UNASSIGNED: Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5) P3) and Phosphatidylinositol 4,5-trisphosphate (PtdIns(4,5) P2] form an insignificant amount of phospholipids but play important roles in controlling membrane-bound signalling. Little attention has been given to visualize and monitor changes or differences in the local generation of PtdIns(4,5) P2 and PtdIns(3,4,5) P3 in the cell membranes of MDA-MB-231 breast cancer cell lines.
UNASSIGNED: PLCδ1-PH-GFP and Btk-PH-GFP were used as biosensors to detected PtdIns(4,5) P2 and PtdIns(3,4,5)P3 respectively. These biosensors and antibodies were transfected, immuostained and then visualized by confocal microscopy on different cell surfaces.
UNASSIGNED: Our results showed that PLCδ1-PH-GFP/mCherry was localized at the cell membrane, while Btk-PH-GFP/mCherry was sometimes localized at the cell membrane but there was also a large amount of fluorescence present in the cytosol and nucleus. Our results also showed that the cells that expressed low levels of Btk-PH-GFP the fluorescence was predominantly localised to the cell membrane. While the cells that expressed high levels of Btk-PH-GFP the fluorescence was localization in the cytosol and cell membrane. Our results demonstrated that both anti-PtdIns(4,5)P2 and anti-PtdIns(3,4,5)P3 antibodies were localized everywhere in cell.
UNASSIGNED: Our results suggest that PLCδ1-PH-GFP and Btk-PH-GFP/mCherry have more specificity, reliability, suitability and accuracy than antibodies in binding with and detecting PtdIns(4,5)P2 and PtdIns(3,4,5)P3 and in studying the molecular dynamics of phospholipids in live and fixed cells.

The present study aimed at investigating possible alterations in the serum lipid profile of euthymic patients with bipolar disorder type I (BD) compared to healthy controls (HC). Thirty-five individuals from both genders were recruited, with 14 diagnosed and treated as BD patients (BD group) and 21 healthy subjects (HC group). Clinical assessment was based on the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I), Young Mania Rating Scale (YMRS), and 17-items of Hamilton Depression Rating Scale (HDRS-17) data, which were used to confirm diagnosis, to verify psychiatric comorbidities, and to estimate the severity of manic and depressive symptoms. Ultra-high performance liquid chromatography (UHPLC) coupled to high resolution mass spectrometry (HRMS) was applied to analyze the lipids extracted from all serum samples from both studied groups. In this pioneer and exploratory study, we observed different serum lipid profiles for BD and HC groups, especially regarding glycerophospholipid, glycerolipid, and sphingolipid distribution. Multivariate statistical analyses indicated that 121 lipids were significantly different between BD and HC. Phosphatidylinositols were identified as the most altered lipids in BD patient sera. The results of this preliminary study reinforce the role of lipid abnormalities in BD and offer additional methodological possibilities for investigation in the field.

The aim of this work was to develop a novel and more efficient platform for sublingual drug delivery using mosapride citrate (MSP) as a model drug. The engineering of this delivery system had two stages, the first stage was tuning of MSP physicochemical properties by complexation with pure phosphatidylcholine or phosphatidylinositol enriched soybean lecithin to form MSP-phospholipid complex (MSP-PLCP). Changes in physicochemical properties were assessed and the optimum MSP-PLCP formula was then used for formulation into a flushing resistant platform using two mucoadhesive polymers; sodium alginates and sodium carboxymethylcellulose at different concentrations. Design of experiment approach was used to characterize and optimize the formulated flushing resistant platform. The optimized formulation was then used in a comparative pharmacokinetics study with the market formulation in human volunteers. Results showed a marked change in MSP physicochemical properties of MSP-PLCP compared to MSP. Addition of mucoadhesive polymers to flushing resistant platform at an optimum concentration balanced between desired mucoadhesive properties and a reasonable drug release rate. The optimized formulation showed significantly a superior bioavailability in humans when compared to the market sublingual product. Finally, the novel developed sublingual flushing resistant platform offers a very promising and efficient tool to extend the use of sublingual route and widen its applications.

In vitro, high-resolution 31P NMR (Nuclear Magnetic Resonance) spectroscopy-based analysis of phospholipids in serum is well recognized in leukemia, lymphoma, non-hematological cancers and renal cell carcinoma. In context of these studies, phospholipids were analyzed in blood of thirty-two (n = 32) patients with Duchenne muscular dystrophy (DMD) (Age, Mean ± SD; 8.0 ± 1.6 years) and sixteen (n = 16) healthy subjects (Age, Mean ± SD; 8.6 ± 2.3 years). Quantity of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and lyso-phosphatidylcholine (Lys-PC) was significantly higher (p < 0.05) in DMD patients as compared to healthy subjects. There were no significant differences (p > 0.05) observed for the quantity of phospholipids in blood of gene deletion positive cases of DMD as compared to negative gene deletion cases of DMD. Quantity of phospholipids in negative gene deletion cases of DMD patients as well as DMD cases with positive gene deletion was significantly higher (p < 0.05) as compared to normal individuals. The present study distinguishes the patients with DMD from the healthy subjects on the basis of the quantity of phospholipids in blood. These observations may be useful in future for the development of new diagnostic method of DMD.

Direct linkage between the plasma membrane and the actin cytoskeleton is controlled by the protein ezrin, a member of the ezrin-radixin-moesin protein family. To function as a membrane-cytoskeleton linker, ezrin needs to be activated in a process that involves binding of ezrin to phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphorylation of a conserved threonine residue. Here, we used colloidal probe microscopy to quantitatively analyze the interaction between ezrin and F-actin as a function of these activating factors. We show that the measured individual unbinding forces between ezrin and F-actin are independent of the activating parameters, in the range of approximately 50 piconewtons. However, the cumulative adhesion energy greatly increases in the presence of PIP2 demonstrating that a larger number of bonds between ezrin and F-actin has formed. In contrast, the phosphorylation state, represented by phosphor-mimetic mutants of ezrin, only plays a minor role in the activation process. These results are in line with in vivo experiments demonstrating that an increase in PIP2 concentration recruits more ezrin to the apical plasma membrane of polarized cells and significantly increases the membrane tension serving as a measure of the adhesion sites between the plasma membrane and the F-actin network.

Research on the secretory pathway in the past three decades accounts for our known knowledge about the composition and architecture of organelles and about the machinery that regulates membrane transport. An emerging topic in the past few years was the discovery that the secretory pathway is regulated by signaling, and in this regard, the Golgi apparatus received major attention. In the current chapter, we will highlight various techniques that are used by us and others to study signaling at the Golgi. We describe methods to study lipid and protein phosphorylation at the Golgi and various techniques for studying spatial activation of GTPases at this organelle. We also discuss how combining these techniques and improving their limitations is important for gaining a better understanding of how the Golgi intersects with various signal transduction pathways.

EPR spectroscopy was applied to investigate the effects of the treatment of Candida albicans cells with fluconazole (FLC) and two newly synthesized azoles (CPA18 and CPA109), in a concentration not altering yeast morphology, on the lipid organization and dynamics of the plasma membrane. Measurements were performed in the temperature range between 0°C and 40°C using 5-doxyl- (5-DSA) and 16-doxyl- (16-DSA) stearic acids as spin probes. 5-DSA spectra were typical of lipids in a highly ordered environment, whereas 16-DSA spectra consisted of two comparable components, one corresponding to a fluid bulk lipid domain in the membrane and the other to highly ordered and motionally restricted lipids interacting with integral membrane proteins. A line shape analysis allowed the relative proportion and the orientational order and dynamic parameters of the spin probes in the different environments to be determined. Smaller order parameters, corresponding to a looser lipid packing, were found for the treated samples with respect to the control one in the region close to the membrane surface probed by 5-DSA. On the other hand, data on 16-DSA indicated that azole treatments hamper the formation of ordered lipid domains hosting integral proteins and/or lead to a decrease in integral protein content in the membrane. The observed effects are mainly ascribable to the inhibition of ergosterol biosynthesis by the antifungal agents, although a direct interaction of the CPA compounds with the membrane bilayer in the region close to the lipid polar head groups cannot be excluded.