Investigating the influence of molybdenum disulfide quantum dots coated with DSPE-PEG-TPP on molecular structures of liver lipids and proteins: An in vivo study.

Molybdenum disulfide (MoS2) quantum dots (QDs) based therapeutic approaches hold great promise for biomedical applications, necessitating a thorough evaluation of their potential effects on biological systems. In this study, we systematically investigated the impact of MoS2 QDs coated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000](DPSE-PEG) linked with (3-carboxypropyl)triphenyl-phosphonium-bromide (TPP) on molecular structures of hepatic tissue lipids and proteins through a multifaceted analysis. The DSPE-PEG-TPP-MoS2 QDs were prepared and administered to the mice daily for 7 weeks. Liver tissues were subjected to a comprehensive examination using various techniques, including Fourier-transform infrared (FTIR) spectroscopy, UV-vis spectroscopy, and liver function tests. FTIR revealed subtle changes in the lipid composition of liver tissues, indicating potential modifications in the cell membrane structure. Also, the (CH stretching and amides I and II regions) analysis unveiled tiny alterations in lipid chain length and fluidity without changes in the protein structures, suggesting a minor influence of DSPE-PEG-TPP-MoS2 QDs on the liver's cellular membrane and no effect on the protein structures. Further scrutiny using UV-vis spectroscopy demonstrated that DSPE-PEG-TPP-MoS2 QDs had no discernible impact on the absorbance intensities of aromatic amino acids and the Soret band. This observation implies that the treatment with SPE-PEG-TPP-MoS2 QDs did not induce significant alterations in helical conformation or the microenvironment surrounding prosthetic groups in liver tissues. The liver function tests, including ALP, ALT, AST, and BIL levels, revealed no statistically significant changes in these key biomarkers despite minor fluctuations in their values, indicating a lack of significant liver dysfunction. This study provides a detailed understanding of the effects of DSPE-PEG-TPP-MoS2 QDs on hepatic lipids and proteins, offering valuable insights into the biocompatibility and limited impact on the molecular and functional aspects of the liver tissue. These findings could be essential for the application of MoS2 QDs-based therapies.

Hyphenation of Liquid Chromatography and Trapped Ion Mobility - Mass Spectrometry for Characterization of Isomeric Phosphatidylethanolamines with Focus on N-Acylated Species.

In prior research, hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) has demonstrated applicability for characterizing regioisomers in lipidomics studies, including phosphatidylglycerols (PG) and bis(monoacyl)glycerophosphates (BMP). However, there are other lipid regioisomers, such as phosphatidylethanolamines (PE) and lyso-N-acyl-PE (LNAPE), that have not been studied as extensively. Therefore, hyphenated mass spectrometric methods are needed to investigate PE and LNAPE regioisomers individually. The asymmetric structure of LNAPE favors isomeric species, which can result in coelution and chimeric MS/MS spectra. One way to address the challenge of chimeric MS/MS spectra is through mobility-resolved fragmentation using trapped ion mobility spectrometry (TIMS). Therefore, we developed a multidimensional HILIC-TIMS-MS/MS approach for the structural characterization of isomeric phosphatidylethanolamines in both negative and positive ionization modes. The study revealed the complementary fragmentation pattern and ion mobility behavior of LNAPE in both ionization modes, which was confirmed by a self-synthesized LNAPE standard. With this knowledge, a distinction of regioisomeric PE and LNAPE was achieved in human plasma samples. Furthermore, regioisomeric LNAPE species containing at least one unsaturated fatty acid were noted to exhibit a change in collision cross-section in positive ionization mode, leading to a lipid characterization with respect to fatty acyl positional level. Similar mobility behavior was also observed for the biological LNAPE precursor N-acyl-PE (NAPE). Application of this approach to plasma and cereal samples demonstrated its effectiveness in regioisomeric LNAPE and NAPE species' elucidation.

Optical and molecular features of negatively curved surfaces created by POPE lipids: A crucial role of the initial conditions.

Membrane fusion is closely related to plasma membrane domains rich in cone-shaped phosphatidylethanolamine (PE) lipids that can reverse membrane curvature under certain conditions. The phase transition of PE-based lipid membranes from the lamellar fluid phase (Lα) to the inverse hexagonal phase (HII) is commonly taken as a general model in reconstructing the membrane fusion pathway, and whose structural features have been mostly described so far using structural and microscopic techniques. The aim of this paper is to decipher the optical and molecular features of Lߠ→ Lα and especially of Lα â†’ HII transition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) lipids at pH = 7.0 when they are initially prepared in the form of both multi- and unilamellar liposomes (MLVs and LUVs). The distinction between optical properties of MLS- and LUVs-derived HII phase, provided from turbidity-sensitive temperature-dependent UV-Vis spectra, was attributed to different formation mechanisms of HII phase. Most importantly, from FTIR spectroscopic data of POPE lipids in Lß (15 °C), Lα (50 °C) and HII (85 °C) phases we identified the changes in molecular features of POPE lipids during phase transitions. Among the latter, by far the most significant is different hydration pattern of POPE lipids in MLVs- and LUVs-derived HII phase which extends from the polar-apolar interface all the way to the terminal amino group of the POPE lipid, along with the changes in the conformation of glycerol backbone as evidenced by the signature of α-methylene groups. Molecular dynamics simulations confirmed higher water penetration in HII phase and provided insight into hydrogen bonding patterns.

Effect of cardiolipin on the lamellarity and elongation of liposomes hydrated in PBS.

Lamellarity and shape are important factors in the formation of vesicles and determine their role in biological systems and pharmaceutical applications. Cardiolipin (CL) is a major lipid in many biological membranes and exerts a great influence on their structural organization due to its particular structure and physico-chemical properties. Here, we used small-angle X-ray and neutron scattering to study the effects of CL with different acyl chain lengths and saturations (CL14:0, CL18:1, CL18:2) on vesicle morphology and lamellarity in membrane models containing mixtures of phosphatidylcholine and phosphatidylethanolamine with different acyl chain lengths and saturations (C14:0 and C 18:1). Measurements were performed in the presence of Phosphate Buffer Saline (PBS), at 37°C, to better reflect physiological conditions, which resulted in strong effects on vesicle morphology, depending on the type and amount of CL used. The presence of small quantities of CL (from 2.5%) reduced inter-membrane correlations and increased perturbation of the membrane, an effect which is enhanced in the presence of matched shorter saturated acyl chains, and mainly unilamellar vesicles (ULV) are formed. In extruded vesicles, employed for SANS experiments, flattened vesicles are observed partly due to the hypertonic effect of PBS, but also influenced by the type of CL added. Our experimental data from SAXS and SANS revealed a strong dependence on CL content in shaping the membrane microstructure, with an apparent optimum in the PC:CL mixture in terms of promoting reduced correlations, preferred curvature and elongation. However, the use of PBS caused distinct differences from previously published studies in water in terms of vesicle shape, and highlights the need to investigate vesicle formation under physiological conditions in order to be able to draw conclusions about membrane formation in biological systems.

Different lateral packing stress in acyl chains alters KcsA orientation and structure in lipid membranes.

The molecular structures of the various intrinsic lipids in membranes regulate lipid-protein interactions. These different lipid structures with unique volumes produce different lipid molecular packing stresses/lateral stresses in lipid membranes. Most studies examining lipid packing effects have used phosphatidylcholine and phosphatidylethanolamine (PE), which are the main phospholipids of eukaryotic cell membranes. In contrast, Gram-negative or Gram-positive bacterial membranes are composed primarily of phosphatidylglycerol (PG) and PE, and the physical and thermodynamic properties of each acyl chain in PG at the molecular level remain unresolved. In this study, we used 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG, 16:0-18:1 PG) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (PAPG, 16:0-20:4 PG) to prepare lipid bilayers (liposome) with the rod-type fluorescence probe DPH. We measured the lipid packing conditions by determining the rotational freedom of DPH in POPG or PAPG bilayers. Furthermore, we investigated the effect of different monoacyl chains on a K+ channel (KcsA) structure when embedded in POPG or PAPG membranes. The results revealed that differences in the number of double bonds and carbon chain length in the monoacyl chain at sn-2 affected the physicochemical properties of the membrane and the structure and orientation of KcsA.

Structural basis of lipid head group entry to the Kennedy pathway by FLVCR1.

Phosphatidylcholine and phosphatidylethanolamine, the two most abundant phospholipids in mammalian cells, are synthesized de novo by the Kennedy pathway from choline and ethanolamine, respectively1-6. Despite the essential roles of these lipids, the mechanisms that enable the cellular uptake of choline and ethanolamine remain unknown. Here we show that the protein encoded by FLVCR1, whose mutation leads to the neurodegenerative syndrome posterior column ataxia and retinitis pigmentosa7-9, transports extracellular choline and ethanolamine into cells for phosphorylation by downstream kinases to initiate the Kennedy pathway. Structures of FLVCR1 in the presence of choline and ethanolamine reveal that both metabolites bind to a common binding site comprising aromatic and polar residues. Despite binding to a common site, FLVCR1 interacts in different ways with the larger quaternary amine of choline in and with the primary amine of ethanolamine. Structure-guided mutagenesis identified residues that are crucial for the transport of ethanolamine, but dispensable for choline transport, enabling functional separation of the entry points into the two branches of the Kennedy pathway. Altogether, these studies reveal how FLVCR1 is a high-affinity metabolite transporter that serves as the common origin for phospholipid biosynthesis by two branches of the Kennedy pathway.

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.

Structure and Activity of Reconstructed Pseudo-Ancestral Cyclotides.

Cyclotides are cyclic peptides that are promising scaffolds for the design of drug candidates and chemical tools. However, despite there being hundreds of reported cyclotides, drug design studies have commonly focussed on a select few prototypic examples. Here, we explored whether ancestral sequence reconstruction could be used to generate new cyclotides for further optimization. We show that the reconstructed 'pseudo-ancestral' sequences, named Ancy-m (for the ancestral cyclotide of the Möbius sub-family) and Ancy-b (for the bracelet sub-family), have well-defined structures like their extant members, comprising the core structural feature of a cyclic cystine knot. This motif underpins efforts to re-engineer cyclotides for agrochemical and therapeutic applications. We further show that the reconstructed sequences are resistant to temperatures approaching boiling, bind to phosphatidyl-ethanolamine lipid bilayers at micromolar affinity, and inhibit the growth of insect cells at inhibitory concentrations in the micromolar range. Interestingly, the Ancy-b cyclotide had a higher oxidative folding yield than its comparator cyclotide cyO2, which belongs to the bracelet cyclotide subfamily known to be notoriously difficult to fold. Overall, this study provides new cyclotide sequences not yet found naturally that could be valuable starting points for the understanding of cyclotide evolution and for further optimization as drug leads.

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.

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).

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.

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.

Cryo-EM structure of human sphingomyelin synthase and its mechanistic implications for sphingomyelin synthesis.

Sphingomyelin (SM) has key roles in modulating mammalian membrane properties and serves as an important pool for bioactive molecules. SM biosynthesis is mediated by the sphingomyelin synthase (SMS) family, comprising SMS1, SMS2 and SMS-related (SMSr) members. Although SMS1 and SMS2 exhibit SMS activity, SMSr possesses ceramide phosphoethanolamine synthase activity. Here we determined the cryo-electron microscopic structures of human SMSr in complexes with ceramide, diacylglycerol/phosphoethanolamine and ceramide/phosphoethanolamine (CPE). The structures revealed a hexameric arrangement with a reaction chamber located between the transmembrane helices. Within this structure, a catalytic pentad E-H/D-H-D was identified, situated at the interface between the lipophilic and hydrophilic segments of the reaction chamber. Additionally, the study unveiled the two-step synthesis process catalyzed by SMSr, involving PE-PLC (phosphatidylethanolamine-phospholipase C) hydrolysis and the subsequent transfer of the phosphoethanolamine moiety to ceramide. This research provides insights into the catalytic mechanism of SMSr and expands our understanding of sphingolipid metabolism.

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.

Cell-penetrating peptide and cationic liposomes mediated siRNA delivery to arrest growth of chronic myeloid leukemia cells in vitro.

Gene silencing through RNA interference (RNAi) is a promising therapeutic approach for a wide range of disorders, including cancer. Non-viral gene therapy, using specific siRNAs against BCR-ABL1, can be a supportive or alternative measure to traditional chronic myeloid leukemia (CML) tyrosine kinase inhibitor (TKIs) therapies, given the prevalence of clinical TKI resistance. The main challenge for such approaches remains the development of the effective delivery system for siRNA tailored to the specific disease model. The purpose of this study was to examine and compare the efficiency of endosomolytic cell penetrating peptide (CPP) EB1 and PEG2000-decorated cationic liposomes composed of polycationic lipid 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2Ð¥3) and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) for anti-bcr-abl siRNA delivery into the K562 human CML cell line. We show that both EB1 and 2Ð¥3-DOPE-DSPE-PEG2000 (0.62 % mol.) liposomes effectively deliver siRNA into K562 cells by endocytic mechanisms, and the use of liposomes leads to more effective inhibition of expression of the targeted gene (BCR-ABL1) and cancer cell proliferation. Taken together, these findings suggest that PEG-decorated cationic liposomes mediated siRNA delivery allows an effective antisense suppression of certain oncogenes, and represents a promising new class of therapies for CML.

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.