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The Database of the Southern Dutch Dialects (DSDD) aims to aggregate and standardise three existing comprehensive lexicographic dialect databases of the Flemish, Brabantic and Limburgian dialects into one integrated dataset (Van Keymeulen, et al., 2019; Van Hout, et al., 2018, De Vriend, et al., 2006). In 2016, the Research Foundation Flanders funded a medium-scale research infrastructure project, the Database of Southern Dutch Dialects (DSDD)[1], which ran from 2017 until 2020 and resulted in a harmonised dataset of concepts (Van den Heuvel, et al., 2016), a user-friendly search engine and a geo-visualisation tool. The application backend provides an Application Programming Interface (API) which will be further developed and made available to researchers to export subsets of the data for analysis using existing digital research tools. At the previous DH Benelux conferences the project team introduced the project (2017), explored the cartographic tools (2018) and demonstrated the prototype (2019). Following the launch of the DSDD platform in 2020, the team will now present the importance of the interdisciplinary approach, the final results of the project, as well as future directions beyond the project lifetime.

It has been proposed that differences may exist between umbilical cord blood (CB) platelets and adult peripheral blood (APB) platelets, including altered protein levels of the main platelet integrins. We have now compared the protein expression profiles of CB and APB platelets employing a label-free comparative proteomics approach. Aggregation studies showed that CB platelets effectively aggregate in the presence of thromboxane A2 analogue, collagen, and peptide agonists of the proteinase-activated receptors 1 and 4. In agreement with previous studies, higher concentrations of the agonists were required to initiate aggregation in the CB platelets. Mass spectrometry analysis revealed no significant difference in the expression levels of critical platelet receptors like glycoprotein (GP)Ib, GPV, GPIX, and integrin αIIbβ3. This was confirmed using flow cytometry-based approaches. Gene ontology enrichment analysis revealed that elevated proteins in CB platelets were in particular enriched in proteins contributing to mitochondrial energy metabolism processes. The reduced proteins were enriched in proteins involved in, among others, platelet degranulation and activation. In conclusion, this study reveals that the CB and APB platelets are distinct. In particular, changes were observed for proteins that belong to metabolic and energy generation processes and not for the critical adhesive platelet integrins and glycoproteins.

Low density lipoprotein receptor-related protein 1 (LRP1) is involved in the catabolism of many ligands, including factor VIII (FVIII) and alpha-2-macroglobulin (α2M). Transfer of FVIII to LRP1 is currently believed to be preceded by pre-concentration on the cell surface, by interacting with a so far unidentified component. In the present study, we used confocal microscopy and flow cytometry to compare endocytosis of FVIII and α2M using U87MG cells. The results show that α2M is rapidly internalized and does not compete for LRP1 mediated internalization of FVIII. FVIII endocytosis did not occur in the presence of receptor-associated-protein (RAP), but FVIII remained visible as a striated fluorescent pattern at the cell borders. In the presence of Von Willebrand Factor (VWF), no FVIII was observed on or within the cells, suggesting that VWF blocks interaction with both cell surface and LRP1. The same dual inhibition has previously been observed for FVIII C1 domain directed monoclonal antibody KM33. Elimination of the KM33 epitope by replacing FVIII C1 residues 2091-2095 and 2155-2160 for the homologues from factor V (FV), however, did not impair FVIII endocytosis. These membrane spikes alone were insufficient for cellular uptake, because FV was neither internalized by U87MG cells nor capable of effectively competing for FVIII endocytosis. These results show that FVIII endocytosis is driven by interaction with LRP1, but at the same time involves the spikes in the C1 domain that have been implicated in lipid binding.

ispartof: Management & Techniek vol:2018 issue:18 pages:5-6 status: published

The vascular endothelium provides a unique interaction plane for plasma proteins and leukocytes in inflammation. The pro-inflammatory cytokines Tumor Necrosis Factor α (TNFα) and interleukin 1β (IL-1β) have a profound effect on endothelial cells, which includes increased levels of adhesion molecules and a disrupted barrier function. To assess the endothelial response to these cytokines at the protein level, we evaluated changes in the whole proteome, cell surface proteome and phosphoproteome after 24 h of cytokine treatment. The effects of TNFα and IL-1β on endothelial cells were strikingly similar and included changes in proteins not previously associated with endothelial inflammation. Temporal profiling revealed time-dependent proteomic changes, including a limited number of early responsive proteins such as adhesion receptors ICAM1 and SELE. In addition, this approach uncovered a greater number of late responsive proteins, including proteins related to self-antigen peptide presentation, and a transient increase in ferritin. Peptide-based cell surface proteomics revealed extensive changes at the cell surface, which were in agreement with the whole proteome. In addition, site-specific changes within ITGA5 and ICAM1 were detected. Combined, our integrated proteomic data provide detailed information on endothelial inflammation, emphasize the role of the extracellular matrix therein, and include potential targets for therapeutic intervention. SIGNIFICANCE: Pro-inflammatory cytokines induce the expression of cell adhesion molecules in vascular endothelial cells. These molecules mediate the adhesion and migration of immune cells across the vessel wall, which is a key process to resolve infections in the underlying tissue. Dysregulation of endothelial inflammation can contribute to vascular diseases and the vascular endothelium is therefore an attractive target to control inflammation. Current strategies targeting endothelial adhesion molecules, including PECAM, CD99, ICAM1 and VCAM1 do not completely prevent transmigration. To identify additional therapeutic targets, we mapped the endothelial proteome after pro-inflammatory cytokine treatment. In addition to the whole proteome, we assessed the surface proteome to focus on cell adhesion molecules, and the phosphoproteome to uncover protein activation states. Here, we present an integrated overview of affected processes which further improves our understanding of endothelial inflammation and may eventually aid in therapeutic intervention of imbalanced inflammation.

Genetics play a significant role in venous thromboembolism (VTE), yet current clinical laboratory-based testing identifies a known heritable thrombophilia (factor V Leiden, prothrombin gene mutation G20210A, or a deficiency of protein C, protein S, or antithrombin) in only a minority of VTE patients. We hypothesized that a substantial number of VTE patients could have lesser-known thrombophilia mutations. To test this hypothesis, we performed whole-exome sequencing (WES) in 64 patients with VTE, focusing our analysis on a novel 55-gene extended thrombophilia panel that we compiled. Our extended thrombophilia panel identified a probable disease-causing genetic variant or variant of unknown significance in 39 of 64 study patients (60.9%), compared with 6 of 237 control patients without VTE (2.5%) (P < .0001). Clinical laboratory-based thrombophilia testing identified a heritable thrombophilia in only 14 of 54 study patients (25.9%). The majority of WES variants were either associated with thrombosis based on prior reports in the literature or predicted to affect protein structure based on protein modeling performed as part of this study. Variants were found in major thrombophilia genes, various SERPIN genes, and highly conserved areas of other genes with established or potential roles in coagulation or fibrinolysis. Ten patients (15.6%) had >1 variant. Sanger sequencing performed in family members of 4 study patients with and without VTE showed generally concordant results with thrombotic history. WES and extended thrombophilia testing are promising tools for improving our understanding of VTE pathogenesis and identifying inherited thrombophilias., C.R.P. was supported by the National Health and Medical Research Council of Australia. A.R.R. was supported by the National Institutes of Health, National Heart, Lung, and Blood Institute R01 HL062565.

LDL receptor-related protein 1 (LRP1) is a highly modular protein and the largest known mammalian endocytic receptor. LRP1 binds and internalizes many plasma components, playing multiple crucial roles as a scavenger and signaling molecule. One major challenge to studying LRP1 has been that it is difficult to express such a large, highly glycosylated, and cysteine-rich protein, limiting structural studies to LRP1 fragments. Here, we report the first recombinant expression of the complete 61 domains of the full-length LRP1 ectodomain. This advance was achieved with a multistep cloning approach and by using DNA dilutions to improve protein yields. We investigated the binding properties of LRP1 using receptor-associated protein (RAP) as a model ligand due to its tight binding interaction. The LRP1 conformation was studied in its bound and unbound state using mass spectrometry, small-angle X-ray scattering, and negative-stain electron microscopy at neutral and acidic pH. Our findings revealed a pH-dependent release of the ligand associated with a conformational change of the receptor. In summary, this investigation of the complete LRP1 ectodomain significantly advances our understanding of this important receptor and provides the basis for further elucidating the mechanism of action of LRP1 in a whole and integrated system.

AgrosupEAGEAPSICT1; Presenting Author Jürgen Vangeyte - ILVO, Agricultural Engineering, Belgium Description Precision fertilization requires accurate techniques for determining the spread pattern of centrifugal fertilizer spreaders. Traditionally, the spread pattern was determined by measuring the fertilizer distribution on the ground. Because such measurements are time consuming and complex, a high speed stereovision setup and dedicated image processing algorithms were developed for position and motion estimation of fertilizer grains leaving a centrifugal spreader. The measurements are combined with a ballistic flight model to predict the landing points of the individual grains and the actual spread pattern. This presentation will show the different development steps taken and the problems encountered the last 10 years to come to this setup. In a first approach, a two-dimensional imaging technique was used with a small field of view (0.33 m on 0.25 m) only measuring horizontal outlet angle and speed. In a second approach, this 2D approach was further refined using a larger field of view (1 m on 1 m) and using motion estimation techniques inspired from Particle Image Velocimetry (PIV). Finally, a small field of view 3D stereovision system using a high irradiance LED system was developed, able to measure 3D motion and position, and which can therefore be used for inclined spreaders or spreaders with conical disks. Together with the improvement of the measuring system, a 3D ballistic flight model was developed not only including gravitational and drag force, but also Magnus force and drag torque. Simulations using this combined experimental/modelling approach were successfully validated against experimental data. Contributors Simon Cool (ILVO, Technology and Food Science Unit, Agricultural Engineering, 9820 Merelbeke, Belgium), Jan Pieters (Department of Biosystems Engineering, Faculty of Bioscience Engineering, Ghent University, Belgium), Koen Mertens (ILVO, Technology and Food Science Unit, Agricultural Engineering, 9820 Merelbeke, Belgium), David Nuyttens (ILVO, Technology and Food Science Unit, Agricultural Engineering, 9820 Merelbeke, Belgium), Bilal Hijazi (ILVO, Technology and Food Science Unit, Agricultural Engineering, 9820 Merelbeke, Belgium), Julien Dubois (AgroSup Dijon, France), Frederic Cointault (AgroSup Dijon, France), Jürgen Vangeyte (ILVO, Technology and Food Science Unit, Agricultural Engineering, 9820 Merelbeke, Belgium)