This study aimed to evaluate the response of HepaRG cells after co-exposure to phthalates and heavy metals, using a high-dimensional biology paradigm (HDB). Liver is the main metabolism site for the majority of xenobiotics. For this reason, the HepaRG cell line was used as an in vitro model, and cells were exposed to two characteristic mixtures of phthalates and heavy metals containing phthalates (DEHP, DiNP, BBzP) and metals (lead, methylmercury, total mercury) in a concentration-dependent manner. The applied chemical mixtures were selected as the most abundant pollutants in the REPRO_PL and PHIME cohorts, which were studied using the exposome-wide approach in the frame of the EU project HEALS. These studies investigated the environmental causation of neurodevelopmental disorders in neonates and across Europe. The INTEGRA computational platform was used for the calculation of the effective concentrations of the chemicals in the liver through extrapolation from human biomonitoring data and this dose (and a ten-times higher one) was applied to the hepatocyte model. Multi-omics analysis was performed to reveal the genes, proteins, and metabolites affected by the exposure to these chemical mixtures. By extension, we could detect the perturbed metabolic pathways. The generated data were analyzed using advanced bioinformatic tools following the HEALS connectivity paradigm for multi-omics pathway analysis. Co-mapped transcriptomics and proteomics data showed that co-exposure to phthalates and heavy metals leads to perturbations of the urea cycle due to differential expression levels of arginase-1 and -2, argininosuccinate synthase, carbamoyl-phosphate synthase, ornithine carbamoyltransferase, and argininosuccinate lyase. Joint pathway analysis of proteomics and metabolomics data revealed that the detected proteins and metabolites, choline phosphate cytidylyltransferase A, phospholipase D3, group XIIA secretory phospholipase A2, α-phosphatidylcholine, and the a 1,2-diacyl-sn-glycero-3-phosphocholine, are responsible for the homeostasis of the metabolic pathways phosphatidylcholine biosynthesis I, and phospholipases metabolism. The urea, phosphatidylcholine biosynthesis I and phospholipase metabolic pathways are of particular interest since they have been identified also in human samples from the REPRO_PL and PHIME cohorts using untargeted metabolomics analysis and have been associated with impaired psychomotor development in children at the age of two. In conclusion, this study provides the mechanistic evidence that co-exposure to phthalates and metals disturb biochemical processes related to mitochondrial respiration during critical developmental stages, which are clinically linked to neurodevelopmental perturbations.

Choline phosphate-based delivery systems can target the acidic tumor microenvironment. In this study, we set out to evaluate the diagnostic value of Choline phosphate cytidylyltransferase-α (CCTα) in laryngeal squamous cell cancer (LSCC). The expression of CCTα was detected using immunohistochemistry in 50 LSCC patients' tissues and 16 vocal polyps as control group. Then, clinical data was collected and we used receiver operating characteristic curve (ROC) to estimate the potential of CCTα as diagnostic biomarker. We found CCTα levels to be significantly high in the tissues derived from LSCC patients, (p < 0.001). Further, we observed a positive correlation of CCTα with tumor size (p < 0.001), TNM stage (p < 0.001), lymph node metastasis (p < 0.001) as well as the grade of LSCC malignancy (p < 0.001). Furthermore, AUC was determined to be 0.939 by ROC, and the optimal cutoff value 3.100, with 76.0% sensitivity and 100% specificity. We also found an epigenetic basis of CCTα over-expression in LSCC tissues with significantly reduced methylation of CCTα in LSCC tissues, compared to vocal polyps (p < 0.001). These results support epigenetically-induced over-expression of CCTα as a potential diagnostic marker for LSCC.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the main membrane phospholipids (PLs) of Plasmodium parasites and can be generated by the de novo (Kennedy) CDP-choline and CDP-ethanolamine pathways and by the CDP-diacylglycerol dependent pathway. The Kennedy pathways initiate from exogenous choline and ethanolamine involving choline kinase (CK) and ethanolamine kinase (EK), followed by the choline-phosphate cytidylyltransferase (CCT) and ethanolamine-phosphate cytidylyltransferase (ECT) that catalyse the formation of CDP-choline and CDP-ethanolamine. Finally, in Plasmodium, PC and PE are apparently synthesized by a common choline/ethanolamine-phosphotransferase (CEPT). Here, we have studied the essential nature of the Kennedy pathways in Plasmodium berghei, a rodent malaria parasite. Sequence analysis of the P. berghei CEPT, CCT, ECT and CK enzymes revealed the presence of all catalytic domains and essential residues and motifs necessary for enzymatic activities. Constructs were designed for the generation of gene knockout and GFP-fusions of the cept, cct, ect and ck genes in P. berghei. We found that all four genes were consistently refractory to knockout attempts. At the same time, successful tagging of these proteins with GFP demonstrated that the loci were targetable and indicated that these genes are essential in P. berghei blood stage parasites. GFP-fusions of CCT, ECT and CK were found in the cytosol whereas the GFP-CEPT mainly localised in the endoplasmic reticulum. These results indicate that both CDP-choline and CDP-ethanolamine de novo pathways are essential for asexual P. berghei development and are non-redundant with other possible sources of PC and PE.

In this study, we investigated whether hepatitis B virus (HBV) causes the alteration of lipid metabolism and composition during acute infection and liver regeneration in a mouse model. The liver controls lipid biogenesis and bile acid homeostasis. Infection of HBV causes various liver diseases and impairs liver regeneration. As there are very few reports available in the literature on lipid alterations by HBV infection or HBV-mediated liver injury, we have analyzed phospholipids that have important roles in liver regeneration by using matrix-assisted laser desorption/ionization (MALDI)-imaging mass spectrometry (IMS) in the livers of HBV model mice. As a result, we identified different phosphatidylcholines (PCs) showing significant changes in their composition as well as cationized ion adduct formation in HBV-infected mouse livers which are associated with virus-mediated regeneration defects. To find the factor of altered PCs, the expression kinetics of enzymes was also examined that regulate PC biosynthesis during liver regeneration. It is noteworthy that the expression of choline-phosphate cytidylyltransferase A (PCYT1A) was significantly delayed in wild type HBV-expressing livers. Moreover, the amount of hepatic total PC was also significantly decreased in wt HBV-expressing mice. These results suggest that infection of HBV alters the composition of PCs which may involve in HBV-mediated regeneration defects and liver disease.

Phosphatidylethanolamine (GPEtn), a major phospholipid component of trypanosome membranes, is synthesized de novo from ethanolamine through the Kennedy pathway. Here the composition of the GPEtn molecular species in the bloodstream form of Trypanosoma brucei is determined, along with new insights into phospholipid metabolism, by in vitro and in vivo characterization of a key enzyme of the Kennedy pathway, the cytosolic ethanolamine-phosphate cytidylyltransferase (TbECT). Gene knockout indicates that TbECT is essential for growth and survival, thus highlighting the importance of the Kennedy pathway for the pathogenic stage of the African trypanosome. Phosphatiylserine decarboxylation, a potential salvage pathway, does not appear to be active in cultured bloodstream form T. brucei, and it is not upregulated even when the Kennedy pathway is disrupted. In vivo metabolic labelling and phospholipid composition analysis by ESI-MS/MS of the knockout cells confirmed a significant decrease in GPEtn species, as well as changes in the relative abundance of other phospholipid species. Reduction in GPEtn levels had a profound influence on the morphology of the mutants and it compromised mitochondrial structure and function, as well as glycosylphosphatidylinositol anchor biosynthesis. TbECT is therefore genetically validated as a potential drug target against the African trypanosome.

The structural challenges faced by eukaryotic cells through the cell cycle are key for understanding cell viability and proliferation. We tested the hypothesis that the biosynthesis of structural lipids is linked to the cell cycle. If true, this would suggest that the cell's structure is important for progress through and perhaps even control of the cell cycle. Lipidomics (31P NMR and MS), proteomics (Western immunoblotting) and transcriptomics (RT-qPCR) techniques were used to profile the lipid fraction and characterise aspects of its metabolism at seven stages of the cell cycle of the model eukaryote, Desmodesmus quadricauda. We found considerable, transient increases in the abundance of phosphatidylethanolamine during the G1 phase (+35%, ethanolamine phosphate cytidylyltransferase increased 2·5×) and phosphatidylglycerol (+100%, phosphatidylglycerol synthase increased 22×) over the G1/pre-replication phase boundary. The relative abundance of phosphatidylcholine fell by ~35% during the G1. N-Methyl transferases for the conversion of phosphatidylethanolamine into phosphatidylcholine were not found in the de novo transcriptome profile, though a choline phosphate transferase was found, suggesting that the Kennedy pathway is the principal route for the synthesis of PC. The fatty acid profiles of the four most abundant lipids suggested that these lipids were not generally converted between one another. This study shows for the first time that there are considerable changes in the biosynthesis of the three most abundant phospholipid classes in the normal cell cycle of D. quadricauda, by margins large enough to elicit changes to the physical properties of membranes.

The potential advantages of recombinant microbes as oral drug carriers for curing diseases have attracted much attention. The use of recombinant oil microbes as living cell liposomes to carry polypeptide drugs may be an ideal polypeptide oral drug delivery system. GM4-ΔTS was constructed by LFH-PCR from Rhodotorula glutinis GM4, which was screened and preserved in our laboratory, and then transferred into choline-phosphate cytidylyltransferase (CCT), which is a rate-limiting enzyme for lecithin synthesis. The results showed that the CCT gene was highly expressed in the GM4-ΔTS strain and could significantly increase fatty acid and lecithin contents in GM4-ΔTS-PGK1-CCT. Moreover, insulin, H22-LP, and α-MSH were successfully introduced into cells in vitro, and the strain no longer proliferated in vivo, for safe and controllable polypeptide drug delivery. In vivo, normal mice were intragastrically administered with recombinant strains carrying insulin and α-MSH, and different levels of polypeptide drugs were detected in serum and tissue, respectively. Then, recombinant strains carrying insulin were administered to type II diabetes mellitus mice. The results showed that the strains could effectively reduce blood glucose levels in mice, which indicated that the recombinant strains could carry insulin into the body, and the drug effect was remarkable. Therefore, recombinant GM4-ΔTS-PGK1-CCT strains were successfully used as living cell liposomes to carry insulin, H22-LP, and α-MSH peptides into the body for the first time; additionally, these strains have enhanced safety, controllability, and efficacy.

Repeated bacterial and viral infections are known to contribute to worsening lung function in several respiratory diseases, including asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). Previous studies have reported alveolar wall cell apoptosis and parenchymal damage in adult pulmonary VEGF gene ablated mice. We hypothesized that VEGF expressed by type II cells is also necessary to provide an effective host defense against bacteria in part by maintaining surfactant homeostasis. Therefore, Pseudomonas aeruginosa (PAO1) levels were evaluated in mice following lung-targeted VEGF gene inactivation, and alterations in VEGF-dependent type II cell function were evaluated by measuring surfactant homeostasis in mouse lungs and isolated type II cells. In VEGF-deficient lungs increased PAO1 levels and pro-inflammatory cytokines, TNFα and IL-6, were detected 24 h after bacterial instillation compared to control lungs. In vivo lung-targeted VEGF gene deletion (57% decrease in total pulmonary VEGF) did not alter alveolar surfactant or tissue disaturated phosphatidylcholine (DSPC) levels. However, sphingomyelin content, choline phosphate cytidylyltransferase (CCT) mRNA, and SP-D expression were decreased. In isolated type II cells an 80% reduction of VEGF protein resulted in decreases in total phospholipids (PL), DSPC, DSPC synthesis, surfactant associated proteins (SP)-B and -D, and the lipid transporters, ABCA1 and Rab3D. TPA-induced DSPC secretion and apoptosis were elevated in VEGF-deficient type II cells. These results suggest a potential protective role for type II cell-expressed VEGF against bacterial initiated infection.

Chronic consumption of a diet enriched with nutritional precursors of phospholipids, including uridine and the polyunsaturated fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), was shown previously to enhance levels of brain phospholipids and synaptic proteins in rodents. Vitamin C, vitamin E, and selenium may directly affect the breakdown or synthesis of membrane phospholipids. The present study investigated the necessity of antioxidants for the effectiveness of supplementation with uridine plus DHA and EPA (as fish oil) in rats. Rats were randomized to four treatment groups and received, for 6 weeks, one of four experimental diets, i.e., a diet low in antioxidants, a diet high in antioxidants, a diet low in antioxidants supplemented with DHA+EPA+uridine, or a diet high in antioxidants supplemented with DHA+EPA+uridine. On completion of dietary treatment, rats were sacrificed, and brain levels of phospholipids, synaptic proteins, and two enzymes involved in phospholipid synthesis (choline-phosphate cytidylyltransferase, PCYT1A, and choline/ethanolamine phosphotransferase, CEPT1) were analyzed. Levels of phospholipids, the pre- and post-synaptic proteins Synapsin-1 and PSD95, and the enzymes PCYT1A and CEPT1 were significantly enhanced by combined supplementation of DHA+EPA+uridine and antioxidants and not enhanced by supplementation of DHA+EPA+uridine with insufficient antioxidant levels. Our data suggest that dietary vitamin C, vitamin E, and selenium are essential for the phospholipid precursors' effects on increasing levels of membrane phospholipids and synaptic proteins, the indirect indicators of synaptogenesis. Their concomitant supply may be relevant in Alzheimer's disease patients, because the disease is characterized by synapse loss and lower plasma and brain levels of phospholipid precursors and antioxidants.