 as well as MMP-dependent cell migration in all cell lines., Conclusions: We conclude that PMPs can fuse into both epithelial-like and mesenchymal-like CRC cells and increase their invasive potential by inducing the expression and release of MMP-2 and MMP-9 via the p38MAPK pathway, whereas CXCR4-related cell motility or the ERK1/2 pathway appears to not be affected by PMPs. Video Abstract., (© 2023. The Author(s).)

Periodontitis is a chronic inflammatory disease characterized by the release of matrix metalloproteinases (MMPs) from resident connective tissue cells in tooth-supporting tissues (periodontium). Platelet activation, and the attendant release of pro-inflammatory chemokines such as platelet factor 4 (CXCL4/PF4), are associated with periodontitis although the associated biochemical pathways remain undefined. Here we report that recombinant PF4 is internalized by cultured human gingival fibroblasts (hGFs), resulting in significant (p < 0.05) upregulation in both the production and release of MMP-2 (gelatinase A). This finding was corroborated by elevated circulating levels of MMP-2 (p < 0.05) in PF4-overexpressing transgenic mice, relative to controls. We also determined that PF4 induces the phosphorylation of NF-κB; notably, the suppression of NF-κB signaling by the inhibitor BAY 11-7082 abrogated PF4-induced MMP-2 upregulation. Moreover, the inhibition of surface glycosaminoglycans (GAGs) blocked both PF4 binding and NF-κB phosphorylation. Partial blockade of PF4 binding to the cells was achieved by treatment with either chondroitinase ABC or heparinase III, suggesting that both chondroitin sulfate and heparan sulfate mediate PF4 signaling. These results identify a novel pathway in which PF4 upregulates MMP-2 release from fibroblasts in an NF-κB- and GAG-dependent manner, and further our comprehension of the role of platelet signaling in periodontal tissue homeostasis., (© 2022. The Author(s).)

Objective: Septic peritonitis is associated with significant morbidity and mortality. As a potential therapeutic agent in the treatment of sepsis, 2-O, 3-O desulfated heparin (ODSH) reduces histones and platelet factor 4 (PF4) in mouse sepsis models. This pilot clinical trial evaluated the safety and effect of ODSH in client-owned dogs with septic peritonitis., Interventions: In an IACUC-approved, open-label, prospective, dose-escalation clinical trial in 6 dogs with spontaneous septic peritonitis, ODSH administration was initiated following surgical explore to achieve source control. Acute patient physiology and laboratory evaluation (APPLE fast and APPLE full ) scores on admission, source of septic peritonitis, requirement for vasopressors, the administration of blood products, and survival to discharge were recorded. Platelet count, cell free DNA (cfDNA) concentration, and platelet factor 4 (PF4) concentrations were measured at the time of each ODSH dosage. A dose of ODSH was administered every 8 hs for a total of 4 doses (maximum total dosage 75 mg/kg) based on a pre-determined escalation protocol. Patients were monitored in the ICU following administration for evidence of clinical hemorrhage., Main Results: The mean APPLE fast and APPLE full scores on admission were 22 +/- 6 and 32 +/-10, respectively. Four dogs received 4 total dosages of ODSH and 2 dogs received 3 total dosages of ODSH intravenously. The mean total dosage of ODSH administered during the study period was 48.3 +/- 21.6 mg/kg. No dog required dose de-escalation or had any evidence of bleeding. Four dogs survived to discharge., Conclusions: No adverse effects of ODSH administration were documented in dogs with septic peritonitis. A randomized controlled trial is necessary to evaluate ODSH as a novel therapeutic in the treatment of septic peritonitis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mauro, Lambert, Kowalska, Thawley, Poncz and Otto.)

Fibrosis is a major cause of mortality worldwide, characterized by myofibroblast activation and excessive extracellular matrix deposition. Systemic sclerosis is a prototypic fibrotic disease in which CXCL4 is increased and strongly correlates with skin and lung fibrosis. Here we aim to elucidate the role of CXCL4 in fibrosis development. CXCL4 levels are increased in multiple inflammatory and fibrotic mouse models, and, using CXCL4-deficient mice, we demonstrate the essential role of CXCL4 in promoting fibrotic events in the skin, lungs, and heart. Overexpressing human CXCL4 in mice aggravates, whereas blocking CXCL4 reduces, bleomycin-induced fibrosis. Single-cell ligand-receptor analysis predicts CXCL4 to affect endothelial cells and fibroblasts. In vitro, we confirm that CXCL4 directly induces myofibroblast differentiation and collagen synthesis in different precursor cells, including endothelial cells, by stimulating endothelial-to-mesenchymal transition. Our findings identify a pivotal role of CXCL4 in fibrosis, further substantiating the potential role of neutralizing CXCL4 as a therapeutic strategy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

During metastasis, cancer cells undergo phenotype changes in the epithelial-mesenchymal transition (EMT) process. Extracellular vesicles (EVs) released by cancer cells are the mediators of intercellular communication and play a role in metastatic process. Knowledge of factors that influence the modifications of the pre-metastatic niche for the migrating carcinoma cells is important for prevention of metastasis. We focus here on how cancer progression is affected by EVs released from either epithelial-like HT29-cells or from cells that are in early EMT stage triggered by Snail transcription factor (HT29-Snail). We found that EVs released from HT29-Snail, as compared to HT29-pcDNA cells, have a different microRNA profile. We observed the presence of interstitial pneumonias in the lungs of mice injected with HT29-Snail cells and the percent of mice with lung inflammation was higher after injection of HT29-Snail-EVs. Incorporation of EVs released from HT29-pcDNA, but not released from HT29-Snail, leads to the increased secretion of IL-8 from macrophages. We conclude that Snail modifications of CRC cells towards more invasive phenotype also alter the microRNA cargo of released EVs. The content of cell-released EVs may serve as a biomarker that denotes the stage of CRC and EVs-specific microRNAs may be a target to prevent cancer progression.

Essentials The frequency of predicted loss-of-function (pLoF) variants in platelet-associated genes is unknown in the general population. Datasets like Genome Aggregation Database allow us to analyze pLoF variants with increased resolution. Expected prevalence of significant pLoF variants in platelet-associated genes in 0.329% in the general population. Platelet-associated genes that cause phenotypes due to haploinsufficiency are significantly depleted for deleterious variation. ABSTRACT: Background Inherited platelet disorders are being recognized more frequently as advanced sequencing technologies become more commonplace in clinical scenarios. The prevalence of each inherited platelet disorder and the disorders in aggregate are not known. This deficit in the field makes it difficult for clinicians to discuss results of sequencing assays and provide appropriate anticipatory guidance. Objectives In this study, we aim to calculate the prevalence of predicted loss-of-function variants in platelet-associated genes in the general population. Methods Here, we leverage the aggregation of exomes from the general population in the form of Genome Aggregation Database to assess 58 platelet-associated genes with phenotypic correlates. We use the loss-of-function transcript effect estimator (LOFTEE) to identify predicted loss-of-function mutations in these platelet-associated genes. These variants are curated and we then quantify the frequency of predicted loss-of-function variants in each gene. Results Our data show that 0.329% of the general population have a clinically meaningful predicted loss-of-function variant in a platelet-associated gene. Thus, these individuals are at risk for bleeding disorders that can range from mild to severe. Conclusions These data provide a novel lens through which clinicians can analyze sequencing results in their patients as well as an additional method to curate newly discovered platelet-associated genes in the future., (© 2020 International Society on Thrombosis and Haemostasis.)

Inherited bone marrow failure (IBMF) syndromes are rare blood disorders characterized by hematopoietic cell dysfunction and predisposition to hematologic malignancies. Despite advances in the understanding of molecular pathogenesis of these heterogeneous diseases, genetic variant interpretation, genotype-phenotype correlation, and outcome prognostication remain difficult. As new IBMF and other myelodysplastic syndrome (MDS) predisposition genes continue to be discovered (frequently in small kindred studies), there is an increasing need for a systematic framework to evaluate penetrance and prevalence of mutations in genes associated with IBMF phenotypes. To address this need, we analyzed population-based genomic data from >125 000 individuals in the Genome Aggregation Database for loss-of-function (LoF) variants in 100 genes associated with IBMF. LoF variants in genes associated with IBMF/MDS were present in 0.426% of individuals. Heterozygous LoF variants in genes in which haploinsufficiency is associated with IBMF/MDS were identified in 0.422% of the population; homozygous LoF variants associated with autosomal recessive IBMF/MDS diseases were identified in only .004% of the cohort. Using age distribution of LoF variants and 2 measures of mutational constraint, LOEUF ("loss-of-function observed/expected upper bound fraction") and pLI ("probability of being loss-of-function intolerance"), we evaluated the pathogenicity, tolerance, and age-related penetrance of LoF mutations in specific genes associated with IBMF syndromes. This analysis led to insights into rare IBMF diseases, including syndromes associated with DHX34, MDM4, RAD51, SRP54, and WIPF1. Our results provide an important population-based framework for the interpretation of LoF variant pathogenicity in rare and emerging IBMF syndromes., (© 2020 by The American Society of Hematology.)

The transcription factor Snail triggers epithelial-to-mesenchymal transition (EMT), endowing cancer cells with invasive properties during tumor progression. Extracellular vesicles (EVs) released from cancer cells at various stages of cancer progression are known to influence the tumor pre-metastatic niche and metastatic potential. The aim of this study was to analyze the effect of Snail on murine colon adenocarcinoma cells (MC38 line) and on the characteristics of their EVs. Stable clones of Snail-overexpressing MC38 cells were investigated in vitro versus Mock cells. Increased expression of matrix metalloproteinase MMP-14 and augmented activity of MMP-9 and -14 were observed in Snail-MC38 cells. There was no change in the transcriptomic profile of proteoglycans in Snail-MC38 cells; however, the protein level of Glypican-1 (GPC1) was enhanced in EVs released from those cells. Our finding that GPC1 protein level was enhanced in EVs released from MC38 cells that overexpressed Snail and were in an early EMT stage might explain the specificity of the GPC1 biomarker in colon cancer diagnosis. Further, our data suggest that Snail, by changing the level of GPC1 on EVs released by colon cancer cells, may affect the generation of a distant premetastatic niche and metastatic organotropism in colon adenocarcinoma.

Thromboembolism complicates disorders caused by immunoglobulin G (IgG)-containing immune complexes (ICs), but the underlying mechanisms are incompletely understood. Prior evidence indicates that induction of tissue factor (TF) on monocytes, a pivotal step in the initiation, localization, and propagation of coagulation by ICs, is mediated through Fcγ receptor IIa (FcγRIIa); however, the involvement of other receptors has not been investigated in detail. The neonatal Fc receptor (FcRn) that mediates IgG and albumin recycling also participates in cellular responses to IgG-containing ICs. Here we asked whether FcRn is also involved in the induction of TF-dependent factor Xa (FXa) activity by IgG-containing ICs by THP-1 monocytic cells and human monocytes. Induction of FXa activity by ICs containing IgG antibodies to platelet factor 4 (PF4) involved in heparin-induced thrombocytopenia (HIT), β-2-glycoprotein-1 implicated in antiphospholipid syndrome, or red blood cells coated with anti-(α)-Rh(D) antibodies that mediate hemolysis in vivo was inhibited by a humanized monoclonal antibody (mAb) that blocks IgG binding to human FcRn. IgG-containing ICs that bind to FcγR and FcRn induced FXa activity, whereas IgG-containing ICs with an Fc engineered to be unable to engage FcRn did not. Infusion of an α-FcRn mAb prevented fibrin deposition after microvascular injury in a murine model of HIT in which human FcγRIIa was expressed as a transgene. These data implicate FcRn in TF-dependent FXa activity induced by soluble and cell-associated IgG-containing ICs. Antibodies to FcRn, now in clinical trials in warm autoimmune hemolytic anemia to lower IgG antibodies and IgG containing ICs may also reduce the risk of venous thromboembolism., (© 2020 by The American Society of Hematology.)

Sepsis is characterized by multiorgan system dysfunction that occurs because of infection. It is associated with high morbidity and mortality and is in need of improved therapeutic interventions. Neutrophils play a crucial role in sepsis, releasing neutrophil extracellular traps (NETs) composed of DNA complexed with histones and toxic antimicrobial proteins that ensnare pathogens, but also damage host tissues. At presentation, patients often have a significant NET burden contributing to the multiorgan damage. Therefore, interventions that inhibit NET release would likely be ineffective at preventing NET-based injury. Treatments that enhance NET degradation may liberate captured bacteria and toxic NET degradation products (NDPs) and likely be of limited therapeutic benefit as well. We propose that interventions that stabilize NETs and sequester NDPs may be protective in sepsis. We showed that platelet factor 4 (PF4), a platelet-associated chemokine, binds and compacts NETs, increasing their resistance to DNase I. We now show that PF4 increases NET-mediated bacterial capture, reduces the release of NDPs, and improves outcome in murine models of sepsis. A monoclonal antibody KKO which binds to PF4-NET complexes, further enhances DNase resistance. However, the Fc portion of this antibody activates the immune response and increases thrombotic risk, negating any protective effects in sepsis. Therefore, we developed an Fc-modified KKO that does not induce these negative outcomes. Treatment with this antibody augmented the effects of PF4, decreasing NDP release and bacterial dissemination and increasing survival in murine sepsis models, supporting a novel NET-targeting approach to improve outcomes in sepsis., (© 2020 by The American Society of Hematology.)

Tumor metastasis, the major problem for clinical oncology in colon cancer treatment, is linked with an epithelial-mesenchymal transition (EMT). The observed cellular transformation in this process is manifested by cell elongation, enhanced cell migration and invasion ability, coordinated by cytoskeleton reorganization. In the present study, we examined the role of tubulin-β4 (TUBB4B) downregulation that occurs during EMT in colon cancer cells, in the modulation of the function of microtubules. Based on biochemical and behavioral analysis (transmigration) we posit that the decrease of the TUBB4B level is critical for microtubule-vimentin interaction and contributes to the maintenance of polarity in migrating cells. The microscopic studies revealed that TUBB4B decrease is accompanied by cell elongation and increased number of matured focal adhesion sites, which is a characteristic of the cell metastatic stage. We also demonstrated faster polymerization of microtubules in cells with a lower level of TUBB4B. Simultaneous TUBB3 upregulation, reported during EMT, acts additively in this process. Our studies suggest that the protein level of TUBB4B could be used as a marker for detection of the preinvasive stages of the colon cancer cells. We also concluded that chemotherapy enriched to increase TUBB4B level and/or to stabilize microtubule polymerization might more effectively prevent metastasis in colon cancer development., Competing Interests: The authors declare no conflict of interest

Cadherins play a major role in mediating cell-cell adhesion, which shares many parallels with platelet-platelet interactions during aggregate formation and clot stabilization. Platelets express epithelial (E)-cadherin, but its contribution to platelet function and/or platelet production is currently unknown. To assess the role of E-cadherin in platelet production and function in vitro and in vivo, we utilized a megakaryocyte-specific E-cadherin knockout mouse model. Loss of E-cadherin in megakaryocytes does not affect megakaryocyte maturation, platelet number or size. However, platelet dysfunction in the absence of E-cadherin is revealed when conditional knockout mice are challenged with acute antibody-mediated platelet depletion. Unlike wild-type mice that recover fully, knockout mice die within 72 hours post-antibody administration, likely from haemorrhage. Furthermore, conditional knockout mice have prolonged tail bleeding times, unstable clot formation, reduced clot retraction and reduced fibrin deposition in in vivo injury models. Murine platelet aggregation in vitro in response to thrombin and thrombin receptor activating peptide is compromised in E-cadherin null platelets, while aggregation in response to adenosine diphosphate (ADP) is not significantly different. Consistent with this, in vitro aggregation of primary human platelets in response to thrombin is decreased by an inhibitory E-cadherin antibody. Integrin activation and granule secretion in response to ADP and thrombin are not affected in E-cadherin null platelets, but Akt and glycogen synthase kinase 3β (GSK3β) activation are attenuated, suggesting a that E-cadherin contributes to aggregation, clot stabilization and retraction that is mediated by phosphoinositide 3-kinase/Akt/GSK3β signalling. In summary, E-cadherin plays a salient role in platelet aggregation and clot stability., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Platelet factor 4 (PF4) is a pleiotropic inflammatory chemokine, which has been implicated in various inflammatory disorders including liver fibrosis. However, its role in acute liver diseases has not yet been elucidated. Here we describe an unexpected, anti-inflammatory role of PF4. Serum concentrations of PF4 were measured in patients and mice with acute liver diseases. Acute liver injury in mice was induced either by carbon tetrachloride or by D-galactosamine hydrochloride and lipopolysaccharide. Serum levels of PF4 were decreased in patients and mice with acute liver diseases. PF4 -/- mice displayed increased liver damage in both models compared to control which was associated with increased apoptosis of hepatocytes and an enhanced pro-inflammatory response of liver macrophages. In this experimental setting, PF4 -/- mice were unable to generate activated Protein C (APC), a protein with anti-inflammatory activities on monocytes/macrophages. In vitro , PF4 limited the activation of liver resident macrophages. Hence, the systemic application of PF4 led to a strong amelioration of experimental liver injury. Along with reduced liver injury, PF4 improved the severity of the pro-inflammatory response of liver macrophages and induced increased levels of APC. PF4 has a yet unidentified direct anti-inflammatory effect in two models of acute liver injury. Thus, attenuation of acute liver injury by systemic administration of PF4 might offer a novel therapeutic approach for acute liver diseases.

During tumor development and ongoing metastasis the acquisition of mesenchymal cell traits by epithelial carcinoma cells is achieved through a programmed phenotypic shift called the epithelial-to-mesenchymal transition, EMT. EMT contributes to increased cancer cell motility and invasiveness mainly through invadosomes, the adhesion structures that accompany the mesenchymal migration. The invadosomes and their associated proteases restrict protease activity to areas of the cell in direct contact with the ECM, thus precisely controlling cell invasion. Our data prove that Snail-overexpressing HT-29 cells that imitate the phenotype of colon cancer cells in the early stage of the EMT showed an increase in the expression and pericellular activity of cathepsin B. It appears that the pericellular localization of cathepsin B, also observed in colon and rectum adenocarcinoma tissue samples, plays a key role in its function., Competing Interests: The authors declare no conflict of interest.

Epithelial-to-mesenchymal transition (EMT) in cancer cells, represents early stages of metastasis and is a promising target in colorectal cancer (CRC) therapy. There have been many attempts to identify markers and key pathways induced throughout EMT but the process is complex and depends on the cancer type and tumour microenvironment. Here we used the colon cancer cell line HT29, which stably overexpressed Snail, the key transcription factor in early EMT, as a model for colorectal adenocarcinoma cells with a pro-metastatic phenotype. We investigated miRNA expression regulation during that phenotypic switching. We found that overexpression of Snail in HT29 cells triggered significant changes in individual miRNA levels but did not change the global efficiency of miRNA processing. Snail abundance repressed the expression of miR-192 and miR-194 and increased miR-205, let-7i and SNORD13 levels. These identified changes correlated with the reported transcriptomic alterations in Snail-overexpressing HT29 cells. We also investigated how Snail affected the miRNA content of extracellular vesicles (EVs) released from HT29 cells. Our data suggest that the presence of Snail significantly alters the complex mRNA/miRNA interactions in the early steps of metastasis and also has an impact on the content of EVs released from HT29 cells.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenic disorder associated with a severe prothrombotic state. We investigated whether neutrophils and neutrophil extracellular traps (NETs) contribute to the development of thrombosis in HIT. Using an endothelialized microfluidic system and a murine passive immunization model, we show that HIT induction leads to increased neutrophil adherence to venous endothelium. In HIT mice, endothelial adherence is enhanced immediately downstream of nascent venous thrombi, after which neutrophils undergo retrograde migration via a CXCR2-dependent mechanism to accumulate into the thrombi. Using a microfluidic system, we found that PF4 binds to NETs, leading them to become compact and DNase resistant. PF4-NET complexes selectively bind HIT antibodies, which further protect them from nuclease digestion. In HIT mice, inhibition of NET formation through Padi4 gene disruption or DNase treatment limited venous thrombus size. PAD4 inactivation did affect arterial thrombi or severity of thrombocytopenia in HIT. Thus, neutrophil activation contributes to the development of venous thrombosis in HIT by enhancing neutrophil-endothelial adhesion and neutrophil clot infiltration, where incorporated PF4-NET-HIT antibody complexes lead to thrombosis propagation. Inhibition of neutrophil endothelial adhesion, prevention of neutrophil chemokine-dependent recruitment of neutrophils to thrombi, or suppression of NET release should be explored as strategies to prevent venous thrombosis in HIT.