Paradigm of biased PAR1 (protease-activated receptor-1) activation and inhibition in endothelial cells dissected by phosphoproteomics

Thrombin is the key serine protease of the coagulation cascade and mediates cellular responses by activation of PARs (protease-activated receptors). The predominant thrombin receptor is PAR1, and in endothelial cells (ECs), thrombin dynamically regulates a plethora of phosphorylation events. However, it has remained unclear whether thrombin signaling is exclusively mediated through PAR1. Furthermore, mechanistic insight into activation and inhibition of PAR1-mediated EC signaling is lacking. In addition, signaling networks of biased PAR1 activation after differential cleavage of the PAR1 N terminus have remained an unresolved issue. Here, we used a quantitative phosphoproteomics approach to show that classical and peptide activation of PAR1 induce highly similar signaling, that low thrombin concentrations initiate only limited phosphoregulation, and that the PAR1 inhibitors vorapaxar and parmodulin-2 demonstrate distinct antagonistic properties. Subsequent analysis of the thrombin-regulated phosphosites in the presence of PAR1 inhibitors revealed that biased activation of PAR1 is not solely linked to a specific G-protein downstream of PAR1. In addition, we showed that only the canonical thrombin PAR1 tethered ligand induces extensive early phosphoregulation in ECs. Our study provides detailed insight in the signaling mechanisms downstream of PAR1. Our data demonstrate that thrombin-induced EC phosphoregulation is mediated exclusively through PAR1, that thrombin and thrombin-tethered ligand peptide induce similar phosphoregulation, and that only canonical PAR1 cleavage by thrombin generates a tethered ligand that potently induces early signaling. Furthermore, platelet PAR1 inhibitors directly affect EC signaling, indicating that it will be a challenge to design a PAR1 antagonist that will target only those pathways responsible for tissue pathology

Buckling Instabilities of a Confined Colloid Crystal Layer

A model predicting the structure of repulsive, spherically symmetric, monodisperse particles confined between two walls is presented. We study the buckling transition of a single flat layer as the double layer state develops. Experimental realizations of this model are suspensions of stabilized colloidal particles squeezed between glass plates. By expanding the thermodynamic potential about a flat state of $N$ confined colloidal particles, we derive a free energy as a functional of in-plane and out-of-plane displacements. The wavevectors of these first buckling instabilities correspond to three different ordered structures. Landau theory predicts that the symmetry of these phases allows for second order phase transitions. This possibility exists even in the presence of gravity or plate asymmetry. These transitions lead to critical behavior and phases with the symmetry of the three-state and four-state Potts models, the X-Y model with 6-fold anisotropy, and the Heisenberg model with cubic interactions. Experimental detection of these structures is discussed.Comment: 24 pages, 8 figures on request. EF508

Stability of Colloidal Quasicrystals

Freezing of charge-stabilized colloidal suspensions and relative stabilities of crystals and quasicrystals are studied using thermodynamic perturbation theory. Macroion interactions are modelled by effective pair potentials combining electrostatic repulsion with polymer-depletion or van der Waals attraction. Comparing free energies -- counterion terms included -- for elementary crystals and rational approximants to icosahedral quasicrystals, parameters are identified for which one-component quasicrystals are stabilized by a compromise between packing entropy and cohesive energy.Comment: 6 pages, 4 figure

До питання фольклоризму та фольклоризації творчості трудових мігрантів

The topicality of the research is caused by the lack of study of creative activities of the Ukrainians who are hunting for a job abroad. Prose, poetry, small genres are not the object of the thorough research yet. At the same time folkloristic processes flow lively among this group of people. This thought is proved by author’s own fieldwork. Dramatic peculiarities of migrants’ creativity (homesickness, loneliness, nostalgia) help laborers to bear their lives. Some of the bright examples of migrant folklore (poems, jokes, and a vocabulary) are given in the article as well

Variant mapping using mass spectrometry–based proteotyping as a diagnostic tool in von Willebrand disease

Background: von Willebrand disease (VWD) is the most common inherited bleeding disorder, characterized by either partial or complete von Willebrand factor (VWF) deficiency or by the occurrence of VWF proteoforms of altered functionality. The gene encoding VWF is highly polymorphic, giving rise to a variety of proteoforms with varying plasma concentrations and clinical significance. Objectives: To address this complexity, we translated genomic variation in VWF to corresponding VWF proteoforms circulating in blood. Methods: VWF was characterized in VWD patients (n = 64) participating in the Willebrand in the Netherlands study by conventional laboratory testing, DNA sequencing and complementary discovery, and targeted mass spectrometry–based plasma proteomic strategies. Results: Unbiased plasma profiling combined with immune enrichment of VWF verified VWF and its binding partner factor VIII as key determinants of VWD and revealed a remarkable heterogeneity in VWF amino acid sequence coverage among patients. Subsequent VWF proteotyping enabled identification of both polymorphisms (eg, p.Thr789Ala, p.Gln852Arg, and p.Thr1381Ala), as well as pathogenic variants (n = 16) along with their corresponding canonical sequences. Targeted proteomics using stable isotope–labeled peptides confirmed unbiased proteotyping for 5 selected variants and suggested differential proteoform quantities in plasma. The variant-to-wild-type peptide ratio was determined in 6 type 2B patients heterozygous for p.Arg1306Trp, confirming the relatively low proteoform concentration of the pathogenic variant. The elevated VWF propeptide/VWF ratio indicated increased clearance of specific VWF proteoforms. Conclusion: This study highlights how VWF proteotyping from plasma could be the first step to bridge the gap between genotyping and functional testing in VWD.</p

SYMPHONY consortium:Orchestrating personalized treatment for patients with bleeding disorders

Background Treatment choices for individual patients with an inborn bleeding disorder are increasingly challenging due to increasing options and rising costs for society. We have initiated an integrated interdisciplinary national research program. Objectives The SYMPHONY consortium strives to orchestrate personalized treatment in patients with an inborn bleeding disorder, by unraveling the mechanisms behind interindividual variations of bleeding phenotype. Patients The SYMPHONY consortium will investigate patients with an inborn bleeding disorder, both diagnosed and not yet diagnosed. Results Research questions are categorized under the themes: (1) diagnosis, (2) treatment, and (3) fundamental research, and consist of work packages addressing specific domains. Importantly, collaborations between patients and talented researchers from different areas of expertise promise to augment the impact of the SYMPHONY consortium, leading to unique interactions and intellectual property. Conclusions SYMPHONY will perform research on all aspects of care, treatment individualization in patients with inborn bleeding disorders, as well as diagnostic innovations and results of molecular genetics and cellular model technology with regard to the hemostatic process. We believe that these research investments will lead to health-care innovations with long-term clinical and societal impact. This consortium has been made possible by a governmental, competitive grant from the Netherlands Organization for Scientific Research (NWO) within the framework of the NWA-ORC Call grant agreement NWA.1160.18.038

Accretion-related properties of Herbig Ae/Be stars. Comparison with T Tauris

We look for trends relating the mass accretion rate (Macc) and the stellar ages (t), spectral energy distributions (SEDs), and disk masses (Mdisk) for a sample of 38 HAeBe stars, comparing them to analogous correlations found for classical T Tauri stars. Our goal is to shed light on the timescale and physical processes that drive evolution of intermediate-mass pre-main sequence objects. Macc shows a dissipation timescale \tau = 1.3^{+1.0}_{-0.5} Myr from an exponential law fit, while a power law yields Macc(t) \propto t^{-\eta}, with \eta = 1.8^{+1.4}_{-0.7}. This result is based on our whole HAeBe sample (1-6 Msun), but the accretion rate decline most probably depends on smaller stellar mass bins. The near-IR excess is higher and starts at shorter wavelengths (J and H bands) for the strongest accretors. Active and passive disks are roughly divided by 2 x 10^{-7} Msun/yr. The mid-IR excess and the SED shape from the Meeus et al. classification are not correlated with Macc. We find Macc \propto Mdisk^{1.1 +- 0.3}. Most stars in our sample with signs of inner dust dissipation typically show accretion rates ten times lower and disk masses three times smaller than the remaining objects. The trends relating Macc with the near-IR excess and Mdisk extend those for T Tauri stars, and are consistent with viscous disk models. The differences in the inner gas dissipation timescale, and the relative position of the stars with signs of inner dust clearing in the Macc-Mdisk plane, could be suggesting a slightly faster evolution, and that a different process - such as photoevaporation - plays a more relevant role in dissipating disks in the HAeBe regime compared to T Tauri stars. Our conclusions must consider the mismatch between the disk mass estimates from mm fluxes and the disk mass estimates from accretion, which we also find in HAeBe stars.Comment: 11 pages, 7 figures, 1 appendix. Accepted in A&

A novel class of microRNA-recognition elements that function only within open reading frames.

MicroRNAs (miRNAs) are well known to target 3' untranslated regions (3' UTRs) in mRNAs, thereby silencing gene expression at the post-transcriptional level. Multiple reports have also indicated the ability of miRNAs to target protein-coding sequences (CDS); however, miRNAs have been generally believed to function through similar mechanisms regardless of the locations of their sites of action. Here, we report a class of miRNA-recognition elements (MREs) that function exclusively in CDS regions. Through functional and mechanistic characterization of these 'unusual' MREs, we demonstrate that CDS-targeted miRNAs require extensive base-pairing at the 3' side rather than the 5' seed; cause gene silencing in an Argonaute-dependent but GW182-independent manner; and repress translation by inducing transient ribosome stalling instead of mRNA destabilization. These findings reveal distinct mechanisms and functional consequences of miRNAs that target CDS versus the 3' UTR and suggest that CDS-targeted miRNAs may use a translational quality-control-related mechanism to regulate translation in mammalian cells