Endogenous Phospholipids Research Articles

Novel phosphanegold(I) thiolate complexes suppress de novo lipid synthesis in human lung cancer

The upregulation of lipid metabolism is considered one of the most common characteristic changes in tumor cells. In this study, the effects of phosphanegold(I) thiolate complexes on the regulation of lipid metabolism in lung cancer cells were investigated. Six novel phosphanegold(I) thiolate complexes capable of inhibiting lung cancer cell growth both in vitro and in vivo were synthesized and characterized. These complexes significantly inhibited the activity of thioredoxin reductases and impaired the normal structure and function of mitochondria, leading to an accumulation of internal reactive oxygen species. In addition, they markedly inhibited key enzymes involved in de novo lipid synthesis, including sterol regulatory element-binding proteins, fatty acid synthase, and adenosine triphosphate citrate lyase, thus reducing endogenous fatty acid and phospholipid synthesis. Both approaches suppressed lipid droplets storage and ultimately induced apoptosis in lung cancer cells. Interestingly, pretreatment with N-acetyl-l-cysteine and free fatty acids partially reversed the inhibitory effect of phosphanegold(I) thiolate complexes on lung cancer cells. These results are the first to indicated that gold(I) complexes are promising candidates for targeting lipid metabolism in lung cancer treatment strategies.

Screening of Phospholipids in Plasma of Large-Artery Atherosclerotic and Cardioembolic Stroke Patients With Hydrophilic Interaction Chromatography-Mass Spectrometry.

Ischemic stroke (IS) is a deadly and debilitating disease with a high incidence and recurrence rate in elderly people worldwide. Large-artery atherosclerotic (LAA) and cardioembolic (CE) stroke are two leading subtypes and require different management. As a complementary biochemistry method for current diagnostic techniques, a sensitive and accurate phospholipid (PL) targeted lipidomic method was developed in this study. Plasma PLs were selectively extracted with titanium dioxide/fibrous silica nanosphere material, then characterized and quantified with hydrophilic interaction chromatography-mass spectrometry. A total of 31 molecular species of PLs were determined and ten biomarkers including seven molecular species of sphingomyelins (SM d18:1/18:1, d18:1/18:0, d18:1/24:1, d18:1/16:1, d18:1/22:1, d18:1/24:2, and d18:1/16:0) and three molecular species of phosphatidylcholines (16:0/18:1, 16:0/18:2 and 16:0/22:6) showed significant differences in LAA, CE, and healthy control (HC) groups. The independent diagnostic capabilities of these PL biomarkers were successfully evaluated and validated with receiver operating characteristic curves. Additionally, the oleic acid-enriched SMs, which can result in atherogenic lipoprotein aggregation, were proved to be positively related to IS and may perform as the potential risk factors in the future. Meanwhile, valuable suggestions for dietary interventions as an essential source of endogenous PLs could be obtained from this study.

A phospholipase B from Pseudomonas aeruginosa with activity towards endogenous phospholipids affects biofilm assembly

Pseudomonas aeruginosa is a severe threat to immunocompromised patients due to its numerous virulence factors and biofilm-mediated multidrug resistance. It produces and secretes various toxins with hydrolytic activities including phospholipases. However, the function of intracellular phospholipases for bacterial virulence has still not been established. Here, we demonstrate that the hypothetical gene pa2927 of P. aeruginosa encodes a novel phospholipase B named PaPlaB. At reaction equilibrium, PaPlaB purified from detergent-solubilized membranes of E. coli released fatty acids (FAs) from sn-1 and sn-2 positions of phospholipids at the molar ratio of 51:49. PaPlaB in vitro hydrolyzed P. aeruginosa phospholipids reconstituted in detergent micelles and phospholipids reconstituted in vesicles. Cellular localization studies indicate that PaPlaB is a cell-bound PLA of P. aeruginosa and that it is peripherally bound to both membranes in E. coli, yet the active form was predominantly associated with the cytoplasmic membrane of E. coli. Decreasing the concentration of purified and detergent-stabilized PaPlaB leads to increased enzymatic activity, and at the same time triggers oligomer dissociation. We showed that the free FA profile, biofilm amount and architecture of the wild type and ΔplaB differ. However, it remains to be established how the PLB activity of PaPlaB is regulated by homooligomerisation and how it relates to the phenotype of the P. aeruginosa ΔplaB. This novel putative virulence factor contributes to our understanding of phospholipid degrading enzymes and might provide a target for new therapeutics against P. aeruginosa biofilms.

The Effect of Platelets on Thrombin Generation in Cord Blood and Peripheral Neonatal Blood in the Premature Infant; The Event Study

IntroductionInfants born very preterm (<32 weeks) are at increased risk of haemorrhage, particularly intraventricular haemorrhage which can result in short and long term morbidity. Routine clinical laboratory tests such as the prothrombin time and activated partial thromboplastin time are frequently abnormal in this patient group but do not appear to correlate with clinical outcomes. Moreover, while reduced levels of circulating coagulation factors and platelet hypo-reactivity have been reported, plasma thrombin generation (TG), has been reported to be similar or even enhanced in very preterm infants when compared to term infants.Extracellular vesicles, comprising of lipid-bound nanoparticles released from cells (including platelets), may potentially play a role in modulating neonatal haemostasis. We aimed to characterize phospholipid (PL)-dependent plasma thrombin generation in platelet-rich (PRP) and platelet-poor plasma (PPP) in premature infants in both umbilical cord blood & peripheral neonatal blood using calibrated automated thrombography (CAT).MethodsIn this prospective observational study, PRP and PPP was prepared by centrifugation from citrated umbilical cord blood and peripheral neonatal blood collected from premature infants (24 - 31 weeks) and healthy term controls (>37 weeks). No samples were collected from heparinised lines. Parameters of plasma thrombin generation in PRP were assessed using CAT, with thrombin generation stimulated by tissue factor only (TF; final concentration 1pM). CAT was also repeated in PPP using only TF and no exogenous PL (rendering the assay dependent upon the endogenous PL content of plasma).ResultsIn the analysis of umbilical cord blood PRP, plasma thrombin generation was accelerated in the preterm infant group with a significantly shorter time to peak TG observed. The other parameters of TG were similar in both groups (Table 1).In the subset of infants from whom peripheral blood samples were available, there was further evidence of enhanced plasma TG in preterm PRP relative to term infants (Table 2). In this subgroup, the lag time and time to peak thrombin were significantly shorter in the preterm group and peak thrombin was significantly higher.TG was also assessed in both PPP and PRP prepared from umbilical cord blood samples in a subgroup of infants (n=10 term, n=6 preterm) using 1pM TF only, rendering the assay dependent on the phospholipid content of plasma. No difference was observed in any CAT parameters, suggesting that neonatal PPP phospholipid content (potentially from circulating extracellular vesicles) is sufficient to support thrombin generation in the absence of exogenous phospholipid.ConclusionThese preliminary data suggest that neonatal PRP, measured in both umbilical cord blood and peripheral neonatal blood, has similar thrombin generation or may even be hypercoagulable, compared with healthy term controls. Moreover, neonatal plasma phospholipid appears to support thrombin generation in the absence of exogenous phospholipid. This ongoing prospective study aims to further characterize the platelet-dependency of neonatal thrombin generation and evaluate the potential role of extracellular vesicles in neonatal haemostasis. [Display omitted] DisclosuresMaguire: Actelion: Research Funding; Bayer Pharma: Research Funding. Ni Ainle: Leo Pharma: Research Funding; Actelion: Research Funding; Daiichi-Sankyo: Research Funding; Bayer Pharma: Research Funding.

Lysophospholipid (LPA) receptors in GtoPdb v.2021.3

Lysophosphatidic acid (LPA) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Lysophospholipid Receptors [55, 19, 82, 129]) are activated by the endogenous phospholipid LPA. The first receptor, LPA1, was identified as ventricular zone gene-1 (vzg-1) [40], This discovery represented the beginning of the de-orphanisation of members of the endothelial differentiation gene (edg) family, as other LPA and sphingosine 1-phosphate (S1P) receptors were found. Five additional LPA receptors (LPA2,3,4,5,6) have since been identified [82] and their gene nomenclature codified for human LPAR1, LPAR2, etc. (HUGO Gene Nomenclature Committee, HGNC) and Lpar1, Lpar2, etc. for mice (Mouse Genome Informatics Database, MGI) to reflect species and receptor function of their corresponding proteins. The crystal structure of LPA1 is solved and indicates that LPA accesses the extracellular binding pocket, consistent with its proposed delivery via autotaxin [13]. These studies have also implicated cross-talk with endocannabinoids via phosphorylated intermediates that can also activate these receptors. The binding affinities to LPA1 of unlabeled, natural LPA and anandamide phosphate (AEAp) were measured using backscattering interferometry (pKd = 9) [83, 104]. Utilization of this method indicated affinities that were 77-fold lower than when measured using radioactivity-based protocols [128]. Targeted deletion of LPA receptors has clarified signalling pathways and identified physiological and pathophysiological roles. Multiple groups have independently published validation of all six LPA receptors described in these tables, and further validation was achieved using a distinct read-out via a novel TGFα "shedding* assay [48]. LPA has been proposed to be a ligand for GPR35 [94], supported by a study revealing that LPA modulates macrophage function through GPR35 [54]. However chemokine (C-X-C motif) ligand 17 (CXCL17) is reported to be a ligand for GPR35/CXCR8 [76]. Moreover, LPA has also been described as an agonist for the transient receptor potential (Trp) ion channels TRPV1 [87] and TRPA1 [58]. All of these proposed non-GPCR receptor identities require confirmation and are not currently recognized as bona fide LPA receptors.

Abstract 1987: Lysophosphatidic acid's unknown role in the initiation and progression of gastric cancer through LPAR2 receptor and beta-catenin signaling pathway by altering the energy metabolism

Abstract Introduction: Lysophosphatidic acid (LPA), a multifunctional endogenous phospholipid, plays a vital role in cellular homeostasis and malignant behavior of the cancer cells through G-protein coupled receptors. Although dysregulated beta-catenin signaling pathway has been reported as a critical mediator of gastric cancer, LPA's role in beta-catenin mediated gastric cancer is unknown. Hence, our study aims to determine LPA's functional role and its downstream signaling mechanism in beta-catenin mediated gastric cancer. Method: We used commercially available tissue lysates from the human normal and cancerous stomach to check the LPA level and the expression of LPA receptors by ELISA, western blotting, and Immunohistochemical analysis. To examine LPA's functional effect in gastric cancer, we performed ECIS proliferation, migration assay, scratch assay, and transwell invasion assay in LPA treated gastric cancer cells. Next, to elucidate LPA's molecular mechanism mediated gastric cancer progression, we performed Western blotting, and Real-time PCR of beta-catenin and its downstream target genes in LPA treated cells. To investigate the LPA-induced beta-catenin activity, Luciferase assay, Immunocytochemistry, and nuclear fractionation were done in LPA-treated cells. TCGA database analysis was carried out to check the mRNA level of beta-catenin and its downstream target genes and LPA receptors. Furthermore, superoxide production and Seahorse analysis were performed in the LPA treated cells to determine the oxidative stress and mitochondrial energy metabolism. Result: LPA ELISA and Western blotting of LPA receptors in human gastric cancer tissues showed that the LPA level is significantly higher (P&amp;lt;0.001), and LPAR2 is robustly increased (P&amp;lt;0.001)in human gastric cancer tissue samples suggesting a possible role of LPA in gastric cancer. In cultured gastric cancer cell line, LPA treatment increased proliferation, migration, and invasion activity, whereas LPA receptor antagonist abrogated the LPA induced effect. Similarly, LPA treatment leads to activate the beta-catenin and increased the expression of the downstream target genes of the beta-catenin signaling pathway. LPA-induced activation of beta-catenin signaling pathway was abrogated with the addition of LPA receptor antagonist and knockdown of LPAR2. Besides, LPA treatment increased oxidative stress and increased glucose uptake and ATP production (P&amp;lt;0.001) by both oxidative phosphorylation and glycolysis, whereas LPA antagonist abrogated this effect. Conclusion: Together, all of our results suggested that LPA mediates gastric cancer initiation and progression through the beta-catenin signaling pathway via the LPAR2 receptor by altering energy metabolism. Our findings will provide a novel insight into gastric cancer treatment. Citation Format: Hosne Ara, Susmita Bhattarai, Sudha Sharma, Utsab Subedi, MD.Shenuarin Bhuiyan, Xiuping Yu, Sumitra Miriyala, Manikandan Panchatcharam. Lysophosphatidic acid's unknown role in the initiation and progression of gastric cancer through LPAR2 receptor and beta-catenin signaling pathway by altering the energy metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1987.

Lysophospholipid (LPA) receptors in GtoPdb v.2021.2

Lysophosphatidic acid (LPA) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Lysophospholipid Receptors [55, 19, 82, 129]) are activated by the endogenous phospholipid LPA. The first receptor, LPA1, was identified as ventricular zone gene-1 (vzg-1) [40], This discovery represented the beginning of the de-orphanisation of members of the endothelial differentiation gene (edg) family, as other LPA and sphingosine 1-phosphate (S1P) receptors were found. Five additional LPA receptors (LPA2,3,4,5,6) have since been identified [82] and their gene nomenclature codified for human LPAR1, LPAR2, etc. (HUGO Gene Nomenclature Committee, HGNC) and Lpar1, Lpar2, etc. for mice (Mouse Genome Informatics Database, MGI) to reflect species and receptor function of their corresponding proteins. The crystal structure of LPA1 is solved and indicates that LPA accesses the extracellular binding pocket, consistent with its proposed delivery via autotaxin [13]. These studies have also implicated cross-talk with endocannabinoids via phosphorylated intermediates that can also activate these receptors. The binding affinities to LPA1 of unlabeled, natural LPA and anandamide phosphate (AEAp) were measured using backscattering interferometry (pKd = 9) [83, 104]. Utilization of this method indicated affinities that were 77-fold lower than when measured using radioactivity-based protocols [128]. Targeted deletion of LPA receptors has clarified signalling pathways and identified physiological and pathophysiological roles. Multiple groups have independently published validation of all six LPA receptors described in these tables, and further validation was achieved using a distinct read-out via a novel TGFα "shedding* assay [48]. LPA LPA has been proposed to be a ligand for GPCR35 [94], supported by a recent study revealing that LPA modulates macrophage function through GPR35 [54]. However chemokine (C-X-C motif) ligand 17 (CXCL17) is reported to be a ligand for GPR35/CXCR8 [76]. Moreover, LPA has also been described as an agonist for the transient receptor potential (Trp) ion channels TRPV1 [87] and TRPA1 [58]. All of these proposed non-GPCR receptor identities require confirmation and are not currently recognized as bona fide LPA receptors.

Towards the Development of Long Circulating Phosphatidylserine (PS)- and Phosphatidylglycerol (PG)-Enriched Anti-Inflammatory Liposomes: Is PEGylation Effective?

The anionic phospholipids (PLs) phosphatidylserine (PS) and phosphatidylglycerol (PG) are endogenous phospholipids with anti-inflammatory and immunomodulatory activity. A potential clinical use requires well-defined systems and for several applications, a long circulation time is desirable. Therefore, we aimed the development of long circulating liposomes with intrinsic anti-inflammatory activity. Hence, PS- and PG-enriched liposomes were produced, whilst phosphatidylcholine (PC) liposomes served as control. Liposomes were either formulated as conventional or PEGylated formulations. They had diameters below 150 nm, narrow size distributions and composition-dependent surface charges. Pharmacokinetics were assessed non-invasively via in vivo fluorescence imaging (FI) and ex vivo in excised organs over 2 days. PC liposomes, conventionally formulated, were rapidly cleared from the circulation, while PEGylation resulted in prolongation of liposome circulation robustly distributing among most organs. In contrast, PS and PG liposomes, both as conventional or PEGylated formulations, were rapidly cleared. Non-PEGylated PS and PG liposomes did accumulate almost exclusively in the liver. In contrast, PEGylated PS and PG liposomes were observed mainly in liver and spleen. In summary, PEGylation of PS and PG liposomes was not effective to prolong the circulation time but caused a higher uptake in the spleen.

The Potential for Phospholipids in the Treatment of Airway Inflammation: An Unexplored Solution

Asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) are major inflammatory respiratory diseases. Current mainstay therapy for asthma, and chronic obstructive pulmonary disease are corticosteroids, which have well-established side effect profiles. Phospholipids (PLs) are ubiquitous, diverse compounds with varying functions such as their structural role in the cell membrane, energy storage, and cell signaling. Recent advances in understanding PLs role as inflammatory mediators in the body as well as their widespread long-standing use as carrier molecules in drug delivery demonstrate the potential application of PLs in modulating inflammatory conditions. This review briefly explains the main mechanisms of inflammation in chronic respiratory diseases, current anti-inflammatory treatments and areas of unmet need. The structural features, roles of endogenous and exogenous phospholipids, including their use as pharmaceutical excipients, are reviewed. Current research on the immunomodulatory properties of PLs and their potential application in inflammatory diseases is the major section of this review. Considering the roles of PLs as inflammatory mediators and their safety profile established in pharmaceutical formulations, these small molecules demonstrate great potential as candidates in respiratory inflammation. Future studies need to focus on the immunomodulatory properties and the underlying mechanisms of PLs in respiratory inflammatory diseases.

Современный взгляд на вчерашнее лекарство (обзор литературы)

Phospholipids (PL) – lipids that are not involved in accumulation of fatty deposits play a significant role and serve various functions. Firstly, as a component of cell membranes of all cells in the organism. In clinical practice, for the past decades, phospholipids were primarily used as hepaprotective substances, going by the name of “essential phospholipids (EPL)”. Becoming a popular, demanded, well-promoted remedy for the treatment of liver diseases. However, despite the many years of approbation, the existing and sufficientclinicalexperience, discussions regarding their benefits, effectiveness, safety and reliability are still underway and have brought up diametrically opposed views. From complete rejection of EPL as medicinal means, to full recognition of the advertised properties of EPL drugs based on the given manufacturer’s descriptions. For this reason, to clarify the issuing situation, we have conducted a literature search on the object of investigation. Purpose of the study. Search and critical analysis of modern literature sources on the topic of work using the principles of evidence-based medicine. Material and Methods. A critical online review of the literature sources chosen for the research has been carried out in the Web of Science Thompson Reuters, Springer Link and Pubmed databases, as well as in research works and online articles 10 years deep. Inclusion criteria: research papers with a high index of evidence base. Exclusion criteria: literature sources with no evidence, low quality works. Results and Discussion. The articleoutlines the natural role of proteins, lipids and the function of the cytolemma of intracellularorganoids in the restoration of damaged membranes of hepatocytes - de novo. Substantiating the obvious uselessness of using essential phospholipids in the treatment of liver diseases in general, fatty hepatosis in particular. The inability of penetration into liver cells for phospholipids in their native form and their alignment in thehepacyte membrane is indicated, since they belong to different biological species and have a mutually closed genetic system. In the literature search undertaken by us on this issue, the analytical and critical review of scientific literature have also shown that when ingested, essential phospholipids have low bioavailability, as the phospholipids in the chylomicrons do not directly enter the liver, but first the lymphatic system through which they are transported to the adipose tissue of the organism, where they are accumulated and metabolized. With parenteral administration, EPL spreading through the bloodstream can accumulate in other organs, systems, without reaching the liver tissue. For example, the largest amounts of EPL are usually found in the cell membranes of the nervous tissue and the brain. Conclusion. Essential phospholipids in the form of soy lecithin products cannot replace the endogenous phospholipids of the human body - in principle. Keywords: essentialphospholipids, liver cell membranes, hepaprotectors.

Development of isotope-enriched phosphatidylinositol-4- and 5-phosphate cellular mass spectrometry probes.

Synthetic phosphatidylinositol phosphate (PtdInsPn) derivatives play a pivotal role in broadening our understanding of PtdInsPn metabolism. However, the development of such tools is reliant on efficient enantioselective and regioselective synthetic strategies. Here we report the development of a divergent synthetic route applicable to the synthesis of deuterated PtdIns4P and PtdIns5P derivatives. The synthetic strategy developed involves a key enzymatic desymmetrisation step using Lipozyme TL-IM®. In addition, we optimised the large-scale synthesis of deuterated myo-inositol, allowing for the preparation of a series of saturated and unsaturated deuterated PtdIns4P and PtdIns5P derivatives. Experiments in MCF7 cells demonstrated that these deuterated probes enable quantification of the corresponding endogenous phospholipids in a cellular setting. Overall, these deuterated probes will be powerful tools to help improve our understanding of the role played by PtdInsPn in physiology and disease.

Mechanism of Inhibition of Cytochrome c Oxidase by Triton X-100.

It is known that Triton X-100 (TX) reversibly inhibits activity of cytochrome c oxidase (CcO). The mechanism of inhibition is analyzed in this work. The action of TX is not directed to the reaction of CcO with cytochrome c, does not cause transition of the enzyme to the "slow" form, and is not associated with monomerization of the enzyme complex. TX completely suppresses oxygen reduction by CcO, but inhibition is prevented and partially reversed by dodecyl-β-D-maltoside (DDM), a detergent used to maintain CcO in solution. A 1/1 stoichiometry competition is shown between DDM and TX for binding to CcO, with Ki = 0.3 mM and affinity of DDM for the enzyme of 1.2 mM. TX interaction with the oxidized enzyme induces spectral response with maximum at 421 nm and [TX]1/2 = 0.28 mM, presumably associated with heme a3. When CcO interacts with excess of H2O2 TX affects equilibrium of the oxygen intermediates of the catalytic center accelerating the FI-607 → FII-580 transition, inhibits generation of O2·- by the enzyme, and, to a lesser extent, suppresses the catalase partial activity. The observed effects can be explained by inhibition of the conversion of the intermediate FII-580 to the free oxidized state during the catalytic cycle. TX suppresses intraprotein electron transfer between hemes a and a3 during enzyme turnover. Partial peroxidase activity of CcO remains relatively resistant to TX under conditions that block oxidase reaction effectively. These features indicate an impairment of the K proton channel conductivity. We suggest that TX interacts with CcO at the Bile Acid Binding Site (BABS) that is located on the subunit I at the K-channel mouth and contacts with amphipathic regulators of CcO [Buhrow et al. (2013) Biochemistry, 52, 6995-7006]. Apparently, TX mimics the physiological ligand of BABS, whereas the DDM molecule mimics an endogenous phospholipid bound at the edge of BABS that controls effective affinity for the ligand.

Processed meat products with added plant antioxidants affect the microbiota and immune response in C57BL/6JRj mice with cyclically induced chronic inflammation

Epidemiological studies have found that there is a correlation between red and processed meat consumption and an increased risk of colorectal cancer. There are numerous existing hypotheses on what underlying mechanisms are causative to this correlation, but the results remain unclear. A common hypothesis is that lipid oxidation, which occurs in endogenous lipids and phospholipids in consumed food, are catalyzed by the heme iron in meat. In this study, five pre-selected plant antioxidant preparations (sea buckthorn leaves and sprouts, summer savory leaves, olive polyphenols, onion skin and lyophilized black currant leaves) were added to a meatball type prone to oxidize (pork meat, 20 % fat, 2% salt, deep-fried and after 2 weeks of storage). Pro-inflammatory markers, neutrophil infiltration and microbiota composition were studied after four months in a chronic inflammation model in C57BL6/J female mice. We found that the bacterial diversity index was affected, as well as initial immunological reactions.

Short communication: Effects of dietary deoiled soy lecithin supplementation on circulating choline and choline metabolites, and the plasma phospholipid profile in Holstein cows fed palm fat

Dietary lecithin is a source of choline. Our objective was to evaluate the effects of dietary deoiled soy lecithin feeding on circulating choline, choline metabolites, and the plasma phospholipid profile in lactating dairy cows fed fractionated palm fatty acids. In a split-plot Latin square design, 16 Holstein cows (160 ± 7 d in milk; 3.6 ± 1.2 parity) were randomly allocated to a main plot receiving a corn silage and alfalfa haylage-based diet with palm fat containing either moderate or high palmitic acid content at 1.75% of ration dry matter (moderate and high palmitic acid containing 72 or 99% palmitic acid in fat supplement, respectively; n = 8/palm fat diet). Within each palm fat group, deoiled soy lecithin was top-dressed at 0, 0.12, 0.24, or 0.36% of ration dry matter in a replicated 4 × 4 Latin square design with 14-d experimental periods. A 14-d covariate period was used to acclimate cows to palm fat feeding without lecithin supplementation. Blood sampling occurred during the final 3 d of each experimental period. Plasma choline and choline metabolites were quantified using liquid chromatography and mass spectrometry. Plasma phospholipids were profiled using time-of-flight mass spectrometry. Whereas no effects of treatments were detected for plasma choline or methionine, lecithin feeding increased the plasma concentrations of choline metabolites trimethylamine N-oxide and dimethylglycine (24 and 11%, respectively). Plasma phosphatidylcholine (PC) and sphingomyelin (SM) concentrations increased with deoiled lecithin feeding (e.g., PC 16:0/22:6 and SM d18:1/18:3). Lecithin supplementation also increased plasma lysophosphatidylcholine (LPC) concentrations (e.g., LPC 18:0) while reducing plasma phosphatidylethanolamine (PE) concentrations (e.g., PE 16:0/20:5). Although increases in microbial-derived trimethylamine N-oxide suggest gastrointestinal lecithin degradation, elevations in plasma dimethylglycine, PC, LPC, and SM suggest that choline availability was improved by lecithin feeding in cows, thus supporting enhanced endogenous phospholipid synthesis.

Lysophospholipid (LPA) receptors (version 2020.5) in the IUPHAR/BPS Guide to Pharmacology Database

Lysophosphatidic acid (LPA) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Lysophospholipid Receptors [54, 18, 80, 125]) are activated by the endogenous phospholipid LPA. The first receptor, LPA1, was identified as ventricular zone gene-1 (vzg-1) [39], leading to deorphanisation of members of the endothelial differentiation gene (edg) family as other LPA receptors along with sphingosine 1-phosphate (S1P) receptors. Additional LPA receptor GPCRs were later identified. Gene names have been codified as LPAR1, etc. to reflect the receptor function of proteins. The crystal structure of LPA1 was solved and demonstrates extracellular LPA access to the binding pocket, consistent with proposed delivery via autotaxin [12]. These studies have also implicated cross-talk with endocannabinoids via phosphorylated intermediates that can also activate these receptors. The identified receptors can account for most, although not all, LPA-induced phenomena in the literature, indicating that a majority of LPA-dependent phenomena are receptor-mediated. Binding affinities of unlabeled, natural LPA and AEAp to LPA1 were measured using backscattering interferometry (pKd = 9) [81, 102]. Binding affinities were 77-fold lower than than values obtained using radioactivity [124]. Targeted deletion of LPA receptors has clarified signalling pathways and identified physiological and pathophysiological roles. Independent validation by multiple groups has been reported in the peer-reviewed literature for all six LPA receptors described in the tables, including further validation using a distinct read-out via a novel TGFα "shedding" assay [47]. LPA LPA has been proposed to be a ligand for GPCR35 [92], supported by a recent study revealing that LPA modulates macrophage function through GPR35 [53]. However CXCL17 is reported to be a ligand for GPR35/CXCR8 [74]. Moreover, LPA has also been described as an agonist for the transient receptor potential (Trp) ion channel TRPV1 [85] and TRPA1 [57]. All of these proposed non-GPCR receptor identities require confirmation and are not currently recognized as bona fide LPA receptors.

Short-term exposure to air pollution and novel markers of cardiovascular effect: a repeated measures study in the Multi-Ethnic Study of Atherosclerosis (MESA)

Background: Several pathophysiologic mechanisms have been proposed to underlie the associations between ambient air pollutants and cardiovascular disease. We employed a repeated measures design to investigate the short-term associations of outdoor concentrations of four air pollutants – particulate matter smaller than 2.5 micrometers in diameter (PM2.5), nitrogen dioxide (NO2), ozone (O3) and sulfur dioxide (SO2) – with two blood markers involved in vascular effects of oxidative stress, soluble lectin-like oxidized LDL receptor-1 (sLOX-1) and nitrite, using data from the MESA study.Methods: 740 participants who submitted blood samples at three MESA exams between 2002 and 2007 were included. sLOX-1 and nitrite were measured in plasma samples. Daily concentrations of PM2.5, NO2, O3 and SO2 0-7 days prior to blood draw at each exam were estimated from central monitors in the six MESA regions, pre-adjusted using splines of meteorology, indicators for day of the week, and splines of site-specific time trend. Unconstrained distributed lag linear mixed effect models and generalized estimating equations were used to estimate net effects over several days with adjustment for demographic, socioeconomic and behavioral factors.Results: Median detectable nitrite and sLOX-1 concentrations were 24.7 umol/L and 94.5 pg/ml, respectively. Higher short-term PM2.5 concentrations, but not other pollutants, were associated with elevated sLOX-1 level analyzed both as a continuous outcome (percent change per interquartile increase: 18.5%, 95%CI: 2.4-37.2%) and dichotomized at the median (odds ratio per interquartile increase: 1.21, 95%CI: 1.01-1.44); the finding was not meaningfully changed after adjustment for additional covariates or in several sensitivity analyses. Pollutant concentrations were not associated with nitrite levels. Conclusion: This study adds to the evidence that short-term PM2.5 exposure is associated with higher sLOX-1 and supports the mechanistic hypothesis that oxidative modification of endogenous phospholipids in the lung by PM2.5 drives vascular endothelial cell activation via cell surface pattern recognition receptors.

Activation of Macrophages by Lysophosphatidic Acid through the Lysophosphatidic Acid Receptor 1 as a Novel Mechanism in Multiple Sclerosis Pathogenesis.

Multiple sclerosis (MS) is a neuroinflammatory disease whose pathogenesis remains unclear. Lysophosphatidic acid (LPA) is an endogenous phospholipid involved in multiple immune cell functions and dysregulated in MS. Its receptor LPA1 is expressed in macrophages and regulates their activation, which is of interest due to the role of macrophage activation in MS in both destruction and repair. In this study, we studied the genetic deletion and pharmaceutical inhibition of LPA1 in the mouse MS model, experimental autoimmune encephalomyelitis (EAE). LPA1 expression was analyzed in EAE mice and MS patient immune cells. The effect of LPA and LPA1 on macrophage activation was studied in human monocyte-derived macrophages. We show that lack of LPA1 activity induces milder clinical EAE course and that Lpar1 expression in peripheral blood mononuclear cells (PBMC) correlates with onset of relapses and severity in EAE. We see the same over-expression in PBMC from MS patients during relapse compared with progressive forms of the disease and in stimulated monocyte-derived macrophages. LPA induced a proinflammatory-like response in macrophages through LPA1, providing a plausible way in which LPA and LPA1 dysregulation can lead to the inflammation in MS. These data show a new mechanism of LPA signaling in the MS pathogenesis, prompting further research into its use as a therapeutic target biomarker.

CLINICAL CHARACTERISTICS OF PATIENTS WITH SEASONAL ALLERGIC RHINOCONJUNCTIVITIS (SARK). ANAMNESTIC DATA AND THE STRUCTURE OF COMORBIDITY

Introduction. Seasonal allergic rhinitis is AZ of mucous membranes (primarily the conjunctiva of the eye and nasal mucosa), due to hypersensitivity to aerosol allergens of plant pollen and fungi spores, the concentration of which in the air periodically becomes prijenosnog. The leading clinical manifestation of SAR is considered as allergic conjunctivitis. According to statistics, about 70-90% of patients with SAR develops Pulawy conjunctivitis, characterized by itching of eyes, eyelids, their redness, photophobia, lacrimation. Clinically important is the combination of SAR and conjunctivitis. Although the SAR attention usually emphasize on nasal symptoms, more than 80% of patients, as practice shows, suffer from symptoms from the eyes. According to the European and North American researchers, more than 70% of patients with SAR suffer from eye and nasal symptoms, and their severity the majority of patients assessed asmoderate or severe.Materials and methods. Clinical and anamnestic study was done in 120 patients aged from 19 to 45 years. All the studied patients were divided according to the degree of severity of the disease: 58 patients with moderate course of seasonal allergic rhinitis and conjunctivitis (EYE), which caused Pilica ambrosia and 62 patients with severe SARK, which pilca caused by ragweed. Of these, 43 (35,8%) women, 77 (64.1%) of men aged 19 to 25 years – 76 (63.3 per cent), from 25 to 45 years – 44 (36.6%) patients. The control group consisted of 30 healthy people.Research results. The differentiation of the history of infectious diseases patients. In the studied patients SARK with moderate current, acute respiratory illness (ARI) was observed in 54 (26,9%) patients, varicella – 16 (8,0%), mumps – 12 (6,0%), measles in 11 (5,5%), viral hepatitis in 4 (1,9%) patients, acute bronchitis 36 (17,9%), pneumonia – 17 (8,5%), the scarlet fever in 6 (2.9%) and intestinal infection in 7 (3,5%), sore throat – 25 (12,4%), otitis in 13 (6,5%) patients. In patients with severe SARK, ARI was observed in 59 (23,3%) patients, varicella – 24 (9,5%), mumps – in 18 (7.1 percent), measles – in 16 (6.3 percent), viral hepatitis in 7 (2.8%) and acute bronchitis – in 43 (17%), pneumonia in 19 (7,5%), scarlet fever in 8 (3,2%), intestinal infection in 9 (3,6%), angina 32 (12,6%), otitis media – in 18 (7.1 per cent) patients.Analyzing the structure of infectious diseases in the anamnesis in the studied patients SARK need to emphasize that their structure is dominated infection in the upper respiratory tract and broncho-pulmonary system that leads to depletion of nonspecific and specific immunity factors, formation of secondary immunodeficiency in these systems.Discussion of research. In the study of biochemical parameters of blood in patients with SARK in the period of aggravation established that the greatest changes are observed from endogenous cholesterol and phospholipids, which may indicate the failure of the macrophage link of immunity. At the same time, β-lipoproteins and NIK was increased only in the group of patients with severe SARK. The increase of phospholipids in serum of patients with severe SARK can point to the implementation of late phase allergic inflammation in cellular tissue structure of the nasal mucosa.Conclusions. Conducted research of history data, the structure of comorbidity, laboratory parameters allergological studies have shown that SARK is formed on the background of progressive sensitization and allergization of the organism, which is implemented in the manifestation of allergic reactions, forming the severity of SARK, and on the strength of the immune response to a variety of ecoalign that define the types of immunopathological reactions in this cohort of patients.

Structure of human GABAB receptor in an inactive state.

Human GABAB G protein-coupled receptor (GPCR), a member of the class C family, mediates inhibitory neurotransmission and is implicated in epilepsy, pain, and addiction1. A unique GPCR known to require heterodimerization for function2–6, its two subunits, GABAB1 and GABAB2, are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligand7,8, while GABAB2 couples with G protein9–14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane (TM) domain, and a cytoplasmic tail15. Whereas the VFT heterodimer structure has been resolved16, the structure of the full-length receptor and its transmembrane signaling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor, captured in an inactive state via cryo-electron microscopy (EM). Our structure reveals multiple ligands pre-associated with the receptor, including two large endogenous phospholipids embedded within the TM domains to maintain receptor integrity and modulate receptor function. We also identify a novel heterodimer interface between TM helices 5 and 3 of both subunits, which serves as a signature of the inactive conformation. A unique ′intersubunit latch′ within this TM interface maintains the inactive state, and its disruption leads to constitutive receptor activity.

Aspects of matrix and analyte effects in clinical pharmacokinetic sample analyses using LC-ESI/MS/MS – Two case examples

The increasing focus on high throughput sample analysis has led to the common practice of using simplest sample preparation method possible (i.e. protein precipitation) and shortest sample run-time possible. This means that there will be two aspects of compromise: the first compromise is made between sample cleanliness and sample preparation speed since protein precipitation does not provide very clean final extract; the second compromise is made between peak separation and run-time, meaning that sometimes overlap or co-elution of some peaks has to be accepted. The first compromise may lead to matrix effect, which is caused by co-eluting endogenous substances such as phospholipids. The second compromise can result in analyte effect, which is caused by co-eluting analyte(s). We have encountered the issue of matrix/analyte-mediated ion suppression in multiple preclinical and clinical pharmacokinetic projects during bioanalytical method development/validation or biological sample analysis of many small molecule drugs. As these matrix/analyte effects could occur in different situations with different “syndromes”, sometimes it can be easily overlooked, leading to unreliable result, poor sensitivity, and prolonged assay development process. To increase the awareness of this important issue, in this paper we presented two real case examples on signal suppression caused by either endogenous phospholipids or co-eluting analyte.