Demonstrating the Applicability of Proton Transfer Reaction Mass Spectrometry to Quantify Volatiles Emitted by the Mycoparasitic Fungus Trichoderma atroviride in Real Time: Monitoring of Trichoderma-Based Biopesticides.

The present study aims to explore the potential application of proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) for real-time monitoring of microbial volatile organic compounds (MVOCs). This investigation can be broadly divided into two parts. First, a selection of 14 MVOCs was made based on previous research that characterized the MVOC emissions of Trichoderma atroviride, which is a filamentous fungus widely used as a biocontrol agent. The analysis of gas-phase standards using PTR-ToF-MS allowed for the categorization of these 14 MVOCs into two groups: the first group primarily undergoes nondissociative proton transfer, resulting in the formation of protonated parent ions, while the second group mainly undergoes dissociative proton transfer, leading to the formation of fragment ions. In the second part of this investigation, the emission of MVOCs from samples of T. atroviride was continuously monitored over a period of five days using PTR-ToF-MS. This also included the first quantitative online analysis of 6-amyl-α-pyrone (6-PP), a key MVOC emitted by T. atroviride. The 6-PP emissions of T. atroviride cultures were characterized by a gradual increase over the first two days of cultivation, reaching a plateau-like maximum with volume mixing ratios exceeding 600 ppbv on days three and four. This was followed by a marked decrease, where the 6-PP volume mixing ratios plummeted to below 50 ppbv on day five. This observed sudden decrease in 6-PP emissions coincided with the start of sporulation of the T. atroviride cultures as well as increasing intensities of product ions associated with 1-octen-3-ol and 3-octanone, whereas both these MVOCs were previously associated with sporulation in T. atroviride. The study also presents the observations and discussion of further MVOC emissions from the T. atroviride samples and concludes with a critical assessment of the possible applications and limitations of PTR-ToF-MS for the online monitoring of MVOCs from biological samples in real time.

Establishing a cell-based screening workflow for determining the efficiency of CYP2C9 metabolism: moving towards the use of breath volatiles in personalised medicine.

The use of volatile biomarkers in exhaled breath as predictors to individual drug response would advance the field of personalised medicine by providing direct information on enzyme activity. This would result in enormous benefits, both for patients and for the healthcare sector. Non-invasive breath tests would also gain a high acceptance by patients. Towards this goal, differences in metabolism resulting from extensive polymorphisms in a major group of drug-metabolizing enzymes, the cytochrome P450 (CYP) family, need to be determined and quantified. CYP2C9 is responsible for metabolising many crucial drugs (e.g., diclofenac) and food ingredients (e.g., limonene). In this paper, we provide a proof-of-concept study that illustrates thein vitrobioconversion of diclofenac in recombinant HEK293T cells overexpressing CYP2C9 to 4'-hydroxydiclofenac. Thisin vitroapproach is a necessary and important first step in the development of breath tests to determine and monitor metabolic processes in the human body. By focusing on the metabolic conversion of diclofenac, we have been able to establish a workflow using a cell-based system for CYP2C9 activity. Furthermore, we illustrate how the bioconversion of diclofenac is limited in the presence of limonene, which is another CYP2C9 metabolising substrate. We show that increasing limonene levels continuously reduce the production of 4'-hydroxydiclofenac. Michaelis-Menten kinetics were performed for the diclofenac 4'-hydroxylation with and without limonene, giving a kinetic constant of the reaction,KM, of 103µM and 94.1µM, respectively, and a maximum reaction rate,Vmax, of 46.8 pmol min-1106cells-1and 56.0 pmol min-1106cells-1with and without the inhibitor, respectively, suggesting a non-competitive or mixed inhibition type. The half-maximal inhibitory concentration value (IC50) for the inhibition of the formation of 4'-hydroxydiclofenace by limonene is determined to be 1413µM.

Procoagulant Extracellular Vesicles Alter Trophoblast Differentiation in Mice by a Thrombo-Inflammatory Mechanism.

Procoagulant extracellular vesicles (EV) and platelet activation have been associated with gestational vascular complications. EV-induced platelet-mediated placental inflammasome activation has been shown to cause preeclampsia-like symptoms in mice. However, the effect of EV-mediated placental thrombo-inflammation on trophoblast differentiation remains unknown. Here, we identify that the EV-induced thrombo-inflammatory pathway modulates trophoblast morphology and differentiation. EVs and platelets reduce syncytiotrophoblast differentiation while increasing giant trophoblast and spongiotrophoblast including the glycogen-rich cells. These effects are platelet-dependent and mediated by the NLRP3 inflammasome. In humans, inflammasome activation was negatively correlated with trophoblast differentiation marker GCM1 and positively correlated with blood pressure. These data identify a crucial role of EV-induced placental thrombo-inflammation on altering trophoblast differentiation and suggest platelet activation or inflammasome activation as a therapeutic target in order to achieve successful placentation.

Placental thromboinflammation impairs embryonic survival by reducing placental thrombomodulin expression.

Excess platelet activation by extracellular vesicles (EVs) results in trophoblast inflammasome activation, interleukin 1ß (IL-1ß) activation, preeclampsia (PE), and partial embryonic lethality. Embryonic thrombomodulin (TM) deficiency, which causes embryonic lethality hallmarked by impaired trophoblast proliferation, has been linked with maternal platelet activation. We hypothesized that placental TM loss, platelet activation, and embryonic lethality are mechanistically linked to trophoblast inflammasome activation. Here, we uncover unidirectional interaction of placental inflammasome activation and reduced placental TM expression: although inflammasome inhibition did not rescue TM-null embryos from lethality, the inflammasome-dependent cytokine IL-1ß reduced trophoblast TM expression and impaired pregnancy outcome. EVs, known to induce placental inflammasome activation, reduced trophoblast TM expression and proliferation. Trophoblast TM expression correlated negatively with IL-1ß expression and positively with platelet numbers and trophoblast proliferation in human PE placentae, implying translational relevance. Soluble TM treatment or placental TM restoration ameliorated the EV-induced PE-like phenotype in mice, preventing placental thromboinflammation and embryonic death. The lethality of TM-null embryos is not a consequence of placental NLRP3 inflammasome activation. Conversely, EV-induced placental inflammasome activation reduces placental TM expression, promoting placental and embryonic demise. These data identify a new function of placental TM in PE and suggest that soluble TM limits thromboinflammatory pregnancy complications.

p45 NF-E2 regulates syncytiotrophoblast differentiation by post-translational GCM1 modifications in human intrauterine growth restriction.

Placental insufficiency jeopardizes prenatal development, potentially leading to intrauterine growth restriction (IUGR) and stillbirth. Surviving fetuses are at an increased risk for chronic diseases later in life. IUGR is closely linked with altered trophoblast and placental differentiation. However, due to a paucity of mechanistic insights, suitable biomarkers and specific therapies for IUGR are lacking. The transcription factor p45 NF-E2 (nuclear factor erythroid derived 2) has been recently found to regulate trophoblast differentiation in mice. The absence of p45 NF-E2 in trophoblast cells causes IUGR and placental insufficiency in mice, but mechanistic insights are incomplete and the relevance of p45 NF-E2 for human syncytiotrophoblast differentiation remains unknown. Here we show that p45 NF-E2 negatively regulates human syncytiotrophoblast differentiation and is associated with IUGR in humans. Expression of p45 NF-E2 is reduced in human placentae complicated with IUGR compared with healthy controls. Reduced p45 NF-E2 expression is associated with increased syncytiotrophoblast differentiation, enhanced glial cells missing-1 (GCM1) acetylation and GCM1 desumoylation in IUGR placentae. Induction of syncytiotrophoblast differentiation in BeWo and primary villous trophoblast cells with 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP) reduces p45 NF-E2 expression. Of note, p45 NF-E2 knockdown is sufficient to increase syncytiotrophoblast differentiation and GCM1 expression. Loss of p45 NF-E2 using either approach resulted in CBP-mediated GCM1 acetylation and SENP-mediated GCM1 desumoylation, demonstrating that p45 NF-E2 regulates post-translational modifications of GCM1. Functionally, reduced p45 NF-E2 expression is associated with increased cell death and caspase-3 activation in vitro and in placental tissues samples. Overexpression of p45 NF-E2 is sufficient to repress GCM1 expression, acetylation and desumoylation, even in 8-Br-cAMP exposed BeWo cells. These results suggest that p45 NF-E2 negatively regulates differentiation and apoptosis activation of human syncytiotrophoblast by modulating GCM1 acetylation and sumoylation. These studies identify a new pathomechanism related to IUGR in humans and thus provide new impetus for future studies aiming to identify new biomarkers and/or therapies of IUGR.