Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear

Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s⁻¹). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A₂ (TxA₂), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.     

Recovery of vinyl chloride from by-streams of polyvinyl chloride production by TPSA in a multitubular adsorber

This work aims at developing an efficient and feasible adsorption-based separation process for the separation of vinyl chloride and nitrogen, on activated carbon, by employing a multitubular packed bed geometry, with adsorbent material inside the tubes. Using this geometry, a 2-dimensional mathematical model of a temperature pressure swing adsorption process was used to developed a 6-step three multitubular adsorbers system capable of separating and purifying an industrial scale gas stream of a 40:60% (v/v) vinyl chloride/nitrogen mixture into a 95% (v/v) vinyl chloride stream and a nitrogen stream with a vinyl chloride limit concentration of 8 ppm (w/w). The process reported energy consumption of 4.88 × 10⁶ J/kg_VCM and recovery capacity of 24.35 kg_VCM/(m³_unit h). The multitubular geometry enabled the use of lower adsorbent loads, shorter cycle times, and lower regeneration temperatures. An equivalent 1-dimensional model has also shown to satisfactorily estimate the performance of the current equipment.     

Synthesis and Characterization of Ti-Nb2O5 Catalysts for Discoloration Reaction of Bromophenol Blue and Indigo Carmine Dyes

Topics in Catalysis - This work investigated the photodegradation of bromophenol blue (BPB) and indigo carmine (IC) dye under UV–Visible light using pure oxides of titanium and niobium, as...     

Label-Free Study of the Global Cell Behavior during Exposure to Environmental Radiofrequency Fields in the Presence or Absence of Pro-Apoptotic or Pro-Autophagic Treatments

It remains controversial whether exposure to environmental radiofrequency signals (RF) impacts cell status or response to cellular stress such as apoptosis or autophagy. We used two label-free techniques, cellular impedancemetry and Digital Holographic Microscopy (DHM), to assess the overall cellular response during RF exposure alone, or during co-exposure to RF and chemical treatments known to induce either apoptosis or autophagy. Two human cell lines (SH-SY5Y and HCT116) and two cultures of primary rat cortex cells (astrocytes and co-culture of neurons and glial cells) were exposed to RF using an 1800 MHz carrier wave modulated with various environmental signals (GSM: Global System for Mobile Communications, 2G signal), UMTS (Universal Mobile Telecommunications System, 3G signal), LTE (Long-Term Evolution, 4G signal, and Wi-Fi) or unmodulated RF (continuous wave, CW). The specific absorption rates (S.A.R.) used were 1.5 and 6 W/kg during DHM experiments and ranged from 5 to 24 W/kg during the recording of cellular impedance. Cells were continuously exposed for three to five consecutive days while the temporal phenotypic signature of cells behavior was recorded at constant temperature. Statistical analysis of the results does not indicate that RF-EMF exposure impacted the global behavior of healthy, apoptotic, or autophagic cells, even at S.A.R. levels higher than the guidelines, provided that the temperature was kept constant.     

Analysis of Integrin αIIb Subunit Dynamics Reveals Long-Range Effects of Missense Mutations on Calf Domains

Integrin α_IIbβ₃, a glycoprotein complex expressed at the platelet surface, is involved in platelet aggregation and contributes to primary haemostasis. Several integrin α_IIbβ₃ polymorphisms prevent the aggregation that causes haemorrhagic syndromes, such as Glanzmann thrombasthenia (GT). Access to 3D structure allows understanding the structural effects of polymorphisms related to GT. In a previous analysis using Molecular Dynamics (MD) simulations of α_IIb Calf-1 domain structure, it was observed that GT associated with single amino acid variation affects distant loops, but not the mutated position. In this study, experiments are extended to Calf-1, Thigh, and Calf-2 domains. Two loops in Calf-2 are unstructured and therefore are modelled expertly using biophysical restraints. Surprisingly, MD revealed the presence of rigid zones in these loops. Detailed analysis with structural alphabet, the Proteins Blocks (PBs), allowed observing local changes in highly flexible regions. The variant P741R located at C-terminal of Calf-1 revealed that the Calf-2 presence did not affect the results obtained with isolated Calf-1 domain. Simulations for Calf-1 + Calf-2, and Thigh + Calf-1 variant systems are designed to comprehend the impact of five single amino acid variations in these domains. Distant conformational changes are observed, thus highlighting the potential role of allostery in the structural basis of GT.     

Growth, survival and inactivation of Pseudomonas aeruginosa and Staphylococcus aureus strains of various origin in the presence of ethanol

The influence of ethanol on the behaviour of Pseudomonas aeruginosa and Staphylococcus aureus strains was evaluated throughout this study. Strains of different origin were used: collection, clinical and industrial strains were selected. Concentrations of ethanol from 0 to 20% (v/v) were evaluated by automated optical density measurements and by enumeration. When growth conditions were observed, predictive microbiology models were used to assess quantitatively for the ethanol effect. Primary modelling of kinetics was performed to determine growth rate values; secondary modelling was performed on these growth rates as influenced by ethanol, and minimum inhibitory concentrations of ethanol were determined for each strain. Staphylococcus aureus strains were more resistant to ethanol than P. aeruginosa strains, in growth conditions as well as in inactivation conditions. Furthermore, clinical S. aureus strains were more resistant than the collection strain. The method was promising for management of microbiological safety in cosmetics.     

APOB CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants

Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the APOB gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry APOB missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous APOB-VUS (p.Leu351Arg), in a FHBL family. We generated APOB knock-out (KO) and APOB-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the APOB expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The APOB expression was decreased by 70% in the heterozygous APOB-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The APOB-p.Leu351Arg homozygous cells presented with a 40% decreased APOB expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of APOB-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.     

Strain-specific polyketide synthase genes of Aspergillus niger

In silico comparison of 34 putative pks genes in Aspergillus niger strain CBS 513.88 versus A. niger strain ATCC 1015 genome revealed significant nucleotide identity (>95% covering a minimum of 99% of the gene sequence) for 31 of these genes (approximately 91%). A. niger CBS 513.88 harbors three putative pks genes (An01g01130, An11g05940, and An15g07920), for which nucleotide identity was not found in A. niger ATCC 1015. To compare the results of the in silico analysis with the in vivo situation, experimental data were obtained for a large number of A. niger strains obtained from different substrates and geographical regions. Three putative pks genes that were found to be variable between the two A. niger strains using bioinformatics tools were in fact strain-specific genes based on experimental data. The PCR amplification signals for the An01g01130, An11g05940, and An15g07920 pks genes were detected in only 97%, 71%, and 26% of the strains, respectively. Southern blot analyses confirmed the PCR data. Because one of the strain-specific pks genes (An15g07920) is located in a putative ochratoxin cluster, we focused our investigation on that region. We assessed the ochratoxin production capability of the 119 A. niger strains and found a positive association between the presence of this pks gene and the capability of the respective strain to produce ochratoxin.     

Anti-inflammatory effect of aqueous extracts of roasted and green Coffea arabica L.

Green coffee contains a large quantity and variety of polyphenols and flavonoids. The roasting affects the composition of the polyphenols in coffee, due to the formation of compounds generated by Maillard reaction, which can have anti-inflammatory or antioxidant potential. The anti-inflammatory and antioxidant effects of aqueous extracts of green (AGCa) and roasted (ARCa) coffee beans were investigated in animal models and using a DPPH radical scavenging test. In the formalin test the extracts reduced licking activity only in the late phase. The inhibitory values of oedema at 3 h post-carrageenan were 53% and 74% for 100 and 300 mg/kg of the AGCa extract and 36% for ARCa (300 mg/kg). Leukocyte recruitment into the peritoneal cavity was inhibited by the extracts. The antioxidant activities of the extracts were higher than the reference antioxidants, ascorbic acid and butylated hydroxytoluene. These results indicate that coffee extracts exhibit anti-inflammatory and antioxidant properties.