Comparative Analysis of Platelet-Derived Extracellular Vesicles Using Flow Cytometry and Nanoparticle Tracking Analysis

Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers (Gallios and CytoFLEX LX) and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA⁺CD41⁺ EVs compared to Gallios. NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA⁺ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.     

Discovery of GPR183 Agonists Based on an Antagonist Scaffold

The G protein-coupled receptor GPR183/EBI2, which is activated by oxysterols, is a therapeutic target for inflammatory and metabolic diseases where both antagonists and agonists are of potential interest. Using the piperazine diamide core of the known GPR183 antagonist (E)-3-(4-bromophenyl)-1-(4-(4-methoxybenzoyl)piperazin-1-yl)prop-2-en-1-one (NIBR189) as starting point, we identified and sourced 79 structurally related compounds that were commercially available. In vitro screening of this compound collection using a Ca²⁺ mobilization assay resulted in the identification of 10 compounds with agonist properties. To enable establishment of initial structure-activity relationship trends, these were supplemented with five in-house compounds, two of which were also shown to be GPR183 agonists. Taken together, our findings suggest that the agonist activity of this compound series is dictated by the substitution pattern of one of the two distal phenyl rings, which functions as a molecular efficacy-switch.     

Influence of the Impaction Angle on the Triboelectric Charging of Aerosol Nanoparticles

A low pressure impactor is used to measure triboelectric charging behavior of metallic nanoparticles. Ag nanoparticles, produced by spark discharge, were impacted onto Pt sputtered targets. The influence of the impaction angle and impaction velocity on the triboelectric charging was investigated. While for perpendicular impaction the charge transfer behavior of previous work was confirmed, the oblique impaction revealed new phenomena. Additional charge transfer was observable, which increases with obliqueness. The possibility of mass transfer between particle and target due to the high-energy collisions was also investigated. SEM characterization and Auger spectroscopy indicate mass transfer from the particle to the target surface.     

Mechanisms of Reciprocal Regulation of Gonadotropin-Releasing Hormone (GnRH)-Producing and Immune Systems: The Role of GnRH,Cytokines and Their Receptors in Early Ontogenesis in Normal and Pathological Conditions

Different aspects of the reciprocal regulatory influence on the development of gonadotropin-releasing hormone (GnRH)-producing- and immune systems in the perinatal ontogenesis and their functioning in adults in normal and pathological conditions are discussed. The influence of GnRH on the development of the immune system, on the one hand, and the influence of proinflammatory cytokines on the development of the hypothalamic-pituitary-gonadal system, on the other hand, and their functioning in adult offspring are analyzed. We have focused on the effects of GnRH on the formation and functional activity of the thymus, as the central organ of the immune system, in the perinatal period. The main mechanisms of reciprocal regulation of these systems are discussed. The reproductive health of an individual is programmed by the establishment and development of physiological systems during critical periods. Regulatory epigenetic mechanisms of development are not strictly genetically controlled. These processes are characterized by a high sensitivity to various regulatory factors, which provides possible corrections for disorders.     

Production of the Carboxylate Reductase from Nocardia otitidiscaviarum in a Soluble,Active Form for in vitro Applications

Accessing aldehydes from carboxylate moieties is often a challenging task. In this regard, carboxylate reductases (CARs) are promising catalysts provided by nature that are able to accomplish this task in just one step, avoiding over-reduction to the alcohol product. However, the heterologous expression of CARs can be quite difficult due to the excessive formation of insoluble protein, thus hindering further characterization and application of the enzyme. Here, the heterologous production of the carboxylate reductase from Nocardia otitidiscaviarum (NoCAR) was optimized by a combination of i) optimized cultivation conditions, ii) post-translational modification with a phosphopantetheinyl transferase and iii) selection of an appropriate expression strain. Especially, the selection of Escherichia coli tuner cells as host had a strong effect on the final 110-fold increase in the specific activity of NoCAR. This highly active NoCAR was used to reduce sodium benzoate to benzaldehyde, and it was successfully assembled with an in vitro regeneration of ATP and NADPH, being capable of reducing about 30 mM sodium benzoate with high selectivity in only 2 h of reaction.     

Evaluation of dynamic modulus measurement for C/C-SiC composites at different temperatures

The determination of elastic properties at application temperature is fundamental for the design of fibre reinforced ceramic composite components. An attractive method to characterize the flexural modulus at room and high temperature under specific atmosphere is the nondestructive Resonant Frequency Damping Analysis (RFDA). The objective of this paper was to evaluate and validate the modulus measurement via RFDA for orthotropic C/C-SiC composites at the application temperature. At room temperature flexural moduli of C/C-SiC with 0/90° reinforcement were measured under quasi-static 4-point bending loads and compared with dynamic moduli measured via RFDA longitudinally to fibre direction. The dynamic modulus of C/C-SiC was then measured via RFDA up to 1250°C under flowing inert gas and showed an increase with temperature which fitted with literature values. The measured fundamental frequencies were finally compared to those resulting from numerical modal analyses. Dynamic and quasi-static flexural moduli are comparable and the numerical analyses proved that bending modes are correctly modeled by means of dynamic modulus measured via RFDA. The nondestructive RFDA as well as the numerical modeling approach are suitable for evaluation of C/C-SiC and may be transferred to other fibre reinforced ceramic composite materials.