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In the last few decades, the preparation of solid-supported lipid bilayers by immersing a solid substrate in an aqueous solution where the lipid is dissolved with the aid of a surfactant, followed by dilution of the solution, has been reported. In this study, we attempted to interpret the evolution of supported surfactant/lipid assemblies towards the supported lipid bilayer in terms of a phase equilibrium between the supported assembly phase and its ambient solution system consisting of the dispersed surfactant/lipid assembly phase and the bulk solution phase comprising monomeric surfactant and lipid. We characterized the supported assembly formed on hydrophilized Ge or mica substrates in equilibrium with aqueous solutions containing various concentrations of the nonionic surfactant, n-octyl-β-Dglucopyranoside (OG) and the amphoteric phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), using interaction-force-profile measurements by atomic force microscopy (AFM), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). We also investigated the ambient solution system using equilibrium dialysis to obtain the partition equilibrium profile of OG between the bulk solution and dispersed assembly phases in the micellar or vesicular states. These studies indicate that the properties of the supported assembly depend on the composition of the dispersed assembly and concentration of monomerically dissolved OG. Further, a type of micellar-bilayer state transition occurs in the supported assembly, roughly synchronized with that in the dispersed assembly. [ABSTRACT FROM AUTHOR]

Medulloblastoma (MB) is the most common malignant brain tumour in children. High-risk MB patients harbouring MYC amplification or overexpression exhibit a very poor prognosis. Aberrant activation of MYC markedly reprograms cell metabolism to sustain tumorigenesis, yet how metabolism is dysregulated in MYC-driven MB is not well understood. Growing evidence unveiled the potential of BET-bromodomain inhibitors (BETis) as next generation agents for treating MYC-driven MB, but whether and how BETis may affect tumour cell metabolism to exert their anticancer activities remains unknown. In this study, we explore the metabolic features characterising MYC-driven MB and examine how these are altered by BET-bromodomain inhibition. To this end, we employed an NMR-based metabolomics approach applied to the MYC-driven MB D283 and D458 cell lines before and after the treatment with the BETi OTX-015. We found that OTX-015 triggers a metabolic shift in both cell lines resulting in increased levels of myo-inositol, glycerophosphocholine, UDP-N-acetylglucosamine, glycine, serine, pantothenate and phosphocholine. Moreover, we show that OTX-015 alters ascorbate and aldarate metabolism, inositol phosphate metabolism, phosphatidylinositol signalling system, glycerophospholipid metabolism, ether lipid metabolism, aminoacyl-tRNA biosynthesis, and glycine, serine and threonine metabolism pathways in both cell lines. These insights provide a metabolic characterisation of MYC-driven childhood MB cell lines, which could pave the way for the discovery of novel druggable pathways. Importantly, these findings will also contribute to understand the downstream effects of BETis on MYC-driven MB, potentially aiding the development of new therapeutic strategies to combat medulloblastoma. [ABSTRACT FROM AUTHOR]

The interfacial behaviour of the amphiphilic drug hexadecyl phosphocholine (HePC, also called miltefosine) was analysed by cyclic voltammetry applied at the water/1,2-dichloroethane interface. HePC is the only oral drug currently approved for the treatment of visceral, mucosal  and cutaneous leishmaniasis. Because of its amphiphilic character, it can interact with biological membranes, solubilizing their compounds and leading to cell disruption. These interactions are responsible for its side effects and toxicity; therefore, HePC quantification in biological fluids and pharmaceutical preparations is extremely important. However, the lack of a chromophore in its structure prevents its spectroscopic determination. For this reason, the main challenge of this work was to propose an electroanalytical method for the quantification of this drug, which constitutes a simpler alternative than liquid chromatography-tandem mass spectrometry already reported. With this aim, in the first part of this work, the mechanism of the electrochemical process occurring after polarizing the interface was studied. By varying the experimental conditions, it was possible to determine that in a first step, at open circuit or at low potential values, HePC spontaneously adsorbed to the interface. Later, as the potential increased, the transfer of the anions present in the organic phase towards the aqueous side of the interface, where the HePC polar head groups were present, occurred thus forming adsorbed "ion pairs" and producing an increase in positive current. Subsequently, in the negative sweep, the "ion pairs" dissociated and desorbed giving rise to a negative peak. In this way, both negative and positive currents were considered useful for quantitative purposes. In the second part of this work, an appropriate experimental procedure was designed and proposed as a quantitative methodology for the HePC determination, which consisted of cleaning the interface and controlling the time at open circuit, followed by the voltammetric analysis. A linear response of both, positive or negative, peak currents with drug concentration was obtained within an acceptable range, providing a simple solution for the HePC quantification problem. Future studies will be carried out to evaluate the quantification and selectivity in real matrices containing polymer micelles working as HePC nanocarriers with the aim of avoiding the adverse effects of HePC when it is orally or intravenously administered. [ABSTRACT FROM AUTHOR]

In the past 30 years there has been a significant increase in the number of publications on phospholipid (PL) metabolism, both for the medical purposes of detection and diagnosis of cancer and for the monitoring of the treatment of human cancers. Most of the work has focused on the pathway that produces phosphatidylcholine, the major component of human cell membranes. The trigger for this research was the advent of applications of NMR spectroscopy in vitro and in vivo in the 1980’s and observations that most cancer cells and tumors had significant increases in the water-soluble PL precursors and breakdown products. Increased phosphocholine (PC) has been focused on as a marker for cancer using Magnetic Resonance Spectroscopy (MRS) and Positron Emission Tomography (PET). MRS is now used clinically to aid in the diagnosis and severity of some brain tumors; and choline PET is used for the diagnosis and staging of recurrent prostate cancer, paid for by medical insurance companies. Another major area of research starting in the 1990’s was the development of specific choline kinase (CK) inhibitors aimed at the isoenzyme CK-a. This isoenzyme is markedly upregulated in cancer cells and unexpectedly was found to have a role in oncogenic transformation independent of its enzyme function.

Previous studies have used neutron diffraction to elucidate the hydration of the ceramide and the phosphatidylcholine headgroup in solution. These solution studies provide bond-length resolution information on the system, but are limited to liquid samples. The work presented here investigates how the hydration of ceramide and phosphatidylcholine headgroups in a solution compares with that found in a lipid bilayer. This work shows that the hydration patterns seen in the solution samples provide valuable insight into the preferential location of hydrating water molecules in the bilayer. There are certain subtle differences in the distribution, which result from a combination of the lipid conformation and the lipid-lipid interactions within the bilayer environment. The lipid-lipid interactions in the bilayer will be dependent on the composition of the bilayer, whereas the restricted exploration of conformational space is likely to be applicable in all membrane environments. The generalized description of hydration gathered from the neutron diffraction studies thus provides good initial estimation for the hydration pattern, but this can be further refined for specific systems. [ABSTRACT FROM AUTHOR]

Membrane-associated enzymes have been found to behave differently qualitatively and quantitatively in terms of activity. These findings were highly debated in the 1970s and many general correlations and reaction specific models have been proposed, reviewed, and discarded. However, new biological applications brought up the need for clarification and elucidation. To address literature shortcomings, we chose the intrinsically water-soluble enzyme a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) and large unilamellar vesicles with a relative broad phase transition. We here present activity measurements of ADAMTS13 in the freely dissolved state and the membrane associated state for phosphocholine lipids with different acyl-chain lengths (13:0, 14:0 and 15:0) and thus main phase transition temperatures. While the freely dissolved enzyme shows a simple Arrhenius behavior, the activity of membrane associated ADAMTS13 in addition shows a peak. This peak temperature correlates with the main phase transition temperature of the used lipids. These findings support an alternative theory of catalysis. This theory predicts a correlation of the membrane associated activity and the heat capacity, as both are susceptibilities of the same surface Gibb's free energy, since the enzyme is attached to the membrane. [ABSTRACT FROM AUTHOR]

A new method to extract the free energy of aggregation versus aggregate size from molecular simulation data is proposed and applied to a united atom model of the zwitterionic surfactant dodecyl phosphocholine in water. This system's slow dissociation rate and low critical micelle concentration (CMC of approximately 1–2 mM) make extraction of cluster free energies directly from simulation results using the "partition‐enabled analysis of cluster histogram" (PEACH) method impractical. The new approach applies PEACH to a model with weakened attractions between aggregants, which allows sampling of a continuous range of cluster sizes, then recovers the free energy of aggregation under the original fully‐attractive force field using the BAR free energy difference method. PEACH‐BAR results are compared with free energy differences calculated via umbrella sampling, and are used to make predictions of CMC, average cluster size, and SAXS scattering profiles that are in fair agreement with experiment. [ABSTRACT FROM AUTHOR]

An important step in the simulation of a membrane protein in a phospholipid bilayer is the correct immersion of the protein in the bilayer. Crystal structures are determined without the bilayer. Particularly for proteins with monotopic domains, it can be unclear how deeply and in which orientation the protein is being inserted in the membrane. We have previously developed a procedure combining coarse-grain (CG) with all-atom (AA) molecular dynamics (MD) simulations to insert and simulate a cytochrome P450 (CYP) possessing an N-terminal transmembrane helix connected by a flexible linker region to a globular domain that dips into the membrane. The CG simulations provide a computationally efficient means to explore different orientations and conformations of the CYP in the membrane. Converged configurations obtained in the CG simulations are then refined in AA simulations. Here, we tested different variants of the MARTINI CG model, differing in the water model, the treatment of long-range non-bonded interactions, and the implementation (GROMACS 4.5.5 vs 5.0.4), for this purpose. We examined the behavior of the models for simulating a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer in water and for the immersion of CYP3A4 in a POPC bilayer, and compared the CG-MD results with the previously reported experimental and simulation results. We also tested the methodology on a set of four other CYPs. Finally, we propose an optimized protocol for modeling such protein-membrane systems that provides the most plausible configurations and is computationally efficient; this incorporates the standard non-polar water model and the GROMACS 5.0.4 implementation with a reaction field treatment of long-range interactions. [ABSTRACT FROM AUTHOR]

We have calculated the electric polarizability of two types of lipid bilayers, formed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2- oleoyl-sn-glycero-3- phosphocholine (POPC) lipids. We demonstrate that despite the very similar chemical structures, the bilayers exhibit dramatically different terahertz infrared (IR) properties. We demonstrate that the chemical structure of the lipids influences the morphology of the bilayers, which in turn impacts their IR responses: interestingly, both structures exhibit a non-uniform absorption. For the case of DPPC, the infrared absorption is mostly driven by the hydrophilic heads, whereas for the POPC lipids, the absorption occurs predominately at the hydrophobic tails. [ABSTRACT FROM AUTHOR]

The study of the ability of drug molecules to enter cells through the membrane is of vital importance in the field of drug delivery. In cases where the transport of the drug molecules through the membrane is not easily accomplishable, other carrier molecules are used. Spherical fullerene molecules have been postulated as potential carriers of highly hydrophilic drugs across the plasma membrane. Here, we report the coarse-grain molecular dynamics study of the translocation of C60 fullerene and its derivatives across a cell membrane modeled as a 1,2-distearoyl-sn-glycero-3-phosphocholine bilayer. Simulation results indicate that pristine fullerene molecules enter the bilayer quickly and reside within it. The addition of polar functionalized groups makes the fullerenes less likely to reside within the bilayer but increases their residence time in bulk water. Addition of polar functional groups to one half of the fullerene surface, in effect creating a Janus particle, offers the most promise in developing fullerene models that can achieve complete translocation through the membrane bilayer. [ABSTRACT FROM AUTHOR]

Molecular dynamics simulations in the NPT ensemble have been carried out to investigate the effect of two room temperature ionic liquids (RTILs), on stacks of phospholipid bilayers in water. We consider RTIL compounds consisting of chloride ([bmim][Cl]) and hexafluorophosphate ([bmim][PF6]) salts of the 1-buthyl-3-methylimidazolium ([bmim]+) cation, while the phospholipid bilayer is made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Our investigations focus on structural and dynamical properties of phospholipid and water molecules that could be probed by inelastic and quasi-elastic neutron scattering measurements. The results confirm the fast incorporation of [bmim]+ into the lipid phase already observed in previous simulations, driven by the Coulomb attraction of the cation for the most electronegative oxygens in the POPC head group and by sizeable dispersion forces binding the neutral hydrocarbon tails of [bmim]+ and of POPC. The [bmim]+ absorption into the bilayer favours the penetration of water into POPC, causes a slight but systematic thinning of the bilayer, and further stabilises hydrogen bonds at the lipid/water interface that already in pure samples (no RTIL) display a lifetime much longer than in bulk water. On the other hand, the effect of RTILs on the diffusion constant of POPC (DPOPC) does not reveal a clearly identifiable trend, since DPOPC increases upon addition of [bmim][Cl] and decreases in the [bmim][PF6] case. Moreover, because of screening, the electrostatic signature of each bilayer is only moderately affected by the addition of RTIL ions in solution. The analysis of long wavelength fluctuations of the bilayers shows that RTIL sorption causes a general decrease of the lipid/water interfacial tension and bending rigidity, pointing to the destabilizing effect of RTILs on lipid bilayers. [ABSTRACT FROM AUTHOR]

Phospholipid synthesis is crucial for membrane proliferation in malaria parasites during the entire cycle in the host cell. The major phospholipid of parasite membranes, phosphatidylcholine (PC), is mainly synthesized through the Kennedy pathway. The phosphocholine required for this synthetic pathway is generated by phosphorylation of choline derived from the catabolism of the lyso-phosphatidylcholine (LPC) scavenged from the host milieu. Here we have characterized a Plasmodium falciparum lysophospholipase (PfLPL20) which showed enzymatic activity on LPC substrate to generate choline. Using GFP-targeting approach, PfLPL20 was localized in vesicular structures associated with the neutral lipid storage bodies present juxtaposed to the food-vacuole. The C-terminal tagged glmS mediated inducible knock-down of PfLPL20 caused transient hindrance in the parasite development, however, the parasites were able to multiply efficiently, suggesting that PfLPL20 is not essential for the parasite. However, in PfLPL20 depleted parasites, transcript levels of enzyme of SDPM pathway (Serine Decarboxylase-Phosphoethanolamine Methyltransferase) were altered along with up-regulation of phosphocholine and SAM levels; these results show up-regulation of alternate pathway to generate the phosphocholine required for PC synthesis through the Kennedy pathway. Our study highlights the presence of alternate pathways for lipid homeostasis/membranebiogenesis in the parasite; these data could be useful to design future therapeutic approaches targeting phospholipid metabolism in the parasite. [ABSTRACT FROM AUTHOR]

The tissue metabolomic characteristics associated with endometrial cancer (EC) at different grades were studied using high resolution (400 MHz) magic angle spinning (HR-MAS) proton spectroscopy. The metabolic profiles were obtained from 64 patients (14 with grade 1 (G1), 33 with grade 2 (G2) and 17 with grade 3 (G3) tumors) and compared with the profile acquired from 10 patients with the benign disorders. OPLS-DA revealed increased valine, isoleucine, leucine, hypotaurine, serine, lysine, ethanolamine, choline and decreased creatine, creatinine, glutathione, ascorbate, glutamate, phosphoethanolamine and scyllo-inositol in all EC grades in reference to the non-transformed tissue. The increased levels of taurine was additionally detected in the G1 and G2 tumors in comparison to the control tissue, while the elevated glycine, N-acetyl compound and lactate—in the G1 and G3 tumors. The metabolic features typical for the G1 tumors are the increased dimethyl sulfone, phosphocholine, and decreased glycerophosphocholine and glutamine levels, while the decreased myo-inositol level is characteristic for the G2 and G3 tumors. The elevated 3-hydroxybutyrate, alanine and betaine levels were observed in the G3 tumors. The differences between the grade G1 and G3 malignances were mainly related to the perturbations of phosphoethanolamine and phosphocholine biosynthesis, inositol, betaine, serine and glycine metabolism. The statistical significance of the OPLS-DA modeling was also verified by an univariate analysis. HR-MAS NMR based metabolomics provides an useful insight into the metabolic reprogramming in endometrial cancer. [ABSTRACT FROM AUTHOR]

SUMMARY: Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most abundant phospholipids in membranes. The biosynthesis of phospholipids occurs mainly via the Kennedy pathway. Recent studies have shown that through this pathway, choline (Cho) moieties are synthesized through the methylation of phosphoethanolamine (PEtn) to phosphocholine (PCho) by phospho‐base N‐methyltransferase. In Arabidopsis thaliana, the phosphoethanolamine/phosphocholine phosphatase1 (PECP1) is described as an enzyme that regulates the synthesis of PCho by decreasing the PEtn level during phosphate starvation to avoid the energy‐consuming methylation step. By homology search, we identified a gene (At4g29530) encoding a putative PECP1 homolog from Arabidopsis with a currently unknown biological function in planta. We found that At4g29530 is not induced by phosphate starvation, and is mainly expressed in leaves and flowers. The analysis of null mutants and overexpression lines revealed that PEtn, rather than PCho, is the substrate in vivo, as in PECP1. Hydrophilic interaction chromatography‐coupled mass spectrometry analysis of head group metabolites shows an increased PEtn level and decreased ethanolamine level in null mutants. At4g29530 null mutants have an early flowering phenotype, which is corroborated by a higher PC/PE ratio. Furthermore, we found an increased PCho level. The choline level was not changed, so the results corroborate that the PEtn‐dependent pathway is the main route for the generation of Cho moieties. We assume that the PEtn‐hydrolyzing enzyme participates in fine‐tuning the metabolic pathway, and helps prevent the energy‐consuming biosynthesis of PCho through the methylation pathway. [ABSTRACT FROM AUTHOR]

The interaction of L-Phe with the membrane components, i.e. , lipids and proteins, has been discussed in the current literature due to the interest to understand the effect of single amino acids in relation to the formation of amyloid aggregates. In the present work, it is shown that L-Phe interacts with 9:1 DMPC (1,2-dimyristoyl- sn -glycero-3 phosphocholine)/DPPC (1,2-dipalmitoyl- sn -glycero-3 phosphocholine) mixtures but not in the 1:9 one. An important observation is that the interaction disappears when DPPC is replaced by diether PC (2-di- O -hexadecyl- sn -glycero-3-phosphocholine) a lipid lacking carbonyl groups (CO). This denotes that CO groups may interact specifically with L-Phe in accordance with the appearance of a new peak observed by Infrared spectroscopy (FTIR-ATR). The interaction of L-Phe affects the compressibility pattern of the 9:1 DMPC/DPPC mixture which is congruent with the changes observed by Raman spectra. The specific interaction of L-Phe with CO, propagates to phosphate and choline groups in this particular mixture as analyzed by FTIR-ATR spectroscopy and is absent when DMPC is dopped with diether PC. [Display omitted] • L-Phe binds to 9:1 DMPC/DPPC mixtures • CO groups in the DPPC component are required. • Effect of L-Phe propagates to PO and choline groups. [ABSTRACT FROM AUTHOR]

The article reports that C-polysaccharide is a phosphocholine-substituted ribitol teichoic acid and, in 1971, Volanakis and Kaplan identified phosphocholine as the structure to which CRP binds. Topics incldue considered that CRP bound to C-polysaccharide and other ligands, and activated the classical complement pathway, and showed that CRP was thereby capable of mediating inflammation.

Mesocotyl elongation of rice is crucial for seedlings pushing out of deep soil. The underlying mechanisms of phospholipid signaling in mesocotyl growth of rice are elusive. Here we report that the rice non-specific phospholipase C6 (OsNPC6) is involved in mesocotyl elongation. Our results indicated that all five OsNPCs (OsNPC1, OsNPC2, OsNPC3, OsNPC4 and OsNPC6) hydrolyzed the substrate phosphatidylcholine to phosphocholine (PCho), and all of them showed plasma membrane localization. Overexpression (OE) of OsNPC6 produced plants with shorter mesocotyls compared to those of Nipponbare and npc6 mutants. Although the mesocotyl growth of npc6 mutants was not much affected without gibberellic acid (GA)3, it was obviously elongated by treatment with GA. Upon GA3 treatment, SLENDER RICE1 (SLR1), the DELLA protein of GA signaling, was drastically increased in OE plants; by contrast, the level of SLR1 was found decreased in npc6 mutants. The GA-enhanced mesocotyl elongation and the GA-impaired SLR1 level in npc6 mutants were attenuated by the supplementation of PCho. Further analysis indicated that the GA-induced expression of phospho-base N-methyltransferase 1 in npc6 mutants was significantly weakened by the addition of PCho. In summary, our results suggest that OsNPC6 is involved in mesocotyl development via modulation of PCho in rice. [ABSTRACT FROM AUTHOR]

Brain metabolism evolves rapidly during early post‐natal development in the rat. While changes in amino acids, energy metabolites, antioxidants or metabolites involved in phospholipid metabolism have been reported in the early stages, neurometabolic changes during the later post‐natal period are less well characterized. Therefore, we aimed to assess the neurometabolic changes in male Wistar rats between post‐natal days 29 and 77 (p29‐p77) using longitudinal magnetic resonance spectroscopy (MRS) in vivo at 9.4 Tesla. 1H MRS was performed in the hippocampus between p29 and p77 at 1‐week intervals (n = 7) and in the cerebellum between p35 and p77 at 2‐week intervals (n = 7) using the SPECIAL sequence at ultra‐short echo‐time. NOE enhanced and 1H decoupled 31P MR spectra were acquired at p35, p48 and p63 (n = 7) in a larger voxel covering cortex, hippocampus and part of the striatum. The hippocampus showed a decrease in taurine concentration and an increase in glutamate (with more pronounced changes until p49), seemingly a continuation of their well‐described changes in the early post‐natal period. A constant increase in myo‐inositol and choline‐containing compounds in the hippocampus (in particular glycero‐phosphocholine as shown by 31P MRS) was measured throughout the observation period, probably related to membrane metabolism and myelination. The cerebellum showed only a significant increase in myo‐inositol between p35 and p77. In conclusion, this study showed important changes in brain metabolites in both the hippocampus and cerebellum in the later post‐natal period (p29/p35‐p77) of male rats, something previously unreported. Based on these novel data, changes in some neurometabolites beyond p28‐35, conventionally accepted as the cut off for adulthood, should be taken into account in both experimental design and data interpretation in this animal model. [ABSTRACT FROM AUTHOR]

Cationic amino acid-based surfactants are known to interact with the lipid bilayer of microorganism resulting in cell death through a disruption of the membrane topology. To elucidate the interaction of a cationic surfactant synthesized in our lab, investigations involving Nα-benzoyl-arginine decyl amide (Bz-Arg-NHC10), and model membranes composed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were done. Bz-Arg-NHC10was able to penetrate into DPPC monolayers up to a critical pressure of 59.6 mN m−1. Differential scanning calorimetry revealed that as the concentration of Bz-Arg-NHC10 increased, the main transition temperature of DPPC slightly decreased. Atomic force microscopy (AFM) in situ experiments performed on supported DPPC bilayers on mica allowed monitoring the changes induced by Bz-Arg-NHC10. DPPC bilayer patches were partially removed, mainly in borders and bilayer defects for 50 µM Bz-Arg-NHC10 solution. Increasing the concentration to 100 µM resulted in a complete depletion of the supported bilayers. Surface plasmon resonance (SPR) experiments, carried out with fully DPPC bilayers covered chips, showed a net increase of the SPR signal, which can be explained by Bz-Arg-NHC10 adsorption. When patchy DPPC bilayers were formed on the substrate, a SPR signal net decrease was obtained, which is consistent with the phospholipids' removal observed in the AFM images. The results obtained suggest that the presence of the benzoyl group attached to the polar head of our compound would be the responsible of the increased antimicrobial activity against gram-negative bacteria when compared with other arginine-based surfactants. [ABSTRACT FROM AUTHOR]

Coarse graining of membrane simulations by translating atomistic dynamics to densities and fields with Milestoning is discussed. The space of the membrane system is divided into cells and the different cells are characterized by order parameters presenting the number densities. The dynamics of the order parameters are probed with Milestoning. The methodology is illustrated here for a phospholipid membrane system (a hydrated bilayer of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) lipid molecules). Significant inhomogeneity in membrane internal number density leads to complex free energy landscape and local maps of transition times. Dynamics and distributions of cavities within the membrane assist the permeation of nonpolar solutes such as xenon atoms. It is illustrated that quantitative and detailed dynamics of water transport through DOPC membrane can be analyzed using Milestoning with fields. The reaction space for water transport includes at least two slow variables: the normal to the membrane plane, and the water density. [ABSTRACT FROM AUTHOR]

Raman scattering spectra were obtained at 25-320 K for bilayers prepared from saturated 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and mono-unsaturated 1-palmitoyl-2-oleoyl-snglycero- 3-phosphocholine (POPC) phospholipids, with and without cholesterol. Raman intensities were measured at modes sensitive to lipid inter-chain interactions and/or intra-chain torsional motion (asymmetric CH2 stretching at 2880 cm-1) and to the conformational state of lipids (C-C stretching at 1130 cm-1). These intensities decreased with temperature, which could be ascribed to increased lipid flexibility. For cholesterol-free and cholesterol-containing DPPC bilayers, the decrease of Raman intensities observed above ∼200 K could be related to the phenomenon of dynamical transition known for biological systems near these temperatures. For a cholesterol-free POPC bilayer, the decrease of intensity for the asymmetric CH2 stretching mode started at a lower temperature, above 100 K, while the addition of cholesterol shifted this starting temperature to a more normal ∼200 K value. The low-temperature lipid flexibility in the case of POPC was related to the abundance of freevolume holes, which disappeared in presence of cholesterol. Near gel-fluid phase transitions, Raman intensities for cholesterol-free bilayers dropped sharply, while for cholesterol-containing bilayers, they changed smoothly. [ABSTRACT FROM AUTHOR]

Coarse-grained model for saturated phospholipids: 1,2-didecanoyl-sn-glycero-3-phosphocholine (DCPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-snglycero- 3-phosphocholine (DSPC) and unsaturated phospholipids: 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC) is introduced within the single chain mean field theory. A single set of parameters adjusted for DMPC bilayers gives an adequate description of equilibrium and mechanical properties of a range of saturated lipid molecules that differ only in length of their hydrophobic tails and unsaturated (POPC, DOPC) phospholipids which have double bonds in the tails. A double bond is modeled with a fixed angle of 120◦, while the rest of the parameters are kept the same as saturated lipids. The thickness of the bilayer and its hydrophobic core, the compressibility, and the equilibrium area per lipid correspond to experimentally measured values for each lipid, changing linearly with the length of the tail. The model for unsaturated phospholipids also fetches main thermodynamical properties of the bilayers. This model is used for an accurate estimation of the free energies of the compressed or stretched bilayers in stacks or multilayers and gives reasonable estimates for free energies. The proposed model may further be used for studies of mixtures of lipids, small molecule inclusions, interactions of bilayers with embedded proteins. [ABSTRACT FROM AUTHOR]

We investigated molecular motions in the 0.3-350 ps time range of D2O-hydrated bilayers of 1- palmitoyl-oleoyl-sn-glycero-phosphocholine and 1,2-dimyristoyl-sn-glycero-phosphocholine in the liquid phase by quasielastic neutron scattering. Model analysis of sets of spectra covering scale lengths from 4.8 to 30 Å revealed the presence of three types of motion taking place on well-separated time scales: (i) slow diffusion of the whole phospholipid molecules in a confined cylindrical region; (ii) conformational motion of the phospholipid chains; and (iii) fast uniaxial rotation of the hydrogen atoms around their carbon atoms. Based on theoretical models for the hydrogen dynamics in phospholipids, the spatial extent of these motions was analysed in detail and the results were compared with existing literature data. The complex dynamics of protons was described in terms of elemental dynamical processes involving different parts of the phospholipid chain on whose motions the hydrogen atoms ride. [ABSTRACT FROM AUTHOR]

Proteus mirabilis is a pathogenic, Gram-negative, rod-shaped bacterium that causes ascending urinary tract infections. Swarming motility, urease production, biofilm formation, and the properties of its lipopolysaccharide (LPS) are all factors that contribute to the virulence of this bacterium. Uniquely, members of the O18 serogroup elaborate LPS molecules capped with O antigen polymers built of pentasaccharide repeats; these repeats are modified with a phosphocholine (ChoP) moiety attached to the proximal sugar of each O unit. Decoration of the LPS with ChoP is an important surface modification of many pathogenic and commensal bacteria. The presence of ChoP on the bacterial envelope is correlated with pathogenicity, as decoration with ChoP plays a role in bacterial adhesion to mucosal surfaces, resistance to antimicrobial peptides and sensitivity to complement-mediated killing in several species. The genome of P. mirabilis O18 is 3.98 Mb in size, containing 3,762 protein-coding sequences and an overall GC content of 38.7%. Annotation performed using the RAST Annotation Server revealed genes associated with choline phosphorylation, uptake and transfer. Moreover, amino acid sequence alignment of the translated licC gene revealed it to be homologous to LicC from Streptococcus pneumoniae encoding CTP:phosphocholine cytidylyltransferase. Recognized homologs are located in the O antigen gene clusters of Proteus species, near the wzx gene encoding the O antigen flippase, which translocates lipid-linked O units across the inner membrane. This study reveals the genes potentially engaged in LPS decoration with ChoP in P. mirabilis O18.

Proteus mirabilis is a pathogenic, Gram-negative, rod-shaped bacterium that causes ascending urinary tract infections. Swarming motility, urease production, biofilm formation, and the properties of its lipopolysaccharide (LPS) are all factors that contribute to the virulence of this bacterium. Uniquely, members of the O18 serogroup elaborate LPS molecules capped with O antigen polymers built of pentasaccharide repeats; these repeats are modified with a phosphocholine (ChoP) moiety attached to the proximal sugar of each O unit. Decoration of the LPS with ChoP is an important surface modification of many pathogenic and commensal bacteria. The presence of ChoP on the bacterial envelope is correlated with pathogenicity, as decoration with ChoP plays a role in bacterial adhesion to mucosal surfaces, resistance to antimicrobial peptides and sensitivity to complement-mediated killing in several species. The genome of P. mirabilis O18 is 3.98 Mb in size, containing 3,762 protein-coding sequences and an overall GC content of 38.7%. Annotation performed using the RAST Annotation Server revealed genes associated with choline phosphorylation, uptake and transfer. Moreover, amino acid sequence alignment of the translated licC gene revealed it to be homologous to LicC from Streptococcus pneumoniae encoding CTP:phosphocholine cytidylyltransferase. Recognized homologs are located in the O antigen gene clusters of Proteus species, near the wzx gene encoding the O antigen flippase, which translocates lipid-linked O units across the inner membrane. This study reveals the genes potentially engaged in LPS decoration with ChoP in P. mirabilis O18. [ABSTRACT FROM AUTHOR]

Abnormal cell membrane metabolism is associated with many neuropsychiatric disorders. Free phosphomonoesters and phosphodiesters, which can be detected by in vivo 31P magnetic resonance spectroscopy (MRS), are important cell membrane building blocks. However, the quantification of phosphoesters has been highly controversial even in healthy individuals due to overlapping signals from macromolecule membrane phospholipids (MP). In this study, high signal-to-noise ratio (SNR) cerebral 31P MRS spectra were acquired from healthy volunteers at both 3 and 7 Tesla. Our results indicated that, with minimal spectral interference from MP, the [phosphocreatine (PCr)]/[phosphocholine (PC) + glycerophosphocholine (GPC)] ratio measured at 7 Tesla agreed with its value expected from biochemical constraints. In contrast, the 3 Tesla [PCr]/[PC+GPC] ratio obtained using standard spectral fitting procedures was markedly smaller than the 7 Tesla ratio and than the expected value. The analysis suggests that the commonly used spectral model for MP may fail to capture its complex spectral features at 3 Tesla, and that additional prior knowledge is necessary to reliably quantify the phosphoester signals at low magnetic field strengths when spectral overlapping is significant. [ABSTRACT FROM AUTHOR]

In this study, we describe the engineering of sub‐100 nm nanomicelles (DTX‐PC NMs) derived from phosphocholine derivative of docetaxel (DTX)‐conjugated lithocholic acid (DTX‐PC) and poly(ethylene glycol)‐tethered lithocholic acid. Administration of DTX‐PC NMs decelerate tumor progression and increase the mice survivability compared to Taxotere (DTX‐TS), the FDA‐approved formulation of DTX. Unlike DTX‐TS, DTX‐PC NMs do not cause any systemic toxicity and slow the decay rate of plasma DTX concentration in rodents and non‐rodent species including non‐human primates. We further demonstrate that DTX‐PC NMs target demethylation of CpG islands of Sparcl1 (a tumor suppressor gene) by suppressing DNA methyltransferase activity and increase the expression of Sparcl1 that leads to tumor regression. Therefore, this unique system has the potential to improve the quality of life in cancer patients and can be translated as a next‐generation chemotherapeutic. [ABSTRACT FROM AUTHOR]

28-O-caffeoyl betulin (B-CA) has been demonstrated to reduce the cerebral infarct volume caused by transient middle cerebral artery occlusion (MCAO) injury. B-CA is a novel derivative of naturally occurring caffeoyl triterpene with little information associated with its pharmacological target(s). To date no data is available regarding the effect of B-CA on brain metabolism. In the present study, a 1H-NMR-based metabolomics approach was applied to investigate the therapeutic effects of B-CA on brain metabolism following MCAO in rats. Global metabolic profiles of the cortex in acute period (9 h after focal ischemia onset) after MCAO were compared between the groups (sham; MCAO + vehicle; MCAO + B-CA). MCAO induced several changes in the ipsilateral cortex of ischemic rats, which consequently led to the neuronal damage featured with the downregulation of NAA, including energy metabolism dysfunctions, oxidative stress, and neurotransmitter metabolism. Treatment with B-CA showed statistically significant rescue effects on the ischemic cortex of MCAO rats. Specifically, treatment with B-CA ameliorated the energy metabolism dysfunctions (back-regulating the levels of succinate, lactate, BCAAs, and carnitine), oxidative stress (upregulating the level of glutathione), and neurotransmitter metabolism disturbances (back-regulating the levels of γ-aminobutyric acid and acetylcholine) associated with the progression of ischemic stroke. With the administration of B-CA, the levels of three phospholipid related metabolites (O-phosphocholine, O-phosphoethanolamine, sn-glycero-3-phosphocholine) and NAA improved significantly. Overall, our findings suggest that treatment with B-CA may provide neuroprotection by augmenting the metabolic changes observed in the cortex following MCAO in rats. [ABSTRACT FROM AUTHOR]

Upregulated choline metabolism, characterized by an increase in phosphocholine (PCho), is a hallmark of oncogenesis and tumor progression. Choline kinase (ChoK), the enzyme responsible for PCho synthesis, has consequently become a promising drug target for cancer therapy and as such a significant number of ChoK inhibitors have been developed over the last few decades. More recently, due to the role of this enzyme in other pathologies, ChoK inhibitors have also been used in new therapeutic approaches against malaria and rheumatoid arthritis. Here, we review research results in the field of ChoKα inhibitors from their synthesis to the molecular basis of their binding mode. Strategies for the development of inhibitors and their selectivity on ChoKα over ChoKβ, the plasticity of the choline‐binding site, the discovery of new exploitable binding sites, and the allosteric properties of this enzyme are highlighted. The outcomes summarized in this review will be a useful guide to develop new multifunctional potent drugs for the treatment of various human diseases. [ABSTRACT FROM AUTHOR]

Flip‐flop movement was investigated via dithionite assay of 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐(7‐nitro‐2‐1,3‐benzoxadiazol‐4‐yl)(ammonium salt) (NBD‐PE) fluorescence in 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine/1,2‐dihexanoyl‐sn‐glycero‐3‐phosphocholine (DMPC/DHPC) vesicles with different DMPC/DHPC ratios. The activation energy was determined by using the Arrhenius plot and the thermodynamic functions were determined by the transition state theory. The activation energy increased upon addition of DHPC into the DMPC bilayer, but all the changes of the thermodynamic functions did not show a similar tendency. The experimental results suggest that the DHPC inserted in DMPC bilayers promote the flip‐flop movement of NBD‐PE in DMPC/DHPC vesicles by disordering the transition state by shorter DHPC molecule. This phenomenon might be related to the increment of the accessibility of water molecules into the ground state of the vesicle bilayer. [ABSTRACT FROM AUTHOR]

Transmembrane anion transport is an important biological process in maintaining cellular functions. Thus, synthetic anion transporters are widely developed for their biological applications. Imidazolinium was introduced as anion recognition site to a multiblock amphiphilic structure that consists of octa(ethylene glycol) and aromatic units. Ion transport assay using halide‐sensitive lucigenin and pH‐sensitive 8‐hydroxypyrene‐1,3,6‐trisulfonate (HPTS) revealed that imidazolinium‐based multiblock amphiphile (IMA) transports anions and showed high selectivity for nitrate, which plays crucial roles in many biological events. Temperature‐dependent ion transport assay using 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) indicated that IMA works as a mobile carrier. 1H NMR titration experiments indicated that the C2 proton of the imidazolinium ring recognizes anions via a (C−H)+⋅⋅⋅X− hydrogen bond. Furthermore, all‐atom molecular dynamics simulations revealed a dynamic feature of IMA within the membranes during ion transportation. [ABSTRACT FROM AUTHOR]

The effect of cationic head-group of ionic liquid on the structure and dynamics of phospholipid bilayer was studied to provide insights into the mechanism of ionic liquid-membrane interaction. The effect was observed using six ionic liquids containing benzimidazolium, imidazolium, pyrrolidinium, piperidinium, ammonium, and morpholinium based amphiphilic cations carrying a dodecyl alkyl chain. Unilamellar and multilamellar vesicles composed of zwitterionic 1-palmitoyl-2-oleoyl- sn - glycero -3-phosphocholine (POPC) were used. Permeability of POPC bilayer was found to have a strong dependence on ionic liquid head-group structure. To probe the structural details of interaction, 31P and 2H based solid-state NMR measurements were performed. The cations differed in terms of their effect on the orientation and disorder in the phosphocholine moiety in lipid head-group as revealed by chemical shift anisotropy of 31P. Cations carrying an unshielded charge like benzimidazolium, imidazolium, and ammonium result in strong reorientation of phosphocholine moiety in lipid head-group. Large sized cations like benzimidazolium and piperidinium result in enhanced lipid chain dynamics as revealed by order parameter calculations of deuterated lipid chains. Relatively polar head-group of morpholinium cation neither impacts the phospholipid head-group nor chain packing. Our results suggest that there exists a direct correlation between ionic liquid head-group induced structural changes in bilayer and their ability to permeabilize/disrupt the membrane and be cytotoxic. [ABSTRACT FROM AUTHOR]

Fine particulate matter (PM 2.5 ) is globally recognized for its adverse implications on human health. Yet, remain limited the individual contribution of particular PM 2.5 components to its toxicity, especially considering regional disparities. Moreover, prevention solutions for PM 2.5 -associated health effects are scarce. In the present study, we comprehensively characterized and compared the primary PM 2.5 constituents and their altered metabolites from two locations: Taiyuan and Guangzhou. Analysis of year-long PM 2.5 samples revealed 84 major components, encompassing organic carbon, elemental carbon, ions, metals, and organic chemicals. PM 2.5 from Taiyuan exhibited higher contamination, associated health risks, dithiothreitol activity, and cytotoxicities than Guangzhou's counterpart. Applying metabolomics, BEAS-2B lung cells exposed to PM 2.5 from both cities were screened for significant alterations. A correlation analysis revealed the metabolites altered by PM 2.5 and the critical toxic PM 2.5 components in both regions. Among the PM 2.5 -down-regulated metabolites, phosphocholine emerged as a promising intervention for PM 2.5 cytotoxicities. Its supplementation effectively attenuated PM 2.5 -induced energy metabolism disorder and cell death via activating fatty acid oxidation and inhibiting Phospho1 expression. The highlighted toxic chemicals displayed combined toxicities, potentially counteracted by phosphocholine. Our study offered a promising functional metabolite to alleviate PM 2.5 -induced cellular disorder and provided insights into the geo-based variability in toxic PM 2.5 components., Competing Interests: Competing interests statement:The authors have patent filings (United States Provisional Patent Title: “Phosphocholine-induced energy source shift alleviates mitochondrial dysfunction in lung cells caused by geo-specific PM2.5 components” Application Number: US63/556,846 Date: 22 Feb 2024).

The aim of this study was to test the hypothesis that C-reactive protein (CRP) protects against the development of atherosclerosis and that a conformational alteration of wild-type CRP is necessary for CRP to do so. Atherosclerosis is an inflammatory cardiovascular disease and CRP is a plasma protein produced by the liver in inflammatory states. The co-localization of CRP and low-density lipoproteins (LDL) at atherosclerotic lesions suggests a possible role of CRP in atherosclerosis. CRP binds to phosphocholine-containing molecules but does not interact with LDL unless the phosphocholine groups in LDL are exposed. However, CRP can bind to LDL, without the exposure of phosphocholine groups, if the native conformation of CRP is altered. Previously, we reported a CRP mutant, F66A/T76Y/E81A, generated by site-directed mutagenesis, that did not bind to phosphocholine. Unexpectedly, this mutant CRP, without any more conformational alteration, was found to bind to atherogenic LDL. We hypothesized that this CRP mutant, unlike wild-type CRP, could be anti-atherosclerotic and, accordingly, the effects of mutant CRP on atherosclerosis in atherosclerosis-prone LDL receptor-deficient mice were evaluated. Administration of mutant CRP into mice every other day for a few weeks slowed the progression of atherosclerosis. The size of atherosclerotic lesions in the aorta of mice treated with mutant CRP for 9 weeks was ~40% smaller than the lesions in the aorta of untreated mice. Thus, mutant CRP conferred protection against atherosclerosis, providing a proof of concept that a local inflammation-induced structural change in wild-type CRP is a prerequisite for CRP to control the development of atherosclerosis.

A monomeric form of C-reactive protein (CRP) which precipitates with cell wall pneumococcal C polysaccharide (CWPS) and retains the ability to reversibly bind to its ligand phosphocholine has been produced through urea-induced dissociation at an optimized concentration of 3 M urea over a 10 weeks period. Dissociated samples were purified via size exclusion chromatography and characterized by western blot, phosphocholine affinity chromatography and CWPS precipitation. Human serum samples from patients with raised CRP levels (>100 mg/L as determined by the clinical laboratory assay) were purified by affinity and size exclusion chromatography and analyzed (n = 40) to determine whether circulating monomeric CRP could be detected ex vivo. All 40 samples tested positive for pentameric CRP via western blot and enzyme linked immunosorbent assay (ELISA) analysis. Monomeric C-reactive protein was also identified in all 40 patient samples tested, with an average level recorded of 1.03 mg/L (SE = ±0.11). Both the in vitro monomeric C-reactive protein and the human serum monomeric protein displayed a molecular weight of approximately 23 kDa, both were recognized by the same anti-CRP monoclonal antibody and both reversibly bound to phosphocholine in a calcium-dependent manner. In common with native pentameric CRP, the in vitro mCRP precipitated with CWPS. These overlapping characteristics suggest that a physiologically relevant, near-native monomeric CRP, which retains the structure and binding properties of native CRP subunits, has been produced through in vitro dissociation of pentameric CRP and also isolated from serum with markedly elevated CRP levels. This provides a clear route toward the in-depth study of the structure and function of physiological monomeric CRP.

Whole wheat flour has a shorter shelf life than refined wheat flour due to off-flavor development. An untargeted liquid chromatography/mass spectrometry (LC/MS) flavoromics approach was applied to identify compounds that negatively impact the flavor liking in whole wheat bread made from aged flours. The chemical profiles of thirteen breads made from aged flours were obtained using LC/MS and modeled by orthogonal partial least squares (OPLS) to predict flavor liking. Top predictive chemical features (negatively correlated) were identified as pinellic acid (9S,12S,13S-trihydroxy-10E-octadecenoic acid), 12,13-dihydroxy-9Z-octadecenoic acid, and 1-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine. The sensory analysis confirmed the three compounds increased the bitterness intensity of the bread samples. The formation of the trihydroxy fatty acid bitter compound, pinellic acid (9S,12S,13S-trihydroxy-10E-octadecenoic acid), was impacted by the lipoxygenase activity of the flour; however, there was no influence on the formation of 12,13-dihydroxy-9Z-octadecenoic acid or 1-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine. Additionally, the concentrations of all bitter compounds were significantly higher in bread made from aged flour versus non-aged flour.

Abstract Background Magnetic resonance spectroscopy (MRS) studies have identified elevated levels of the phospholipid precursor phosphocholine (PC) and phosphoethanolamine (PE) as metabolic hallmarks of cancer. Unusually, however, PC and PE levels are reduced in mutant isocitrate dehydrogenase 1 (IDHmut) gliomas that produce the oncometabolite 2-hydroxyglutarate (2-HG) relative to wild-type IDH1 (IDHwt) gliomas. The goal of this study was to determine the molecular mechanism underlying this unusual metabolic reprogramming in IDHmut gliomas. Methods Steady-state PC and PE were quantified using 31P-MRS. To quantify de novo PC and PE synthesis, we used 13C-MRS and measured flux to 13C-PC and 13C-PE in cells incubated with [1,2-13C]-choline and [1,2-13C]-ethanolamine. The activities of choline kinase (CK) and ethanolamine kinase (EK), the enzymes responsible for PC and PE synthesis, were quantified using 31P-MR-based assays. To interrogate the role of 2-HG, we examined IDHwt cells incubated with 2-HG and, conversely, IDHmut cells treated with the IDHmut inhibitor AGI-5198. To examine the role of hypoxia-inducible factor 1-α (HIF-1α), we silenced HIF-1α using RNA interference. To confirm our findings in vivo and in the clinic, we studied IDHwt and IDHmut orthotopic tumor xenografts and glioma patient biopsies. Results De novo synthesis of PC and PE was reduced in IDHmut cells relative to IDHwt. Concomitantly, CK activity and EK activity were reduced in IDHmut cells. Pharmacological manipulation of 2-HG levels established that 2-HG was responsible for reduced CK activity, EK activity, PC and PE. 2-HG has previously been reported to stabilize levels of HIF-1α, a known regulator of CK activity. Silencing HIF-1α in IDHmut cells restored CK activity, EK activity, PC and PE to IDHwt levels. Our findings were recapitulated in IDHmut orthotopic tumor xenografts and, most importantly, in IDHmut patient biopsies, validating our findings in vivo and in the clinic. Conclusions This study identifies, to our knowledge for the first time, a direct role for 2-HG in the downregulation of CK and EK activity, and thereby, PC and PE synthesis in IDHmut gliomas. These results highlight the unusual reprogramming of phospholipid metabolism in IDHmut gliomas and have implications for the identification of MRS-detectable metabolic biomarkers associated with 2-HG status.

The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTa) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease. [ABSTRACT FROM AUTHOR]

Background Bacterial toxins disrupt plasma membrane integrity with multitudinous effects on host cells. The secreted pore-forming toxin listeriolysin O (LLO) of the intracellular pathogen Listeria monocytogenes promotes egress of the bacteria from vacuolar compartments into the host cytosol often without overt destruction of the infected cell. Intracellular LLO activity is tightly controlled by host factors including compartmental pH, redox, proteolytic, and proteostatic factors, and inhibited by cholesterol. Methods Combining infection studies of L. monocytogenes wild type and isogenic mutants together with biochemical studies with purified phospholipases, we investigate the effect of their enzymatic activities on LLO. Results Here, we show that phosphocholine (ChoP), a reaction product of the phosphatidylcholine-specific phospholipase C (PC-PLC) of L. monocytogenes , is a potent inhibitor of intra- and extracellular LLO activities. Binding of ChoP to LLO is redox-independent and leads to the inhibition of LLO-dependent induction of calcium flux, mitochondrial damage, and apoptosis. ChoP also inhibits the hemolytic activities of the related cholesterol-dependent cytolysins (CDC), pneumolysin and streptolysin. Conclusions Our study uncovers a strategy used by L. monocytogenes to modulate cytotoxic LLO activity through the enzymatic activity of its PC-PLC. This mechanism appears to be widespread and also used by other CDC pore-forming toxin-producing bacteria. [ABSTRACT FROM AUTHOR]

Background: Bacterial toxins disrupt plasma membrane integrity with multitudinous effects on host cells. The secreted pore-forming toxin listeriolysin O (LLO) of the intracellular pathogen Listeria monocytogenes promotes egress of the bacteria from vacuolar compartments into the host cytosol often without overt destruction of the infected cell. Intracellular LLO activity is tightly controlled by host factors including compartmental pH, redox, proteolytic, and proteostatic factors, and inhibited by cholesterol.Methods: Combining infection studies of L. monocytogenes wild type and isogenic mutants together with biochemical studies with purified phospholipases, we investigate the effect of their enzymatic activities on LLO.Results: Here, we show that phosphocholine (ChoP), a reaction product of the phosphatidylcholine-specific phospholipase C (PC-PLC) of L. monocytogenes, is a potent inhibitor of intra- and extracellular LLO activities. Binding of ChoP to LLO is redox-independent and leads to the inhibition of LLO-dependent induction of calcium flux, mitochondrial damage, and apoptosis. ChoP also inhibits the hemolytic activities of the related cholesterol-dependent cytolysins (CDC), pneumolysin and streptolysin.Conclusions: Our study uncovers a strategy used by L. monocytogenes to modulate cytotoxic LLO activity through the enzymatic activity of its PC-PLC. This mechanism appears to be widespread and also used by other CDC pore-forming toxin-producing bacteria. [ABSTRACT FROM AUTHOR]

While most of the articles in this issue review the workings of integral membrane enzymes, in this review, we describe the catalytic mechanism of an enzyme that contains a soluble catalytic domain but appears to catalyze its reaction on the membrane surface, anchored and assisted by a separate regulatory amphipathic helical domain and inter-domain linker. Membrane partitioning of CTP: phosphocholine cytidylyltransferase (CCT), a key regulatory enzyme of phosphatidylcholine metabolism, is regulated chiefly by changes in membrane phospholipid composition, and boosts the enzyme's catalytic efficiency > 200-fold. Catalytic enhancement by membrane binding involves the displacement of an auto-inhibitory helix from the active site entrance-way and promotion of a new conformational ensemble for the inter-domain, allosteric linker that has an active role in the catalytic cycle. We describe the evidence for close contact between membrane lipid, a compact allosteric linker, and the CCT active site, and discuss potential ways that this interaction enhances catalysis. Unlabelled Image • CCT is activated by membrane binding. • CCT seeks negatively charged surfaces and lipid packing voids for insertion of an amphipathic helix. • The amphipathic helix is a silencing device in the CCT soluble form. • Membrane binding facilitates remodeling of a malleable inter-domain allosteric linker. • Linker folding pulls the active site close to the membrane and shields it from solvent. [ABSTRACT FROM AUTHOR]

The aim of this study was to test the hypothesis that C-reactive protein (CRP) protects against the development of atherosclerosis and that a conformational alteration of wild-type CRP is necessary for CRP to do so. Atherosclerosis is an inflammatory cardiovascular disease and CRP is a plasma protein produced by the liver in inflammatory states. The co-localization of CRP and low-density lipoproteins (LDL) at atherosclerotic lesions suggests a possible role of CRP in atherosclerosis. CRP binds to phosphocholine-containing molecules but does not interact with LDL unless the phosphocholine groups in LDL are exposed. However, CRP can bind to LDL, without the exposure of phosphocholine groups, if the native conformation of CRP is altered. Previously, we reported a CRP mutant, F66A/T76Y/E81A, generated by site-directed mutagenesis, that did not bind to phosphocholine. Unexpectedly, this mutant CRP, without any more conformational alteration, was found to bind to atherogenic LDL. We hypothesized that this CRP mutant, unlike wild-type CRP, could be anti-atherosclerotic and, accordingly, the effects of mutant CRP on atherosclerosis in atherosclerosis-prone LDL receptor-deficient mice were evaluated. Administration of mutant CRP into mice every other day for a few weeks slowed the progression of atherosclerosis. The size of atherosclerotic lesions in the aorta of mice treated with mutant CRP for 9 weeks was ~40% smaller than the lesions in the aorta of untreated mice. Thus, mutant CRP conferred protection against atherosclerosis, providing a proof of concept that a local inflammation-induced structural change in wild-type CRP is a prerequisite for CRP to control the development of atherosclerosis. [ABSTRACT FROM AUTHOR]

In this study, the interfacial behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPC/DPPG) mixed monolayers with fluoxetine (FLX) in the subphase was investigated by a combination of the Langmuir-Blodgett technique and atomic force microscopy (AFM). It was found that DPPC/DPPG mixed monolayers showed different interfacial behaviors before and after addition of FLX in the subphase. The electrostatic interaction between FLX and lipids molecules destroys the homogeneity of the mixed monolayers and changes the arrangement of lipids molecules at the interface after addition of FLX in the subphase, thereby leading to an increase of compressibility and miscibility and a decrease in the stability of the mixed monolayers. The surface morphology of the mixed monolayers observed by AFM was different between without and with FLX in the subphase, indicating the penetration of FLX into the mixed monolayers. The present study has provided detailed information for further understanding the interactions of drugs with membrane lipids in other lipid monolayers. [ABSTRACT FROM AUTHOR]

Alkylphospholipids are synthetic analogues of endogenous phosphatidylcholines with a remarkable ability: induce the selective apoptosis of exponentially growing tumor cells. One hypothesis concerning their mechanism of action is the inhibition of cytidine triphosphate:phosphocholine cytidyltransferase (CCT), which would significantly suppress the phosphatidylcholine biosynthesis to trigger apoptosis. Herein, homology modeling, docking simulations, and the analyses of molecular interaction fields are used to suggest the most probable binding modes of four alkylphospholipids (edelfosine, erucylphosphocholine, perifosine, and miltefosine) and lysophosphatidylcholine at the catalytic domain of human CCT. All compounds display bind modes in agreement with the corresponding groups found in the CCT substrate, phosphocholine, while their binding strengths are increased because of the interaction of the alkyl chains with hydrophobic residues from the M domain of the protein. Analyses of the geometry of the CCT binding‐site also suggest that small groups, such as benzyl/2‐phenylethyl ethers or equivalent heterocycles, could replace the O‐methyl group in edelfosine to yield even better inhibitors. It is believed this study can guide the development of new alkylphospholipids with an improved profile for the inhibition of phosphatidylcholine biosynthesis, a critical component for cell cycle progression that can be explored in cancer chemotherapy. Practical Applications: Studies focusing on the interactions between small ligands and their protein targets are decisive for the comprehension of how conformational changes in the macromolecular structure dictates the biological activity and, consequently, how they can be explored in drug discovery. Most of the current studies on alkylphospholipids focuses on their physicochemical interactions with cholesterol and sphingolipids in lipid rafts that, because of the variability and complexity of the membrane phases, hardly can provide structural data in the X‐ray crystallography assays necessary for molecular modeling studies. Therefore, by exploring the inhibition of human cytidine triphosphate:phosphocholine cytidyltransferase as an alternative and, probably, complementary hypothesis to the membrane rafts, a helpful strategy can be provided to overcome the clinical limitations of alkylphospholipids. [ABSTRACT FROM AUTHOR]

Phosphocholine phosphatase-1 (PHOSPHO1) is a phosphocholine phosphatase that catalyzes the hydrolysis of phosphocholine (PC) to choline. Here we demonstrate that the PHOSPHO1 transcript is highly enriched in mature brown adipose tissue (BAT) and is further induced by cold and isoproterenol treatments of BAT and primary brown adipocytes. In defining the functional relevance of PHOPSPHO1 in BAT thermogenesis and energy metabolism, we show that PHOSPHO1 knockout mice are cold-tolerant, with higher expression of thermogenic genes in BAT, and are protected from high-fat dietinduced obesity and development of insulin resistance. Treatment of mice with the PHOSPHO1 substrate phosphocholine is sufficient to induce cold tolerance, thermogenic gene expression, and allied metabolic benefits. Our results reveal a role of PHOSPHO1 as a negative regulator of BAT thermogenesis, and inhibition of PHOSPHO1 or enhancement of phosphocholine represent innovative approaches to manage the metabolic syndrome. [ABSTRACT FROM AUTHOR]

The article explores the enzymatic mechanism of Legionella effector, AnkX and the family of filamentation induced by cyclic adenosine monophosphate (FIC) proteins. It discusses about the causative agent of Legionnaires disease, Legionella pneumophila, translocate the phosphocholine transferase AnkX during infection and thereby post translationally modifies the small guanosine triphosphatase (GTPase) Rab1.

Cancer cell lines are often used for cancer research. However, continuous genetic instability-induced heterogeneity of cell lines can hinder the reproducibility of cancer research. Molecular profiling approaches including transcriptomics, chromatin modification profiling, and proteomics are used to evaluate the phenotypic characteristics of cell lines. However, these do not reflect the metabolic function at the molecular level. Metabolic phenotyping is a powerful tool to profile the biochemical composition of cell lines. In the present study, ¹H-NMR spectroscopy-based metabolic phenotyping was used to detect metabolic differences among five cancer cell lines, namely, lung (A549), colonic (Caco2), brain (H4), renal (RCC), and ovarian (SKOV3) cancer cells. The concentrations of choline, creatine, lactate, alanine, fumarate and succinate varied remarkably among different cell types. The significantly higher intracellular concentrations of glutathione, myo-inositol, and phosphocholine were found in the SKOV3 cell line relative to other cell lines. The concentration of glutamate was higher in both SKOV3 and RCC cells compared with other cell lines. For cell culture media analysis, isopropanol was found to be the highest in RCC media, followed by A549 and SKOV3 media, while acetone was the highest in A549, followed by RCC and SKOV3. These results demonstrated that ¹H-NMR-based metabolic phenotyping approach allows us to characterize specific metabolic signatures of cancer cell lines and provides phenotypical information of cellular metabolism. [ABSTRACT FROM AUTHOR]

The lipid profiles, including lipid classes, phospholipid (PL) classes, fatty acid (FA) composition, and glycerophospholipid (GP) molecular species, in the two edible parts (edible viscera and muscles) of two species of cooked crabs (Portunus trituberculatus and Portunus pelagicus) were systematically characterized for the first time to reveal their nutritional values. For the muscles, PL (66.84–82.23% of total lipids) was the major component of lipids. While for the edible viscera, triacylglycerol (79.04–96.66% of total lipids) was the dominant lipid class. For all lipid samples, eicosapentaenoic acid (EPA) (3.47–22.02% of total FA) and docosahexaenoic acid (DHA) (7.30–16.52% of total FA) were the predominant FAs. Among PL from the crab muscles, phosphocholine (61.51–63.27 mol%) and phosphoethanolamine (21.58–23.43 mol%) were abundant. About 250 GP molecular species belonging to six classes, including glycerophosphocholine, lysoglycerophosphocholine, glycerophosphoethanolamine, lysoglycerophosphoethanolamine, glycerophosphoserine, and lysoglycerophosphoserine, were identified in crab muscles using direct infusion mass spectrometric method. A majority of the dominant molecular species of GP contained omega-3 polyunsaturated fatty acid, especially EPA and DHA. The useful information obtained in this study makes it possible to use crab lipid for producing higher value-added products for commercial exploitation. [ABSTRACT FROM AUTHOR]

Background: Choline is an essential nutrient for brain growth and other processes in the developing neonate. The impact of a maternal plant-based diet on the choline composition of breast milk is unknown.Objective: We assessed the water-soluble choline content of milk from lactating women in the United States following 3 dietary patterns: vegan, vegetarian, and nonvegetarian.Methods: We conducted a cross-sectional study of 74 healthy lactating women who provided a single breast-milk sample using a standardized collection protocol. Participants completed a food-frequency screener and were classified as follows: nonvegetarians (NONVEG) consumed meat; vegetarians (VEGT) consumed milk, dairy, and/or fish; and vegans (VEGAN) consumed animal products less than monthly. Primary outcomes measured were the concentration (in milligrams per liter) and distribution (percentage) of choline from the following water-soluble forms: free choline, phosphocholine (PCho), and glycerophosphocholine (GPC). Differences between diet groups were evaluated with ANOVA.Results: There was a wide range in breast-milk total water-soluble choline (4-301 mg/L), with no significant difference (P > 0.05) by maternal diet pattern. There were differences in choline forms, with VEGAN having a greater mean ± SD concentration and distribution of choline derived from GPC (62.7 ± 25.3 mg/L) than VEGT (47.7 ± 21.2 mg/L) and NONVEG (42.4 ± 14.9 mg/L) (P = 0.0052). There was a lower mean ± SD percentage of choline from PCho (P = 0.0106) in VEGAN (32.5% ± 18.3%) than in VEGT (46.1% ± 18.3%) and NONVEG (44.8% ± 15.7%). Lactation stage and maternal BMI were significantly associated with some choline forms.Conclusions: There was a wide range of water-soluble choline concentrations in the milk of healthy lactating women following vegan, vegetarian, and nonvegetarian diets, with no observed difference in total water-soluble choline concentration by maternal diet. This suggests that maternal plant-based diet by itself is not a risk factor for low breast-milk choline. [ABSTRACT FROM AUTHOR]