Critical shifts in lipid metabolism promote megakaryocyte differentiation and proplatelet formation.

During megakaryopoiesis, megakaryocytes (MK) undergo cellular morphological changes with strong modification of membrane composition and lipid signaling. Here we adopt a lipid-centric multiomics approach to create a quantitative map of the MK lipidome during maturation and proplatelet formation. Data reveal that MK differentiation is driven by an increased fatty acyl import and de novo lipid synthesis, resulting in an anionic membrane phenotype. Pharmacological perturbation of fatty acid import and phospholipid synthesis blocked membrane remodeling and directly reduced MK polyploidization and proplatelet formation resulting in thrombocytopenia. The anionic lipid shift during megakaryopoiesis was paralleled by lipid-dependent relocalization of the scaffold protein CKIP-1 and recruitment of the kinase CK2α to the plasma membrane, which seems to be essential for sufficient platelet biogenesis. Overall, this study provides a framework to understand how the MK lipidome is altered during maturation and the impact of MK membrane lipid remodeling on MK kinase signaling involved in thrombopoiesis.

Citrus essential oils - Based nano-emulsions: Functional properties and potential applications.

Citrus essential oils are natural products with various bioactive properties (e.g., antimicrobial, antioxidant, and antimutagenic activities), that are generally recognized as safe (GRAS) by Food and Drug Administration (FDA) to be used as flavorings and food additives. Nonetheless, due to their high volatility, low solubility in water, low thermal stability, susceptibility to oxidation, and strong flavor, their applications in the food industry are limited. Nanotechnology allows the incorporation of citrus essential oils into nano-emulsion systems, thus protecting them from the deterioration caused by external factors and maintaining or even improving their functional properties. This study aims to summarize the antioxidant, antimicrobial, and antimutagenic effects of the nano-emulsions based on essential oils from citrus peels with emphasis on their mechanisms of action and potential applications in, e.g., foods, pharmaceuticals, and cosmetics.

Accurate Sphingolipid Quantification Reducing Fragmentation Bias by Nonlinear Models.

Quantitative sphingolipid analysis is crucial for understanding the roles of these bioactive molecules in various physiological and pathological contexts. Molecular sphingolipid species are typically quantified using sphingoid base-derived fragments relative to a class-specific internal standard. However, the commonly employed "one standard per class" strategy fails to account for fragmentation differences presented by the structural diversity of sphingolipids. To address this limitation, we developed a novel approach for quantitative sphingolipid analysis. This approach utilizes fragmentation models to correct for structural differences and thus overcomes the limitations associated with using a limited number of standards for quantification. Importantly, our method is independent of the internal standard, instrumental setup, and collision energy. Furthermore, we integrated this method into a user-friendly KNIME workflow. The validation results illustrate the effectiveness of our approach in accurately quantifying ceramide subclasses from various biological matrices. This breakthrough opens up new avenues for exploring sphingolipid metabolism and gaining insights into its implications.

Removal and degradation of sodium diclofenac via radical-based mechanisms using S. sclerotiorum laccase.

The recently isolated Sclerotinia sclerotiorum laccase was used for the degradation of sodium diclofenac, a nonsteroidal anti-inflammatory drug widely found in the aquatic environment. The Michaelis-Menten parameters, half-life of diclofenac at different pH values in presence of this enzyme and potential inhibitors were evaluated. Diclofenac-based radicals formed in presence of laccase were spin-trapped and detected using EPR spectroscopy. Almost complete diclofenac degradation (> 96%) occurred after a 30-h treatment via radical-based generated oligomers and their rapid precipitation, thus ensuring an unprecedented green formula suitable not only for degradation but also for straightforward removal of the degradation products. High performance liquid chromatography coupled with atmospheric pressure chemical ionization-ion trap mass spectrometry (HPLC-APCI-MS) analyses of the degradation products of diclofenac in aqueous dosage revealed the presence of at least seven products while HR Orbitrap MS analysis showed that the enzymatic treatment produced high molecular weight metabolites through a radical oligomerization mechanism of diclofenac. The enzymatically formed products precipitated and its constituting components were also characterized using UV-vis spectroscopy, infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA).

LipidSpace: Simple Exploration, Reanalysis, and Quality Control of Large-Scale Lipidomics Studies.

Lipid analysis gained significant importance due to the enormous range of lipid functions, e.g., energy storage, signaling, or structural components. Whole lipidomes can be quantitatively studied in-depth thanks to recent analytical advancements. However, the systematic comparison of thousands of distinct lipidomes remains challenging. We introduce LipidSpace, a standalone tool for analyzing lipidomes by assessing their structural and quantitative differences. A graph-based comparison of lipid structures is the basis for calculating structural space models and subsequently computing lipidome similarities. When adding study variables such as body weight or health condition, LipidSpace can determine lipid subsets across all lipidomes that describe these study variables well by utilizing machine-learning approaches. The user-friendly GUI offers four built-in tutorials and interactive visual interfaces with pdf export. Many supported data formats allow an efficient (re)analysis of data sets from different sources. An integrated interactive workflow guides the user through the quality control steps. We used this suite to reanalyze and combine already published data sets (e.g., one with about 2500 samples and 576 lipids in one run) and made additional discoveries to the published conclusions with the potential to fill gaps in the current lipid biology understanding. LipidSpace is available for Windows or Linux (https://lifs-tools.org).

Flexible Microtube Plasma for the Consecutive-Ionization of Cholesterol in Nano-Electrospray Mass Spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) is an established method for the identification of biomarkers. By nano-ESI (nESI), the polar molecular fraction of complex biological samples can be successfully ionized. In contrast, the less-polar free cholesterol, which serves as an important biomarker for several human diseases, is barely accessible by nESI. Although, complex scan functions of modern high-resolution MS devices are able to increase the signal-to-noise ratio, they are limited by the ionization efficiency of the nESI. One possible method to increase the ionization efficiency is the derivatization with acetyl chloride, however interferences with cholesteryl esters must be considered, so chromatographic separation or complex scan functions may be required. A novel approach to increase the yield of cholesterol ions of the nESI could be the application of a second consecutive-ionization process. This publication presents the flexible microtube plasma (FµTP) as a consecutive-ionization source, which allows the determination of cholesterol in nESI-MS analysis. Focusing on the analytical performance, the nESI-FµTP approach increases the cholesterol signal yield in a complex liver extract by a factor of 49. The repeatability and long-term stability could be successfully evaluated. A linear dynamic range of 1.7 orders of magnitude, a minimum detectability of 5.46 mg/L, and a high accuracy (deviation, -8.1%) demonstrates the nESI-FµTP-MS as an excellent approach for a derivatization-free determination of cholesterol.

Niemann-Pick C1 protein regulates platelet membrane-associated calcium ion signaling in thrombo-occlusive diseases in mice.

BACKGROUND: Pathophysiologic platelet activation leads to thrombo-occlusive diseases such as myocardial infarction or ischemic stroke. Niemann-Pick C1 protein (NPC1) is involved in the regulation of lysosomal lipid trafficking and calcium ion (Ca2+) signaling, and its genetic mutation causes a lysosomal storage disorder. Lipids and Ca2+ are key players in the complex orchestration of platelet activation. OBJECTIVES: The present study aimed to determine the impact of NPC1 on Ca2+ mobilization during platelet activation in thrombo-occlusive diseases. METHODS: Using MK/platelet-specific knockout mice of Npc1 (Npc1Pf4∆/Pf4∆), ex vivo and in vitro approaches as well as in vivo models of thrombosis, we investigated the effect of Npc1 on platelet function and thrombus formation. RESULTS: We showed that Npc1Pf4∆/Pf4∆ platelets display increased sphingosine levels and a locally impaired membrane-associated and SERCA3-dependent Ca2+ mobilisation compared to platelets from wildtype littermates (Npc1lox/lox). Further, we observed decreased platelet. CONCLUSION: Our findings highlight that NPC1 regulates membrane-associated and SERCA3-dependent Ca2+ mobilization during platelet activation and that MK/platelet-specific ablation of Npc1 protects against experimental models of arterial thrombosis and myocardial or cerebral ischemia/reperfusion injury.

Effects of polystyrene nanoplastics exposure on in vitro-grown Stevia rebaudiana plants.

Nanoplastics (NPs) as environmental contaminants have received increased attention in recent years. Numerous studies have suggested possible negative effects of plants exposure to NPs, but more data are needed with various plants under different exposure conditions to clarify the underlying phytotoxicity mechanisms. In this study, we investigated the effect of polystyrene nanoplastics (PSNPs; 28.65 nm average diameter) exposure (10, 100 and 250 mg/L) on plant morphology and production of relevant metabolites (steviol glycosides, chlorophylls, carotenoids, and vitamins) of in vitro-grown Stevia rebaudiana plantlets. Additionally, we used dark field microscopy combined with fluorescence hyperspectral imaging for the visualization of internalized PSNPs inside plant tissues. At higher concentrations (>100 mg/L), PSNPs were shown to aggregate in roots and to be transported to leaves, having a significantly negative impact on plant growth (reduced size and biomass), while increasing the production of metabolites compared to controls, most probably because of response to stress. The production of steviol glycosides presented a biphasic dose-response suggestive of hormesis, with the highest values at 10 mg/L PSNPs (1.5-2.2-fold increase compared to controls), followed by a decline in production at higher concentrations (100 and 250 mg/L), but with values comparable to controls. These results are promising for future in vivo studies evaluating the effect of NP exposure on the production of steviol glycosides, the natural sweeteners from stevia.

Nano-Encapsulation of Citrus Essential Oils: Methods and Applications of Interest for the Food Sector.

Citrus essential oils possess many health-promoting benefits and properties of high interest in the food and agri-food sector. However, their large-scale application is limited by their sensitivity to environmental factors. Nanostructures containing citrus essential oils have been developed to overcome the high volatility and instability of essential oils with respect to temperature, pH, UV light, etc. Nanostructures could provide protection for essential oils and enhancement of their bioavailability and biocompatibility, as well as their biological properties. Nano-encapsulation is a promising method. The present review is mainly focused on methods developed so far for the nano-encapsulation of citrus essential oils, with emphasis on lipid-based (including liposomes, solid lipid nanoparticles, nanostructured lipid particles, and nano- and micro-emulsions) and polymer-based nanostructures. The physico-chemical characteristics of the obtained structures, as well as promising properties reported, with relevance for the food sector are also discussed.

Gadolinium Accumulation and Toxicity on In Vitro Grown Stevia rebaudiana: A Case-Study on Gadobutrol.

Gadolinium-based contrast agents are molecular complexes which are extensively used for diagnostic purposes. Apart from their tremendous contribution to disease diagnostics, there are several issues related to their use. They are extremely stable complexes and potential contaminants of surface and ground waters, an issue which is documented worldwide. The irrigation of fields with contaminated surface waters or their fertilization with sludge from wastewater treatment plants can lead to the introduction of Gd into the human food supply chain. Thus, this study focused on the potential toxicity of Gd on plants. For this purpose, we have studied the molecular effects of gadobutrol (a well-known MRI contrast agent) exposure on in vitro-grown Stevia rebaudiana. The effects of gadobutrol on plant morphology, on relevant plant metabolites such as chlorophylls, carotenoids, ascorbic acids (HPLC), minerals (ICP-OES), and on the generation of free radical species (MDA assay and EPR) were assessed. Exposures of 0.01, 0.05, 0.1, 1, and 3 mM gadobutrol were used. We found a correlation between the gadobutrol dose and the plant growth and concentration of metabolites. Above the 0.1. mM dose of gadobutrol, the toxic effects of Gd+3 ions became significant.

The effect of 100-200 nm ZnO and TiO2 nanoparticles on the in vitro-grown soybean plants.

Engineered nanomaterials are increasingly used in everyday life applications and, in consequence, significant amounts are being released into the environment. From soil, water, and air they can reach the organelles of edible plants, potentially impacting the food chain and human health. The potential environmental and health impact of these nanoscale materials is of public concern. TiO2 and ZnO are among the most significant nanomaterials in terms of production amounts. Our study aimed at evaluating the effects of large-scale TiO2 (~100 nm) and ZnO (~200 nm) nanoparticles on soybean plants grown in vitro. The effect of different concentrations of nanoparticles (10, 100, 1000 mg/L) was evaluated regarding plant morphology and metabolic changes. ZnO nanoparticles showed higher toxicity compared to TiO2 in the experimental set-up. Overall, elevated levels of chlorophylls and proteins were observed, as well as increased concentrations of ascorbic and dehydroascorbic acids. Also, the decreasing stomatal conductance to water vapor and net CO2 assimilation rate show higher plant stress levels. In addition, ZnO nanoparticle treatments severely affected plant growth, while TEM analysis revealed ultrastructural changes in chloroplasts and rupture of leaf cell walls. By combining ICP-OES and TEM results, we were able to show that the nanoparticles were metabolized, and their internalization in the soybean plant tissues occurred in ionic forms. This behavior most likely is the main driving force of nanoparticle toxicity.

Targeting early stages of cardiotoxicity from anti-PD1 immune checkpoint inhibitor therapy.

AIMS: Cardiac immune-related adverse events (irAEs) from immune checkpoint inhibition (ICI) targeting programmed death 1 (PD1) are of growing concern. Once cardiac irAEs become clinically manifest, fatality rates are high. Cardio-oncology aims to prevent detrimental effects before manifestation of severe complications by targeting early pathological changes. We therefore aimed to investigate early consequences of PD1 inhibition for cardiac integrity to prevent the development of overt cardiac disease. METHODS AND RESULTS: We investigated cardiac-specific consequences from anti-PD1 therapy in a combined biochemical and in vivo phenotyping approach. Mouse hearts showed broad expression of the ligand PDL1 on cardiac endothelial cells as a main mediator of immune-crosstalk. Using a novel melanoma mouse model, we assessed that anti-PD1 therapy promoted myocardial infiltration with CD4+ and CD8+ T cells, the latter being markedly activated. Left ventricular (LV) function was impaired during pharmacological stress, as shown by pressure-volume catheterization. This was associated with a dysregulated myocardial metabolism, including the proteome and the lipidome. Analogous to the experimental approach, in patients with metastatic melanoma (n = 7) receiving anti-PD1 therapy, LV function in response to stress was impaired under therapy. Finally, we identified that blockade of tumour necrosis factor alpha (TNFα) preserved LV function without attenuating the anti-cancer efficacy of anti-PD1 therapy. CONCLUSIONS: Anti-PD1 therapy induces a disruption of cardiac immune homeostasis leading to early impairment of myocardial functional integrity, with potential prognostic effects on the growing number of treated patients. Blockade of TNFα may serve as an approach to prevent the manifestation of ICI-related cardiotoxicity.

Multiomics of synaptic junctions reveals altered lipid metabolism and signaling following environmental enrichment.

Membrane lipids and their metabolism have key functions in neurotransmission. Here we provide a quantitative lipid inventory of mouse and rat synaptic junctions. To this end, we developed a multiomics extraction and analysis workflow to probe the interplay of proteins and lipids in synaptic signal transduction from the same sample. Based on this workflow, we generate hypotheses about novel mechanisms underlying complex changes in synaptic connectivity elicited by environmental stimuli. As a proof of principle, this approach reveals that in mice exposed to an enriched environment, reduced endocannabinoid synthesis and signaling is linked to increased surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in a subset of Cannabinoid-receptor 1 positive synapses. This mechanism regulates synaptic strength in an input-specific manner. Thus, we establish a compartment-specific multiomics workflow that is suitable to extract information from complex lipid and protein networks involved in synaptic function and plasticity.

ANXA7 Regulates Platelet Lipid Metabolism and Ca2+ Release in Arterial Thrombosis.

[Figure: see text].

Targeted Phosphoinositides Analysis Using High-Performance Ion Chromatography-Coupled Selected Reaction Monitoring Mass Spectrometry.

Phosphoinositides are minor components of cell membranes, but play crucial roles in numerous signal transduction pathways. To obtain quantitative measures of phosphoinositides, sensitive, accurate, and comprehensive methods are needed. Here, we present a quantitative targeted ion chromatography-mass spectrometry-based workflow that separates phosphoinositide isomers and increases the quantitative accuracy of measured phosphoinositides. Besides testing different analytical characteristics such as extraction and separation efficiency, the reproducibility of the developed workflow was also investigated. The workflow was verified in resting and stimulated human platelets, fat cells, and rat hippocampal brain tissue, where the LOD and LOQ for phosphoinositides were at 312.5 and 625 fmol, respectively. The robustness of the workflow is shown with different applications that confirms its suitability to analyze multiple less-abundant phosphoinositides.

Simple Targeted Assays for Metabolic Pathways and Signaling: A Powerful Tool for Targeted Proteomics.

We introduce STAMPS, a pathway-centric web service for the development of targeted proteomics assays. STAMPS guides the user by providing several intuitive interfaces for a rapid and simplified method design. Applying our curated framework to signaling and metabolic pathways, we reduced the average assay development time by a factor of ∼150 and revealed that the insulin signaling is actively controlled by protein abundance changes in insulin-sensitive and -resistance states. Although at the current state STAMPS primarily contains mouse data, it was designed for easy extension with additional organisms.

A multi-omics analysis reveals the unfolded protein response regulon and stress-induced resistance to folate-based antimetabolites.

Stress response pathways are critical for cellular homeostasis, promoting survival through adaptive changes in gene expression and metabolism. They play key roles in numerous diseases and are implicated in cancer progression and chemoresistance. However, the underlying mechanisms are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of a stress response pathway, the unfolded protein response (UPR), probing in parallel the transcriptome, the proteome, and changes to translation. Stringent filtering reveals the induction of 267 genes, many of which have not previously been implicated in stress response pathways. We experimentally demonstrate that UPR-mediated translational control induces the expression of enzymes involved in a pathway that diverts intermediate metabolites from glycolysis to fuel mitochondrial one-carbon metabolism. Concomitantly, the cells become resistant to the folate-based antimetabolites Methotrexate and Pemetrexed, establishing a direct link between UPR-driven changes to gene expression and resistance to pharmacological treatment.

LipidCreator workbench to probe the lipidomic landscape.

Mass spectrometry (MS)-based targeted lipidomics enables the robust quantification of selected lipids under various biological conditions but comprehensive software tools to support such analyses are lacking. Here we present LipidCreator, a software that fully supports targeted lipidomics assay development. LipidCreator offers a comprehensive framework to compute MS/MS fragment masses for over 60 lipid classes. LipidCreator provides all functionalities needed to define fragments, manage stable isotope labeling, optimize collision energy and generate in silico spectral libraries. We validate LipidCreator assays computationally and analytically and prove that it is capable to generate large targeted experiments to analyze blood and to dissect lipid-signaling pathways such as in human platelets.

Cellular Internalization of Beta-Carotene Loaded Polyelectrolyte Multilayer Capsules by Raman Mapping.

Raman mapping is becoming a very useful tool in investigating cells and cellular components, as well as bioactive molecules intracellularly. In this study, we have encapsulated beta-carotene using a layer-by-layer technique, as a way to enhance its stability and bioavailability. Further, we have used Raman mapping to characterize the as-obtained capsules and monitor their uptake by the human retinal epithelial D407 cells. We were able to successfully map the beta-carotene distribution inside the capsules, to localize the capsules intracellularly, and distinguish between capsules and other cellular components.