Dynamic Simulations of Interaction of the PEG-DPPE Micelle-Encapsulated Short-Chain Ceramides with the Raft-Included Membrane.

The lipid raft subdomains in cancer cell membranes play a key role in signal transduction, biomolecule recruitment, and drug transmembrane transport. Augmented membrane rigidity due to the formation of a lipid raft is unfavorable for the entry of drugs, a limiting factor in clinical oncology. The short-chain ceramide (CER) has been reported to promote drug entry into membranes and disrupt lipid raft formation, but the underlying mechanism is not well understood. We recently explored the carrier-membrane fusion dynamics of PEG-DPPE micelles in delivering doxorubicin (DOX). Based on the phase-segregated membrane model composed of DPPC/DIPC/CHOL/GM1/PIP2, we aim to explore the dynamic mechanism of the PEG-DPPE micelle-encapsulating DOXs in association with the raft-included cell membrane modulated by C8 acyl tail CERs. The results show that the lipid raft remains integrated and DOX-resistant subjected to free DOXs and the micelle-encapsulating ones. Addition of CERs disorganizes the lipid raft by pushing CHOL aside from DPPC. It subsequently allows for a good permeability for PEG-DPPE micelle-encapsulated DOXs, which penetrate deeper as CER concentration increases. GM1 is significant in guiding drugs' redistributing between bilayer phases, and the anionic PIP2 further helps DOXs attain the inner bilayer surface. These results elaborate on the perturbing effect of CERs on lipid raft stability, which provides a new comprehensive approach for further design of drug delivery systems.

Elucidating Structural Configuration of Lipid Assemblies for mRNA Delivery Systems.

The development of mRNA delivery systems utilizing lipid-based assemblies holds immense potential for precise control of gene expression and targeted therapeutic interventions. Despite advancements in lipid-based gene delivery systems, a critical knowledge gap remains in understanding how the biophysical characteristics of lipid assemblies and mRNA complexes influence these systems. Herein, we investigate the biophysical properties of cationic liposomes and their role in shaping mRNA lipoplexes by comparing various fabrication methods. Notably, an innovative fabrication technique called the liposome under cryo-assembly (LUCA) cycle, involving a precisely controlled freeze-thaw-vortex process, produces distinctive onion-like concentric multilamellar structures in cationic DOTAP/DOPE liposomes, in contrast to a conventional extrusion method that yields unilamellar liposomes. The inclusion of short-chain DHPC lipids further modulates the structure of cationic liposomes, transforming them from multilamellar to unilamellar structures during the LUCA cycle. Furthermore, the biophysical and biological evaluations of mRNA lipoplexes unveil that the optimal N/P charge ratio in the lipoplex can vary depending on the structure of initial cationic liposomes. Cryo-EM structural analysis demonstrates that multilamellar cationic liposomes induce two distinct interlamellar spacings in cationic lipoplexes, emphasizing the significant impact of the liposome structures on the final structure of mRNA lipoplexes. Taken together, our results provide an intriguing insight into the relationship between lipid assembly structures and the biophysical characteristics of the resulting lipoplexes. These relationships may open the door for advancing lipid-based mRNA delivery systems through more streamlined manufacturing processes.

Toxin-triggered liposomes for the controlled release of antibiotics to treat infections associated with the gram-negative bacterium, Aggregatibacter actinomycetemcomitans.

Antibiotic resistance has become an urgent threat to health care in recent years. The use of drug delivery systems provides advantages over conventional administration of antibiotics and can slow the development of antibiotic resistance. In the current study, we developed a toxin-triggered liposomal antibiotic delivery system, in which the drug release is enabled by the leukotoxin (LtxA) produced by the Gram-negative pathogen, Aggregatibacter actinomycetemcomitans. LtxA has previously been shown to mediate membrane disruption by promoting a lipid phase change in nonlamellar lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (N-methyl-DOPE). In addition, LtxA has been observed to bind strongly and nearly irreversibly to membranes containing large amounts of cholesterol. Here, we designed a liposomal delivery system composed of N-methyl-DOPE and cholesterol to take advantage of these interactions. Specifically, we hypothesized that liposomes composed of N-methyl-DOPE and cholesterol, encapsulating antibiotics, would be sensitive to LtxA, enabling controlled antibiotic release. We observed that liposomes composed of N-methyl-DOPE were sensitive to the presence of low concentrations of LtxA, and cholesterol increased the extent and kinetics of content release. The liposomes were stable under various storage conditions for at least 7 days. Finally, we showed that antibiotic release occurs selectively in the presence of an LtxA-producing strain of A. actinomycetemcomitans but not in the presence of a non-LtxA-expressing strain. Together, these results demonstrate that the designed liposomal vehicle enables toxin-triggered delivery of antibiotics to LtxA-producing strains of A. actinomycetemcomitans.

Stabilization of all-natural water-in-oil high internal phase pickering emulsion by using diosgenin/soybean phosphatidylethanolamine complex: Characterization and application in 3D printing.

This study aimed to prepare an all-natural water-in-oil high internal phase Pickering emulsion (W/O-HIPPE) using diosgenin/soybean phosphatidylethanolamine complex (DGSP) and investigate the 3D printing performance. Results suggested that the self-assembly of diosgenin crystal was modified by SP in DGSP (diosgenin-SP ratios at 3:1 and 1:1), revealing a variation from large-size outward radiating needle-like to small-size granular-like shape, which facilitated closely packing at the interface. Hydrophilicity of DGSP was also increased (contact angle varying from 133.3 o to 106.4 o), ensuring more adequate interfacial adsorption to reduce interfacial tension more largely (6.5 mN/m). Thus, the W/O-HIPPE made by DGSP with diosgenin-SP = 1:1, exhibited smaller droplets and better freeze/thawing stability. The W/O-HIPPE was also measured improved rheological properties for 3D printing: satisfied shear-thinning behavior, higher recovery and self-supporting (viscoelasticity and deformation resistance). Consequently, the W/O-HIPPE allowed for printing more delicate patterns. This work provided guidance to prepare W/O-HIPPE for 3D printing.

Maribacter halichondriae sp. nov., isolated from the marine sponge Halichondria panicea, displays features of a sponge-associated life style.

A new member of the family Flavobacteriaceae (termed Hal144T) was isolated from the marine breadcrumb sponge Halichondria panicea. Sponge material was collected in 2018 at Schilksee which is located in the Kiel Fjord (Baltic Sea, Germany). Phylogenetic analysis of the full-length Hal144T 16S rRNA gene sequence revealed similarities from 94.3 to 96.6% to the nearest type strains of the genus Maribacter. The phylogenetic tree of the 16S rRNA gene sequences depicted a cluster of strain Hal144T with its closest relatives Maribacter aestuarii GY20T (96.6%) and Maribacter thermophilus HT7-2T (96.3%). Genome phylogeny showed that Maribacter halichondriae Hal144T branched from a cluster consisting of Maribacter arenosus, Maribacter luteus, and Maribacter polysiphoniae. Genome comparisons of strain Maribacter halichondriae Hal144T with Maribacter sp. type strains exhibited average nucleotide identities in the range of 75-76% and digital DNA-DNA hybridisation values in the range of 13.1-13.4%. Compared to the next related type strains, strain Hal144T revealed unique genomic features such as phosphoenolpyruvate-dependent phosphotransferase system pathway, serine-glyoxylate cycle, lipid A 3-O-deacylase, 3-hexulose-6-phosphate synthase, enrichment of pseudogenes and of genes involved in cell wall and envelope biogenesis, indicating an adaptation to the host. Strain Hal144T was determined to be Gram-negative, mesophilic, strictly aerobic, flexirubin positive, resistant to aminoglycoside antibiotics, and able to utilize N-acetyl-ß-D-glucosamine. Optimal growth occurred at 25-30 °C, within a salinity range of 2-6% sea salt, and a pH range between 5 and 8. The major fatty acids identified were C17:0 3-OH, iso-C15:0, and iso-C15:1 G. The DNA G + C content of strain Hal144T was 41.4 mol%. Based on the polyphasic approach, strain Hal144T represents a novel species of the genus Maribacter, and we propose the name Maribacter halichondriae sp. nov. The type strain is Hal144T (= DSM 114563T = LMG 32744T).

Molecular dynamics simulations support a preference of cyclotide kalata B1 for phosphatidylethanolamine phospholipids.

Kalata B1 (kB1), a naturally occurring cyclotide has been shown experimentally to bind lipid membranes that contain phosphatidylethanolamine (PE) phospholipids. Here, molecular dynamics simulations were used to explore its interaction with two phospholipids, palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), and a heterogeneous membrane comprising POPC/POPE (90:10), to understand the basis for the selectivity of kB1 towards PE phospholipids. The simulations showed that in the presence of only 10 % POPE lipid, kB1 forms a stable binding complex with membrane bilayers. An ionic interaction between the E7 carboxylate group of kB1 and the ammonium group of PE headgroups consistently initiates binding of kB1 to the membrane. Additionally, stable noncovalent interactions such as hydrogen bonding (E7, T8, V10, G11, T13 and N15), cation-π (W23), and CH-π (W23) interactions between specific residues of kB1 and the lipid membrane play an important role in stabilizing the binding. These findings are consistent with a structure-activity relationship study on kB1 where lysine mutagenesis on the bioactive and hydrophobic faces of the peptide abolished membrane-dependent bioactivities. In summary, our simulations suggest the importance of residue E7 (in the bioactive face) in enabling kB1 to recognize and bind selectively to PE-containing phospholipids bilayers through ionic and hydrogen bonding interactions, and of W23 (in the hydrophobic face) for the association and insertion of kB1 into the lipid bilayer through cation-π and CH-π interactions. Overall, this work enhances our understanding of the molecular basis of the membrane binding and bioactivity of this prototypic cyclotide.

Poly(N-methyl-N-vinylacetamide): A Strong Alternative to PEG for Lipid-Based Nanocarriers Delivering siRNA.

Lipid-based nanocarriers have demonstrated high interest in delivering genetic material, exemplified by the success of Onpattro and COVID-19 vaccines. While PEGylation imparts stealth properties, it hampers cellular uptake and endosomal escape, and may trigger adverse reactions like accelerated blood clearance (ABC) and hypersensitivity reactions (HSR). This work highlights the great potential of amphiphilic poly(N-methyl-N-vinylacetamide) (PNMVA) derivatives as alternatives to lipid-PEG for siRNA delivery. PNMVA compounds with different degrees of polymerization and hydrophobic segments, are synthesized. Among them, DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)-PNMVA efficiently integrates into lipoplexes and LNP membranes and prevents protein corona formation around these lipid carriers, exhibiting stealth properties comparable to DSPE-PEG. However, unlike DSPE-PEG, DSPE-PNMVA24 shows no adverse impact on lipoplexes cell uptake and endosomal escape. In in vivo study with mice, DSPE-PNMVA24 lipoplexes demonstrate no liver accumulation, indicating good stealth properties, extended circulation time after a second dose, reduced immunological reaction, and no systemic pro-inflammatory response. Safety of DSPE-PNMVA24 is confirmed at the cellular level and in animal models of zebrafish and mice. Overall, DSPE-PNMVA is an advantageous substitute to DSPE-PEG for siRNA delivery, offering comparable stealth and toxicity properties while improving efficacy of the lipid-based carriers by minimizing the dilemma effect and reducing immunological reactions, meaning no ABC or HSR effects.

Structural analysis of the NK-lysin-derived peptide NK-2 upon interaction with bacterial membrane mimetics consisting of phosphatidylethanolamine and phosphatidylglycerol.

NK-2 is an antimicrobial peptide derived from helices 3 and 4 of the pore-forming protein of natural killer cells, NK-lysin. It has potent activities against Gram-negative and Gram-positive bacteria, fungi and protozoan parasites without being toxic to healthy human cells. In biophysical assays its membrane activities were found to require phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), lipids which dominate the composition of bacterial membranes. Here the structure and activities of NK-2 in binary mixtures of different PE/PG composition were investigated. CD spectroscopy reveals that a threshold concentration of 50 % PG is needed for efficient membrane association of NK-2 concomitant with a random coil - helix transition. Association with PE occurs but is qualitatively different when compared to PG membranes. Oriented solid-state NMR spectroscopy of NK-2 specifically labelled with 15N indicates that the NK-2 helices are oriented parallel to the PG bilayer surface. Upon reduction of the PG content to 20 mol% interactions are weaker and/or an in average more tilted orientation is observed. Fluorescence spectroscopy of differently labelled lipids is in agreement of an interfacial localisation of both helices where the C-terminal end is in a less hydrophobic environment. By inserting into the membrane interface and interacting differently with PE and PG the peptides probably induce high curvature strain which result in membrane openings and rupture.

Effects of a N-Maleimide-derivatized Phosphatidylethanolamine on the Architecture and Properties of Lipid Bilayers.

N-maleimide-derivatized phospholipids are often used to facilitate protein anchoring to membranes. In autophagy studies, this is applied to the covalent binding of Atg8, an autophagy protein, to a phosphatidylethanolamine (PE) in the nascent autophagosome. However, the question remains on how closely the N-maleimide PE derivative (PE-mal) mimicks the native PE in the bilayer. In the present paper, spectroscopic and calorimetric techniques have been applied to vesicles containing either PE or PE-mal (together with other phospholipids) to compare the properties of the native and derivatized forms of PE. According to differential scanning calorimetry, and to infrared spectroscopy, the presence of PE-mal did not perturb the fatty acyl chains in the bilayer. Fluorescence spectroscopy and microscopy showed that PE-mal did not alter the bilayer permeability either. However, fluorescence emission polarization of the Laurdan and DPH probes indicated an increased order, or decreased fluidity, in the bilayers containing PE-mal. In addition, the infrared spectral data from the phospholipid phosphate region revealed a PE-mal-induced conformational change in the polar heads, accompanied by increased hydration. Globally considered, the results suggest that PE-mal would be a reasonable substitute for PE in model membranes containing reconstituted proteins.

Robiginitalea aurantiaca sp. nov. and Algoriphagus sediminis sp. nov., isolated from coastal sediment.

Two novel rod-shaped, Gram-stain-negative, aerobic and non-motile bacterial strains, designated M39T and C2-7T, were isolated from the coastal sediment of Xiaoshi Island, Weihai, PR China. Growth of strain M39T occurred at 15-37 °C, at pH 6.0-9.0 and in the presence of 1.0-9.0 % (w/v) NaCl. Strain C2-7T grew at 15-40 °C, at pH 6.0-8.0 and in the presence of 0.5-8.0 % (w/v) NaCl. Phylogenetic analysis based 16S rRNA gene sequences revealed that strains M39T and C2-7T belong to the phylum Bacteroidota. Based on the results of 16S rRNA gene sequence analysis, the closest relative of strain M39T was Robiginitalea marina KCTC 92035T (95.4 %), and the closest relative of strain C2-7T was Algoriphagus namhaensis DPG-3T (97.0 %). The percentage of conserved protein and average nucleotide identity values between strain M39T and some species of the genus Robiginitalea were 66.9-77.6% and 69.3-71.0 %, respectively, while those between strain C2-7T and some species of the genus Algoriphagus were 68.0-70.1% and 56.1-72.6 %, respectively. The major cellular fatty acids (>10 %) of strain M39T consisted of iso-C15 : 1 F, iso-C15 : 0 and iso-C17 : 0 3-OH, while those of strain C2-7T were iso-C15 : 0 and C16 : 1 ω7c/C16 : 1 ω6c. MK-6 was the only respiratory quinone that was compatible with the genus of strain M39T. The predominant menaquinone of strain C2-7T was MK-7. The major polar lipids of strain M39T were phosphatidylethanolamine and glycolipids, and those of strain C2-7T were phosphatidylethanolamine, one unidentified aminolipid and four unidentified lipids. The DNA G+C contents of strains M39T and C2-7T were 46.9 and 40.8 mol%, respectively. Based upon the results presented in this study, strains M39T and C2-7T represent novel species of the genera Robiginitalea and Algoriphagus, respectively, for which the names Robiginitalea aurantiaca sp. nov. and Algoriphagus sediminis sp. nov. are proposed with the type strains M39T (=MCCC 1H00498T=KCTC 92014T) and C2-7T (=MCCC 1H00414T=KCTC 92027T).

Membrane regulation of 15LOX-1/PEBP1 complex prompts the generation of ferroptotic signals, oxygenated PEs.

Ferroptosis is a regulated form of cell death, the mechanism of which is still to be understood. 15-lipoxygenase (15LOX) complex with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) catalyzes the generation of pro-ferroptotic cell death signals, hydroperoxy-polyunsaturated PE. We focused on gaining new insights into the molecular basis of these pro-ferroptotic interactions using computational modeling and liquid chromatography-mass spectrometry experiments. Simulations of 15LOX-1/PEBP1 complex dynamics and interactions with lipids revealed that association with the membrane triggers a conformational change in the complex. This conformational change facilitates the access of stearoyl/arachidonoyl-PE (SAPE) substrates to the catalytic site. Furthermore, the binding of SAPE promotes tight interactions within the complex and induces further conformational changes that facilitate the oxidation reaction. The reaction yields two hydroperoxides as products, 15-HpETE-PE and 12-HpETE-PE, at a ratio of 5:1. A significant effect of PEBP1 is observed only on the predominant product. Moreover, combined experiments and simulations consistently demonstrate the significance of PEBP1 P112E mutation in generating ferroptotic cell death signals.

A rhein-huprine hybrid protects erythrocyte membrane integrity against Alzheimer's disease related Aß(1-42) peptide.

Alzheimer's disease remains largely unknown, and currently there is no complete cure for the disease. New synthetic approaches have been developed to create multi-target agents, such as RHE-HUP, a rhein-huprine hybrid which can modulate several biological targets that are relevant to the development of the disease. While RHE-HUP has shown in vitro and in vivo beneficial effects, the molecular mechanisms by which it exerts its protective effect on cell membranes have not been fully clarified. To better understand RHE-HUP interactions with cell membranes, we used synthetic membrane models and natural models of human membranes. For this purpose, human erythrocytes and molecular model of its membrane built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were used. The latter correspond to classes of phospholipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively. X-ray diffraction and differential scanning calorimetry (DSC) results indicated that RHE-HUP was able to interact mainly with DMPC. In addition, scanning electron microscopy (SEM) analysis showed that RHE-HUP modified the normal biconcave shape of erythrocytes inducing the formation of echinocytes. Moreover, the protective effect of RHE-HUP against the disruptive effect of Aß(1-42) on the studied membrane models was tested. X-ray diffraction experiments showed that RHE-HUP induced a recovery in the ordering of DMPC multilayers after the disruptive effect of Aß(1-42), confirming the protective role of the hybrid.

PE, or not PE, that is the question: The case of overlooked lyso-N-acylphosphatidylethanolamines.

RATIONALE: Lyso derivatives of N-acyl-1,2-diacylglycero-3-phosphoethanolamines (L-NAPEs) are a lipid class mostly expressed in vegetables during stress and tissue damage that is involved in the synthesis of the lipid mediator N-acylethanolamines. L-NAPEs can be challenging to distinguish from isomeric phosphatidylethanolamines (PEs), especially in extracted complex samples where they could be confused with abundant PEs. METHODS: In this study, hydrophilic interaction liquid chromatography with electrospray ionization hyphenated with (tandem) mass spectrometry (MS) was proposed to distinguish L-NAPEs and PEs as deprotonated molecules, [M - H]─ , using both high-resolution/accuracy Fourier transform MS and low-resolution linear ion trap (LIT) mass analyzers. MS3 experiments of [M - H - KE]─ as precursor ions (KE, ketene loss) using the LIT instrument allowed us to distinguish between isomeric L-NAPE and PE species. RESULTS: Regiochemical rules were proposed working on enzymatically synthesized L-NAPEs. A few key differences in MS/MS spectra, including abnormal intensity of acyl chain losses as fatty acids, the presence of N-acylphosphoethanolamine ions, and diagnostic ions of the polar head, were disclosed. Additionally, MS3 spectra of [M - H - KE]─ as precursor ions allowed us to confirm the identification of L-NAPE species. The proposed rules were applied to samples extracted from tomato by-products including stems and leaves. CONCLUSIONS: Overall, our methodology is demonstrated as a robust approach to recognizing L-NAPEs in complex samples. L-NAPEs 18:2-N-18:2, 18:2-N-18:3, 18:3-N-18:2, and 18:2-N-18:1 were the prevailing compounds in the analyzed tomato samples, accounting for more than 90%. In summary, a reliable method for identifying L-NAPEs in complex samples is described. The proposed method could prevent overlooking L-NAPEs and overestimating isomeric PE species in future lipid analyses.

The effect of methylated phosphatidylethanolamine derivatives on the ionization properties of signaling phosphatidic acid.

Phosphatidylethanolamine (PE) and Phosphatidylcholine (PC) are the most abundant glycerophospholipids in eukaryotic membranes. The differences in the physicochemical properties of their headgroups have contrasting modulatory effects on their interaction with intracellular macromolecules. As such, their overall impact on membrane structure and function differs significantly. Enzymatic methylation of PE's amine headgroup produces two methylated derivatives namely monomethyl PE (MMPE) and dimethyl PE (DMPE) which have physicochemical properties that generally range between that of PE and PC. Additionally, their influence on membrane properties differs from both PE and PC. Although variations in headgroup methylation have been reported to affect signaling pathways, the direct influence that these differences exert on the ionization properties of signaling phospholipids have not been investigated. Here, we briefly review membrane function and structure that are mediated by the differences in headgroup methylation between PE, MMPE, DMPE and PC. In addition, using 31P MAS NMR, we investigate the effect of these four phospholipids on the ionization properties of the ubiquitous signaling anionic lipid phosphatidic acid (PA). Our results show that PA's ionization properties are differentially affected by changes in phospholipid headgroup methylation. This could have important implications for PA-protein binding and hence physiological functions in cells where signaling events lead to changes in abundance of methylated PE derivatives in the membrane.

Unusual Robustness of Neurotransmitter Vesicle Membranes against Serotonin-Induced Perturbations.

Nature confines hundreds of millimolar of amphiphilic neurotransmitters, such as serotonin, in synaptic vesicles. This appears to be a puzzle, as the mechanical properties of lipid bilayer membranes of individual major polar lipid constituents of synaptic vesicles [phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS)] are significantly affected by serotonin, sometimes even at few millimolar concentrations. These properties are measured by atomic force microscopy, and their results are corroborated by molecular dynamics simulations. Complementary 2H solid-state NMR measurements also show that the lipid acyl chain order parameters are strongly affected by serotonin. The resolution of the puzzle lies in the remarkably different properties displayed by the mixture of these lipids, at molar ratios mimicking those of natural vesicles (PC:PE:PS:Cholesterol = 3:5:2:5). Bilayers constituting of these lipids are minimally perturbed by serotonin, and show only a graded response at physiological concentrations (>100 mM). Significantly, the cholesterol (up to 33% molar ratio) plays only a minor role in dictating these mechanical perturbations, with PC:PE:PS:Cholesterol = 3:5:2:5 and 3:5:2:0 showing similar perturbations. We infer that nature uses an emergent mechanical property of a specific mixture of lipids, all individually vulnerable to serotonin, to appropriately respond to physiological serotonin levels.

Temperature and oxidation-sensitive dioleoylphosphatidylethanolamine liposome stabilized with poly(ethyleneimine)/(phenylthio)acetic acid ion pair.

Temperature and oxidation-sensitive liposomes were prepared by stabilizing dioleoylphosphatidylethanolamine (DOPE) bilayers with the ion pair of poly(ethyleneimine)/(phenylthio)acetic acid (PEI/PTA). An upper critical solution temperature (UCST) behavior was observed when PEI/PTA ion pair was suspended in an aqueous solution. It was observed that the UCST increased with increasing PTA content. The ion pair was self-assembled into nanospheres owing to its amphiphilic property which was confirmed by transmission electron microscopy. The FT-IR spectroscopic spectrum showed that the ion pair formed a salt bridge between the amino group and the carboxyl group and the PTA content in the ion pair was readily oxidized by H2O2. Further, DOPE liposomal membranes could be stabilized with PEI/PTA ion pair. Due to the amphiphilic property, the ion pair played a role as a stabilizer for the formation of DOPE liposomes. The liposome released its payload in a temperature-responsive manner, possibly because when the temperature is raised, the ion pair loses its amphiphilic property and can be detached from the liposomal membrane. The liposome was also oxidation-responsive in terms of release, possibly because the amphiphilic property of the ion pair disappears when the PTA is oxidized.

Radiometric Assay of NAPE-PLD Activity.

N-Acyl-phosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD) is a prominent enzyme involved in the biosynthesis of fatty acid amides, a family of bioactive lipids including anandamide as the prototypical member. Here, we describe a NAPE-PLD assay based on radioactive substrates and product separation by thin layer chromatography (TLC).

Liposome Formulation for Tumor-Targeted Drug Delivery Using Radiation Therapy.

Targeted delivery of drugs or other therapeutic agents through internal or external triggers has been used to control and accelerate the release from liposomal carriers in a number of studies, but relatively few utilize energy of therapeutic X-rays as a trigger. We have synthesized liposomes that are triggered by ionizing radiation (RTLs) to release their therapeutic payload. These liposomes are composed of natural egg phosphatidylethanolamine (PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000), and the mean size of the RTL was in the range of 114 to 133 nm, as measured by nanoparticle tracking analysis (NTA). The trigger mechanism is the organic halogen, chloral hydrate, which is known to generate free protons upon exposure to ionizing radiation. Once protons are liberated, a drop in internal pH of the liposome promotes destabilization of the lipid bilayer and escape of the liposomal contents. In proof of principle studies, we assessed RTL radiation-release of fluorescent tracers upon exposure to a low pH extracellular environment or exposure to X-ray irradiation. Biodistribution imaging before and after irradiation demonstrated a preferential uptake and release of the liposomes and their cargo at the site of local tumor irradiation. Finally, a potent metabolite of the commonly used chemotherapy irinotecan, SN-38, was loaded into RTL along with near infrared (NIR) fluorescent dyes for imaging studies and measuring tumor cell cytotoxicity alone or combined with radiation exposure, in vitro and in vivo. Fully loaded RTLs were found to increase tumor cell killing with radiation in vitro and enhance tumor growth delay in vivo after three IV injections combined with three, 5 Gy local tumor radiation exposures compared to either treatment modality alone.

Parachryseolinea silvisoli gen. nov., sp. nov., isolated from forest soil, and reclassification of Chryseolinea flava as Pseudochryseolinea flava gen. nov., comb. nov.

A lemon-chiffon strain, designated QH1ED-6-2T, was isolated from a soil sample collected from Qinghai Virgin Forests, Qinghai Province, PR China. The strain was Gram-stain-negative, aerobic, rod-shaped and motile by gliding. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain QH1ED-6-2T belongs to the family Fulvivirgaceae, and has the highest similarity values of 93.6-92.0 % to Ohtaekwangia koreensis CCUG 58939T, Ohtaekwangia kribbensis CCUG 58938T, Chryseolinea flava SDU1-6T and Chryseolinea serpens DSM 24574T, respectively. The major cellular fatty acids included iso-C15 : 0, C16 : 1 ω5c, iso-C17 : 0 3-OH and summed feature 3. The major polar lipid was phosphatidylethanolamine. The predominant respiratory quinone was menaquinone-7. The average amino acid identity values and percentages of conserved proteins between QH1ED-6-2T and its closely related genera were 66.4-69.6 % and 58.9-64.9 %, respectively, which are interspersed in the intra-genera cutoff values. The digital DNA-DNA hybridization values were 17.6-19.2 %. The draft genome size of strain QH1ED-6-2T was 7.98 Mbp with a DNA G+C content of 51.4 mol%. Based on phenotypic, chemotaxonomic, phylogenetic data, genomic DNA G+C content, as well as AAI, POCP and dDDH results, strain QH1ED-6-2T represents a novel species of a new genus in the family Fulvivirgaceae, for which the name Parachryseolinea silvisoli sp. nov. is proposed. The type strain is QH1ED-6-2T (=GDMCC 1.2318T=JCM 35041T). We also propose the reclassification of Chryseolinea flava as Pseudochryseolinea flava gen. nov., comb. nov. (type strain SDU1-6T=CGMCC 1.13492T=JCM 32520T).

Modelling Hyperglycaemia in an Epithelial Membrane Model: Biophysical Characterisation.

Biomimetic models are valuable platforms to improve our knowledge on the molecular mechanisms governing membrane-driven processes in (patho)physiological conditions, including membrane permeability, transport, and fusion. However, current membrane models are over simplistic and do not include the membrane's lipid remodelling in response to extracellular stimuli. Our study describes the synthesis of glycated dimyristoyl-phosphatidylethanolamine (DMPE-glyc), which was structurally characterised by mass spectrometry (ESI-MS) and quantified by NMR spectroscopy to be further incorporated in a complex phospholipid (PL) membrane model enriched in cholesterol (Chol) and (glyco)sphingolipids (GSL) designed to mimic epithelial membranes (PL/Chol/GSL) under hyperglycaemia conditions. Characterisation of synthesised DMPE-glyc adducts by tandem mass spectrometry (ESI-MS/MS) show that synthetic DMPE-glyc adducts correspond to Amadori products and quantification by 1H NMR spectroscopy show that the yield of glycation reaction was 8%. The biophysical characterisation of the epithelial membrane model shows that excess glucose alters the thermotropic behaviour and fluidity of epithelial membrane models likely to impact permeability of solutes. The epithelial membrane models developed to mimic normo- and hyperglycaemic scenarios are the basis to investigate (poly)phenol-lipid and drug-membrane interactions crucial in nutrition, pharmaceutics, structural biochemistry, and medicinal chemistry.