Acyl Chain Length Research Articles

Serinc5 Restricts HIV Membrane Fusion by Altering Lipid Order and Heterogeneity in the Viral Membrane.

The host restriction factor, Serinc5, incorporates into budding HIV particles and inhibits their infection by an incompletely understood mechanism. We have previously reported that Serinc5 but not its paralogue, Serinc2, blocks HIV cell entry by membrane fusion, specifically by inhibiting fusion pore formation and dilation. A body of work suggests that Serinc5 may alter the conformation and clustering of the HIV fusion protein, Env. To contribute an additional perspective to the developing model of Serinc5 restriction, we assessed Serinc2 and Serinc5's effects on HIV pseudoviral membranes. By measuring pseudoviral membrane thickness via cryo-electron microscopy and order via the fluorescent dye, FLIPPER-TR, Serinc5 was found to increase membrane heterogeneity, skewing the distribution toward a larger fraction of the viral membrane in an ordered phase. We also directly observed for the first time the coexistence of membrane domains within individual viral membrane envelopes. Using a total internal reflection fluorescence-based single particle fusion assay, we found that treatment of HIV pseudoviral particles with phosphatidylethanolamine (PE) rescued HIV pseudovirus fusion from restriction by Serinc5, which was accompanied by decreased membrane heterogeneity and order. This effect was specific for PE and did not depend on acyl chain length or saturation. Together, these data suggest that Serinc5 alters multiple interrelated properties of the viral membrane─lipid chain order, rigidity, line tension, and lateral pressure─which decrease the accessibility of fusion intermediates and disfavor completion of fusion. These biophysical insights into Serinc5 restriction of HIV infectivity could contribute to the development of novel antivirals that exploit the same weaknesses.

The Road to Quantitative Lipid Biochemistry in Living Cells.

Traditional cell biological techniques are not readily suitable for studying lipid signaling events because genetic perturbations are much slower than the interconversion of lipids in complex metabolic networks. For this reason, novel chemical biological approaches have been developed. One approach is to chemically modify a lipid with a so-called "caging group" that renders it inactive, but this cage can be removed photochemically inside cells to release the bioactive molecule. These caged compounds offer unique advantages for studying the kinetics of cellular biochemistry and have been extensively used in the past. However, a limitation of conventional caged compounds is their ability to diffuse freely inside the cell, which does not permit localized activation below optical precision. This poses a challenge for studying lipid signaling as lipid function inside cells is tightly linked to their parent membrane. An ideal lipid probe should, therefore, be restricted to a single organelle membrane or preferentially to a single leaflet. We first demonstrated the plasma-membrane-specific photorelease of fatty acids by employing sulfonated caging groups. Using these caged fatty acid probes we demonstrated that lipid localization determines signaling outcome. Generalizing this approach, we designed a so-called "click cage" that can be coupled to lipids and offers the possibility to attach organelle targeting groups via click chemistry. Using this strategy, we have synthesized plasma membrane, lysosomal, mitochondria, and endoplasmic-reticulum-targeted lipids that can be used to dissect organelle-specific signaling events. To reduce the synthetic effort associated with generating caged compounds, we designed a coumarin triflate reagent that allows the direct functionalization of phosphate- or carboxylate-containing compounds. With this novel reagent, we synthesized a small library of photocaged G-protein-coupled receptor (GPCR) ligands to study the underlying lipid signaling dynamics. Most recently, we have focused on quantifying the kinetics of lipid signaling for different diacylglycerol (DAG) species using plasma-membrane-targeted caged DAGs. Using this approach, we quantitatively measured lipid-protein affinities and lipid transbilayer dynamics in living cells. After analyzing DAGs with different acyl chain length and saturation degree, we discovered that affinities can vary by up to an order of magnitude. This finding clearly shows that cells are able to distinguish between individual DAG species, thereby demonstrating that lipid diversity matters in cellular signal processing. Although the recent advances have yielded valuable tools to study lipid signaling, challenges remain on specifically targeting the different leaflets of organelle membranes. Furthermore, it is necessary to simplify the experimental approaches required for parametrizing and corroborating quantitative kinetic models of lipid signaling. In the future, we envision that the application of leaflet-specific caged lipids to model membrane systems will be of crucial importance for understanding lipid asymmetry.

Structural variations and phospholipid binding characteristics of Streptomyces klenkii phospholipase D at the lipid-water interface

The lack of knowledge about adsorption characteristics limits the acquisition of phospholipase D (PLD) selectivity information for various phospholipids. In this study, we found that the adsorption equilibrium coefficient (KAds) of Streptomyces klenkii PLD (SkPLD) adsorption onto phosphatidylcholine (PC) monolayer films were inversely proportional to the acyl chain length of PC. SkPLD showed the greatest KAds value of 2.7 × 106 L mol−1 to 12:0/12:0-PC monolayer film, which was associated with the highest adsorption rate constant (ka) value (6.9 × 103 L mol−1 s−1). The high KAds value helped to enhance the specific activity (81.86 U/mg) of SkPLD toward 12:0/12:0-PC. Molecular dynamics simulation revealed that SkPLD was more dynamic in 12:0/12:0-PC, whereas more rigid in other membrane protein systems. When the conformation is too rigid, the enzyme activity will be reduced. Compared with other membrane protein systems, the suitable number of 18 hydrogen bonds between SkPLD and 12:0/12:0-PC monolayer film might contribute to an increase in KAds value, which would modulate the binding affinity of PLD molecules to the lipid-water interface, and it subsequently affected the catalytic activity. This study provided important information on the role of interfacial adsorption prosperities in controlling substrate recognition and enzyme activity of Streptomyces PLDs.

Optimized enzymatic synthesis of (epi)gallocatechin (EGC) monolaurate and the antioxidant evaluation of its ester analogs

The fatty acid ester of catechins such as (epi)gallocatechin (EGC) occur naturally in green tea but reports on their enzymatic synthesis and antioxidant evaluation is scarce. In this study, EGC esters were prepared via a one-step enzymatic transesterification. The main O-acylation products were 8 EGC monoesters consisting of varying acyl groups ranging from 2 to 18 carbon atoms (acetate, butyrate, caproate, caprylate, caprate, laurate, myristate, and stearate). The dimyristate and distearate esters of EGC were also exemplified as minor production during EGC's enzymatic esterification. LC-ToF and NMR HSQC were used to characterize the structures of purified EGC esters. Besides, the antioxidant activity of purified EGC esters were evaluated and systematically compared via several classical chemical (DPPH, ABTS, FRAP, Fe2+ chelation assays), food (β-carotene bleaching assay), and biological (LDL and DNA oxidation assays) methods/models. The lipophilicity of EGC derivatives increased upon O-acylation and was found to positively correlate with the chain length of their acyl groups. The dual effect of acyl chain length on altering EGC monoesters' antioxidant efficacies also corresponded well with previous data of the fatty acid esters of quercetin, gallic acid, EGCG, and resveratrol. Finally, response surface design was used to optimize the reaction condition to achieve the ultimate reaction yield of EGC monolaurate in a laboratory scale. Overall, the study results support that the acylated EGCs can serve as functional/health-beneficial ingredients for use in lipid-based matrices of cosmetics, supplements, and nutraceuticals. The enzymatic synthesis with optimized reaction conditions may be utilized for further pilot-scale manufacturing of similar analogs of EGC monolaurate.

Structures of an unusual 3-hydroxyacyl dehydratase (FabZ) from a ladderane-producing organism with an unexpected substrate preference

The genomes of anaerobic ammonium-oxidizing (anammox) bacteria contain a gene cluster comprising genes of unusual fatty acid biosynthesis enzymes that were suggested to be involved in the synthesis of the unique "ladderane" lipids produced by these organisms. This cluster encodes an acyl carrier protein (denoted as "amxACP") and a variant of FabZ, an ACP-3-hydroxyacyl dehydratase. In this study, we characterize this enzyme, which we call anammox-specific FabZ ("amxFabZ"), to investigate the unresolved biosynthetic pathway of ladderane lipids. We find that amxFabZ displays distinct sequence differences to "canonical" FabZ, such as a bulky, apolar residue on the inside of the substrate binding tunnel, where the canonical enzyme has a glycine. Additionally, substrate screens suggest that amxFabZ efficiently converts substrates with acyl chain lengths of up to eight carbons, whereas longer substrates are converted much more slowly under the conditions used. We also present crystal structures of amxFabZs, mutational studies and the structure of a complex between amxFabZ and amxACP, which show that the structures alone cannot explain the apparent differences from canonical FabZ. Moreover, we find that while amxFabZ does dehydrate substrates bound to amxACP, it does not convert substrates bound to canonical ACP of the same anammox organism. We discuss the possible functional relevance of these observations in the light of proposals for the mechanism for ladderane biosynthesis.

Direct Chiral Supercritical Fluid Chromatography-Mass Spectrometry Analysis of Monoacylglycerol and Diacylglycerol Isomers for the Study of Lipase-Catalyzed Hydrolysis of Triacylglycerols.

The fast and selective separation method of intact monoacylglycerol (MG) and diacylglycerol (DG) isomers using chiral supercritical fluid chromatography-mass spectrometry (SFC-MS) was developed and employed to study lipase selectivity in the hydrolysis of triacylglycerols (TGs). The synthesis of 28 enantiomerically pure MG and DG isomers was performed in the first stage using the most commonly occurring fatty acids in biological samples such as palmitic, stearic, oleic, linoleic, linolenic, arachidonic, and docosahexaenoic acids. To develop the SFC separation method, different chromatographic conditions such as column chemistry, mobile phase composition and gradient, flow rate, backpressure, and temperature were carefully assessed. Our SFC-MS method used a chiral column based on a tris(3,5-dimethylphenylcarbamate) derivative of amylose and neat methanol as a mobile phase modifier, which provides baseline separation of all the tested enantiomers in 5 min. This method was used to evaluate hydrolysis selectivity of lipases from porcine pancreas (PPL) and Pseudomonas fluorescens (PFL) using nine TGs differing in acyl chain length (14-22 carbon atoms) and number of double bonds (0-6) and three DG regioisomer/enantiomers as hydrolysis intermediate products. PFL exhibited preference of the fatty acyl hydrolysis from the sn-1 position of TG more pronounced for the substrates with long polyunsaturated acyls, while PPL did not show considerable stereoselectivity to TGs. Conversely, PPL preferred hydrolysis from the sn-1 position of prochiral sn-1,3-DG regioisomer, whereas PFL exhibited no preference. Both lipases showed selectivity for the hydrolysis of outer positions of DG enantiomers. The results show complex reaction kinetics of lipase-catalyzed hydrolysis given by different stereoselectivities for substrates.

Aortic Regeneration is Promoted by Intermittent Fasting in Aged Rats

Intermittent fasting (IF) plays important role in health. The regeneration that starts at the cellular level is reflected in all tissues and organs. In this study, molecular changes in the aortic tissue of 12-month-old male Wistar rats that underwent intermittent fasting for 18 hours a day for 35 days were determined by spectrochemical analysis and machine learning algoritm. While IF did not significantly affect body weights or blood glucose levels, it led to increased food and water consumption. Spectrochemical analysis revealed significant differences in the forms of DNA, specifically an increase in the A-DNA form in aortic samples. This form of DNA plays an essential role in cellular defense systems and biological processes. There was also an increase in the Amid I band, providing information about hydration status and lipid molecule interactions. Notably, a significant decrease was observed in protein phosphorylation markers, which could impact a wide range of cellular activities. IF also led to reductions in protein carbonylation, a marker of oxidative damage, and changes in the acyl chain length of fatty acids, impacting membrane fluidity. These findings suggest that IF may offer several health benefits, including improved membrane dynamics, reduced oxidative stress, and potential cellular regeneration through autophagy. Further research is needed to confirm these observations and understand their implications for human health.

Hydrolysis and transport characteristics of tyrosol-SCFA esters in rat intestine and blood: Two-step release of tyrosol and SCFAs to enhance the beneficial effects

The models of rat everted gut sac and hydrolysis by rat plasma were used to clarify the hydrolysis and transport characteristics of tyrosol-SCFA esters (TYr-SEs). HPLC-UV results indicated that TYr-SEs could be hydrolyzed by intestinal lipase, which showed sustained release of SCFAs and TYr. Meanwhile, TYr-SEs and the liberated SCFAs and TYr could cross the membrane and were transported into blood circulation. TYr-SEs were further hydrolyzed by carboxylesterase in plasma. Obviously, the hydrolysis of TYr-SEs in blood also showed sustained release of SCFAs and TYr. Especially, the rates of hydrolysis and transport correlated positively with the acyl chain lengths. Besides, the above rates of the TYr-SE with a straight chain were greater than those of its isomer with a branched chain. Therefore, the above-mentioned two-step release of SCFAs and TYr clearly demonstrated that TYr-SEs would be an effective approach to enhance the beneficial health effects of SCFAs and TYr.

A combined X-ray fluorescence and infrared microspectroscopy study for new insights into elemental-biomolecular obesity-induced changes in rat brain structures

The objective of our research was to determine the brain changes at the molecular and elemental levels typical of early-stage obesity. Therefore a combined approach using Fourier transform infrared micro-spectroscopy (FTIR-MS) and synchrotron radiation induced X-ray fluorescence (SRXRF) was introduced to evaluate some brain macromolecular and elemental parameters in high-calorie diet (HCD)- induced obese rats (OB, n = 6) and in their lean counterparts (L, n = 6). A HCD was found to alter the lipid- and protein- related structure and elemental composition of the certain brain areas important for energy homeostasis. The increased lipid unsaturation in the frontal cortex and ventral tegmental area, the increased fatty acyl chain length in the lateral hypothalamus and substantia nigra as well as the decreased both protein α helix to protein β- sheet ratio and the percentage fraction of β-turns and β-sheets in the nucleus accumbens were revealed in the OB group reflecting obesity-related brain biomolecular aberrations. In addition, the certain brain elements including P, K and Ca were found to differentiate the lean and obese groups at the best extent. We can conclude that HCD-induced obesity triggers lipid- and protein- related structural changes as well as elemental redistribution within various brain structures important for energy homeostasis. In addition, an approach applying combined X-ray and infrared spectroscopy was shown to be a reliable tool for identifying elemental-biomolecular rat brain changes for better understanding the interplay between the chemical and structural processes involved in appetite control.

Self-assembled Trehalose Amphiphiles as Molecular Gels: A Unique Formulation to Wax-free Cosmetics.

Trehalose has been used as an emollient and antioxidant in cosmetics. However, we aimed to explore trehalose amphiphiles as oil structuring agents for the preparation of gel-based lip balms as part of wax-free cosmetics. This article describes the synthesis of trehalose fatty acyl amphiphiles and their corresponding oleogel-based lip balms. Trehalose dialkanoates were synthesized by esterifying the two primary hydroxyls of trehalose with fatty acids (C4-C12) using a facile, regioselective lipase catalysis. The gelation potential of as-synthesized amphiphiles was evaluated in organic solvents and vegetable oils. Stable oleogels were subjected to X-ray diffraction (XRD), thermal (DSC), and rheological studies and further used for the preparation of lip balms. Trehalose dioctanoate (Tr8), trehalose didecanoate (Tr10) were found to be super gelators as their minimum gelation concentration is ≤ 0.2 wt%. XRD studies revealed their hexagonal columnar molecular packing while forming the fibrillar networks. Rheometry proved that the fatty acyl chain length of amphiphiles can influence the strength and flow properties of oleogels. Further rheometry (at 25 °C, 37 °C, and 50 °C) and DSC studies have validated that Tr8- and Tr10-based oleogels are stable for commercial applications. Tr8- and Tr10-based olive oil oleogels were used for the preparation of lip balms. The preliminary results suggested that the cumulative effect of trehalose's emolliency and vegetable oil gelling nature can be achieved with trehalose amphiphiles, specifically, Tr8 and Tr10. This study has also demonstrated that Tr8- and Tr10-based lip balms can be used as an alternative to beeswax and plant wax lip balms, indicating their huge potential to succeed as a new paradigm to formulate wax-free cosmetics.

Membrane thickness affects ELIC activity & M4 helix structure.

Pentameric ligand gated ion channels (pLGICs) are a superfamily of ion channels involved in fast neural transmission. Some examples include the GABA(A) receptor and nicotinic acetylcholine receptor (nAchR). In the nAchR, it has been shown that thick membranes promote coupling of agonist binding to channel activation. However, the effect of membrane thickness on the ion transport activity of any pLGIC is not known. To investigate this, we reconstituted the model pLGIC, Erwinia ligand gated ion channel (ELIC), in liposomes consisting of di-monounsaturated phosphatidylcholine (PC) of varying acyl chain length (16-22), and assayed ion flux using a sequential mixing fluorescence quenching assay. ELIC exhibited maximal agonist responses in di-20:1PC liposomes with a dramatic decrease in thinner or thicker membranes. To verify that differences in agonist responses are not a function of changes in channel orientation in the liposomes, we further assayed an ELIC construct with a quenchable fluorescent label in the N-terminal ECD. Minimal differences in the amount of outwardly facing channels were observed in different membrane conditions confirming that changes in agonist responses are primarily a function of channel activity. To investigate the structural basis of ELIC sensitivity to membrane thickness, we obtained apo and agonist-bound structures of ELIC in a circularized nanodisc with di16:1PC, di20:1PC or di22:1PC using single particle cryo-EM. In the agonist-bound structures, the membrane facing helix, M4, is poorly resolved in thin (di16:1PC) and thick (di22:1PC) membranes but clearly resolved in di20:1PC, indicating a correlation between the structure and dynamics of M4 and the optimal membrane thickness that supports channel function. These findings indicate that ELIC function is promoted by thicker membranes and suggests that sensing of membrane thickness is mediated by M4, as has been proposed for the nAchR.

Proteolysis modification targeting protein corona affects ultrasound-induced membrane homeostasis of saccharomyces cerevisiae: Analysis of lipid relative contributions on membrane properties.

Protein corona (PCN) adsorbed on the surface of nanoparticles has brought new research perspectives for the interaction between nanoparticles and microorganisms. In this study, the responses of saccharomyces cerevisiae' membrane lipid composition, the average length of the fatty acyl chains and the average number of unsaturation of fatty acids to ultrasound combined with nano-Fe3O4@PCN with time-limited proteolysis (nano-Fe3O4@TLP-PCN) was investigated. Lipidomic data was obtained using Ultra-high performance liquid chromatography coupled with a Q-Exactive plus mass spectrometer. The membrane potential, proton motive force assay and the membrane lipid oxidation were measured using Di-BAC4(3), DISC3(5) and C11-BODIPY581/591 as the probes. Combined with the approach of feasible virtual samples generation, the back propagation artificial neural network (BP-ANN) model was adopted to establish the mapping relationship between lipids and membrane properties. The time-limited proteolysis targeting wheat PCN-coated Fe3O4 nanoparticles resulted in regular changes of hydrodynamic diameters, ζ-potentials, and surface hydrophobicity. In addition, with the prolongation of PCN proteolysis time, disturbances of 3 S.cerevisiae membrane characteristics, and membrane lipidomic remodeling in response to ultrasound+ nano-Fe3O4@PCN were observed. The analysis of relative importance which followed revealed that ergosterol, phosphatidylserine, and phosphatidylinositol phosphate had the greatest influence on membrane potential. For membrane lipid oxidation, ceramide, phosphatidylethanolamine, and sitosterol ester contribute 16.2, 14.9, and 13.1%, respectively. The relative contributions of six lysolecithins to the dissipation of proton motive force remained limited. An adaptation mechanism of cell membrane to proteolyzed PCN, wherein lipidome remodeling could preserved functional membrane phenotypes was revealed. Furthermore, it is highlighted that the relative importances of SiE, Cer, PE and PIP in determining membrane potential, PMF dissipation and membrane lipid oxidation by establishing FVSG-BP-ANN model.

The Important Role of Membrane Fluidity on the Lytic Mechanism of the α-Pore-Forming Toxin Sticholysin I.

Actinoporins have emerged as archetypal α-pore-forming toxins (PFTs) that promote the formation of pores in membranes upon oligomerization and insertion of an α-helix pore-forming domain in the bilayer. These proteins have been used as active components of immunotoxins, therefore, understanding their lytic mechanism is crucial for developing this and other applications. However, the mechanism of how the biophysical properties of the membrane modulate the properties of pores generated by actinoporins remains unclear. Here we studied the effect of membrane fluidity on the permeabilizing activity of sticholysin I (St I), a toxin that belongs to the actinoporins family of α-PFTs. To modulate membrane fluidity we used vesicles made of an equimolar mixture of phosphatidylcholine (PC) and egg sphingomyelin (eggSM), in which PC contained fatty acids of different acyl chain lengths and degrees of unsaturation. Our detailed single-vesicle analysis revealed that when membrane fluidity is high, most of the vesicles are partially permeabilized in a graded manner. In contrast, more rigid membranes can be either completely permeabilized or not, indicating an all-or-none mechanism. Altogether, our results reveal that St I pores can be heterogeneous in size and stability, and that these properties depend on the fluid state of the lipid bilayer. We propose that membrane fluidity at different regions of cellular membranes is a key factor to modulate the activity of the actinoporins, which has implications for the design of different therapeutic strategies based on their lytic action.

A mini review on the role of phosphatidylcholine metabolism in obesity

Phosphatidylcholine (PC) is one of the major building blocks of biological membranes in mammalian cells. In PC, two fatty acyl chains are linked to the sn-1 and sn-2 positions of the glycerol backbone, and a head group containing a phosphate and choline is linked to the sn-3 position. The length and saturation of the fatty acyl chains of PC vary significantly and determine the biophysical and chemical properties of PC. In mammalian cells, active metabolism of PC takes place primarily through de novo synthesis, the remodelling cycle of deacylation and reacylation, and further modification of lysophosphatidylcholine. PC metabolism undergoes remarkable changes in obesity. In this mini review, we discuss PC metabolism and summarize recent advances about the important involvement of PC metabolism in obesity and obesity-related diseases.

Determinants of substrate specificity in a catalytically diverse family of acyl-ACP thioesterases from plants

BackgroundACYL-LIPID THIOESTERASES (ALTs) are a subclass of plastid-localized, fatty acyl-acyl carrier protein (ACP) thioesterase enzymes from plants. They belong to the single hot dog-fold protein family. ALT enzymes generate medium-chain (C6-C14) and C16 fatty acids, methylketone precursors (β-keto fatty acids), and 3-hydroxy fatty acids when expressed heterologously in E. coli. The diverse substrate chain-length and oxidation state preferences of ALTs set them apart from other plant acyl-ACP thioesterases, and ALTs show promise as metabolic engineering tools to produce high-value medium-chain fatty acids and methylketones in bacterial or plant systems. Here, we used a targeted motif-swapping approach to explore connections between ALT protein sequence and substrate specificity. Guided by comparative motif searches and computational modelling, we exchanged regions of amino acid sequence between ALT-type thioesterases from Arabidopsis thaliana, Medicago truncatula, and Zea mays to create chimeric ALT proteins.ResultsComparing the activity profiles of chimeric ALTs in E. coli to their wild-type counterparts led to the identification of interacting regions within the thioesterase domain that shape substrate specificity and enzyme activity. Notably, the presence of a 31-CQH[G/C]RH-36 motif on the central α-helix was shown to shift chain-length specificity towards 12–14 carbon chains, and to be a core determinant of substrate specificity in ALT-type thioesterases with preference for 12–14 carbon 3-hydroxyacyl- and β-ketoacyl-ACP substrates. For an ALT containing this motif to be functional, an additional 108-KXXA-111 motif and compatible sequence spanning aa77–93 of the surrounding β-sheet must also be present, demonstrating that interactions between residues in these regions of the catalytic domain are critical to thioesterase activity. The behaviour of chimeric enzymes in E. coli also indicated that aa77–93 play a significant role in dictating whether an ALT will prefer ≤10-carbon or ≥ 12-carbon acyl chain-lengths, and aa91–96 influence selectivity for substrates of fully or partially reduced oxidation states. Additionally, aa64–67 on the hot dog-fold β-sheet were shown to be important for enabling an ALT to act on 3-hydroxy fatty acyl-ACP substrates.ConclusionsBy revealing connections between thioesterase sequence and substrate specificity, this study is an advancement towards engineering recombinant ALTs with product profiles suited for specific applications.

Molecular Dynamics of Glycolipids in Liposomes.

Glycosphingolipids (GSLs) in the mammalian plasma membrane are essential for various biological events as they form glycolipid-rich membrane domains, such as lipid rafts. GSLs consist of a certain oligosaccharide head group and a ceramide tail with various lengths of acyl chains. The structure of the head group as well as the carbon number and degree of the unsaturation of the acyl chain are known to regulate the membrane distributions and interleaflet couplings of GSLs by altering physicochemical properties, such as dynamics, interactions, and cluster sizes. This chapter provides the detailed use of time-resolved fluorescence measurement for investigating the membrane properties of lactosylceramide (LacCer)-enriched domains in bilayer membranes. LacCer belongs to the neutral GSLs and is believedin forming a highly ordered phase in model membranes and biological membranes, while the details of the domain remain unclear. Here, we suggest using trans-parinaric acid (tPA) and tPA-LacCer fluorescent probes to reveal the dynamics and size of the GSL domains since they prefer to bedistributed in the GSL-rich ordered phase. The fluorescence lifetime in the nanosecond timescale reveals the difference in the surrounding membrane environments, which relates to hydrocarbon chain ordering, membrane hydration, and submicrometer domain size. The fluorescence lifetime of these probes can thus provide important information on submicron- to nano-scale small GSL domains not only in model membranes but also in biological membranes.

Reducing the endotoxic activity or enhancing the vaccine immunogenicity by altering the length of lipid A acyl chain in Salmonella

The balance of the attenuation and reactogenicity is an issue in the development of recombinant attenuated Salmonella vaccines (RASV). Some reactogenic strains produced side effects are partially induced by lipid A. As reported, the number of lipid A acyl chains influence the strength and outcome of immune responses. However, there is rarely any study to investigate the modifications of acyl chain length on the effect of the toxicity and immunogenicity in Salmonella. In this study, foreign acyltransferase genes lpxA and lpxD were introduced into S. Typhimurium, which produced the S006 (ΔaraBAD::PlppCtlpxAC10) or S007 (ΔproBA::PlppSslpxDC16) strains with C10 or C16 acyl chains respectively. The results showed that the increased polymyxin B susceptibility, reduced swimming and invasion capabilities were observed in the S006. In addition, it also exhibited a lower endotoxicity and colonization ability compared to the parent strain. The result indicated the introduction of C10 acyl chains could be as a candidate choice for lipid A detoxifying strategy in engineering bacteria. However, the longer acyl chain modification didn't obviously change these abilities. Parallelly, these modifications were introduced into a Salmonella vaccine strain to determine their influences on the immune responses against Pneumonia. After inoculation by the strain V003 (ΔaraBAD ΔproBA::PlppSslpxDC16 χ9241), the mice produced robust levels of anti-PspA IgG, and a balanced Th1/Th2 immunity, which resulted in a significant survival improvement of mice with challenging against Streptococcus pneumonia. Therefore, the combination of lipid A modification with C16 acyl chain may be a better strategy for the development of ideal RASVs.

Controlling lipid crystallization across multiple length scales by directed shear flow.

The crystallization behavior of lipids is relevant in many fields such as adipose tissue formation and regeneration, forensic investigations and food production. Using a lipid model system composed of triacylglycerols, we study the formation of crystalline structures under laminar shear flows across various length scales by polarized light-, scanning electron-, and atomic force microscopy, as well as laser diffraction spectroscopy. The shear rate during crystallization γ̇cryst influences the acyl-chain length structure and promotes domain growth into the flow direction thereby transforming the crystallites from oblate into prolate particles. Concentration dependent aggregation of crystallites into clusters is the rate limiting step for floc and floc network formation. At high γ̇cryst, fast crystallite cluster formation at smaller equilibrium diameters is promoted. The high crystallite cluster concentration induces their aggregation into flocs which form weak networks. At low γ̇cryst, floc generation is limited by the low amount of crystallite clusters leading to slow growth of larger flocs and forming of strong networks. The findings in this work have potential implications ranging from the design of injectable soft tissue fillers for adipose tissue regeneration, to the crystalline network formation in microorganism derived lipids, up to a more energy-efficient production of chocolate confectionery.

Early microglial response, myelin deterioration and lethality in mice deficient for very long chain ceramide synthesis in oligodendrocytes.

The sphingolipids galactosylceramide (GalCer), sulfatide (ST) and sphingomyelin (SM) are essential for myelin stability and function. GalCer and ST are synthesized mostly from C22-C24 ceramides, generated by Ceramide Synthase 2 (CerS2). To clarify the requirement for C22-C24 sphingolipid synthesis in myelin biosynthesis and stability, we generated mice lacking CerS2 specifically in myelinating cells (CerS2ΔO/ΔO ). At 6 weeks of age, normal-appearing myelin had formed in CerS2ΔO/ΔO mice, however there was a reduction in myelin thickness and the percentage of myelinated axons. Pronounced loss of C22-C24 sphingolipids in myelin of CerS2ΔO/ΔO mice was compensated by greatly increased levels of C18 sphingolipids. A distinct microglial population expressing high levels of activation and phagocytic markers such as CD64, CD11c, MHC class II, and CD68 was apparent at 6 weeks of age in CerS2ΔO/ΔO mice, and had increased by 10 weeks. Increased staining for denatured myelin basic protein was also apparent in 6-week-old CerS2ΔO/ΔO mice. By 16 weeks, CerS2ΔO/ΔO mice showed pronounced myelin atrophy, motor deficits, and axon beading, a hallmark of axon stress. 90% of CerS2ΔO/ΔO mice died between 16 and 26 weeks of age. This study highlights the importance of sphingolipid acyl chain length for the structural integrity of myelin, demonstrating how a modest reduction in lipid chain length causes exposure of a denatured myelin protein epitope and expansion of phagocytic microglia, followed by axon pathology, myelin degeneration, and motor deficits. Understanding the molecular trigger for microglial activation should aid the development of therapeutics for demyelinating and neurodegenerative diseases.

Does Sulfoquinovosyl Diacylglycerol Synthase OsSQD1 Affect the Composition of Lipids in Rice Phosphate-Deprived Root?

Lipids are the essential components of the cell intracellular and plasma membranes. Sulfoquinovosyldiacylglycerol (SQDG) is a glycolipid; glycolipids can replace phospholipids in maintaining phosphate (Pi) homeostasis in plants which are undergoing Pi starvation. Sulfoquinovosyl diacylglycerol synthase 1 (OsSQD1) is a critical enzyme in the first step of catalyzation in the formation of SQDG in rice. In this study, the expression pattern of different zones in roots of OsSQD1 in response to different Pi conditions is examined, and it is found that OsSQD1 is highly expressed in lateral roots under Pi-sufficient and -deficient conditions. The root phenotype observation of different OsSQD1 transgenic lines suggests that the knockout/down of OsSQD1 inhibits the formation and growth of lateral roots under different Pi conditions. Additionally, the lipid concentrations in OsSQD1 transgenic line roots indicate that OsSQD1 knockout/down decreases the concentration of phospholipids and glycolipids in Pi-starved roots. The OsSQD1 mutation also changes the composition of different lipid species with different acyl chain lengths, mainly under Pi-deprived conditions. The relative transcript expression of genes relating to glycolipid synthesis and phospholipid degradation is estimated to help study the mechanism by which OsSQD1 exerts an influence on the alteration of lipid composition and concentration in Pi-starved roots. Moreover, in Pi-starved roots, the knockout of OsSQD1 decreases the unsaturated fatty acid content of phospholipids and glycolipids. To summarize, the present study demonstrates that OsSQD1 plays a key role in the maintenance of phospholipid and glycolipid composition in Pi-deprived rice roots, which may influence root growth and development under Pi-deprived conditions.