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This research develops diacylglycerol (DAG) based Pickering emulsions with enhanced oxidative stability stabilized by self-assembled quercetin/DAG/β-cyclodextrin (β-CD) complexes (QDCCs) using a one-step agitation method. Influence of DAG content (5%, 15%, 40%, and 80%, w/w) on the self-assembly behavior, interfacial properties, and emulsifying ability of complex particles was investigated. SEM, XRD and ATR-FTIR studies confirmed the formation of ternary composite particles. QDCCs in 80% DAG oil had the highest quercetin encapsulation efficiency (6.09 ± 0.01%), highest DPPH radical scavenging rate and ferric reducing antioxidant property (FRAP). β-CD and quercetin adsorption rates in emulsion with 80% DAG oil were 88.4 ± 2.53% and 98.34 ± 0.15%, respectively. Pickering emulsions with 80% DAG had the smallest droplet size (8.90 ± 1.87 μm) and excellent oxidation stability. This research develops a novel approach to regulate the physicochemical stability of DAG-based emulsions by anchoring natural antioxidants at the oil-water interface through a one-pot self-assembly method., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Diacylglycerol (DAG) is generally considered one of the precursors of 3-chloropropanol esters (3-MCPDE) and glycidyl esters (GEs). This study aimed to evaluate static heating and stir-frying properties of peanut oil (PO) and PO based 58% and 82% DAG oils (PDAG-58 and PDAG-82). Observations revealed that, phytonutrient levels notably diminished during static heating, with PDAG exhibiting reduced oxidative stability, but maintaining a stability profile similar to PO over a short period. During stir-frying, 3-MCPDE content initially increased and then decreased whereas the opposite was observed for GEs. Furthermore, as temperature, and NaCl concentration increased, there was a corresponding increase in the levels of 3-MCPDE and GEs, although remained within safe limits. When used in suitable concentrations, these findings underscore the potential of DAG, as a nutritionally rich and oxidatively stable alternative to conventional cooking oils, promoting the use of DAG edible oil in heat-cooked food systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The composition and morphology of lipid-based nanoparticles can influence their overall in vivo behavior. Previously, we demonstrated that phase separation in liposomes composed of DSPC and a diacylglycerol lipid analogue (DOaG) drives the in vivo biodistribution towards a specific subset of endothelial cells in zebrafish embryos. In the absence of traditional targeting functionalities ( e.g. , antibodies, ligands), this selectivity is mediated solely by the unique liposome morphology and composition, characterized by a DOaG-rich lipid droplet within the DSPC-rich phospholipid bilayer. The phase separation is induced due to the geometry of DOaG lipid and its ability to create non-bilayer phases in lipid membranes. To investigate the underlying principles of phase separation and to optimize the liposome colloidal stability, we performed a structure-function relationship study by synthesizing a library of DOaG analogues with varying molecular properties, such as the number, length and sn -position of the acyl chains, as well as the degree of saturation or carbonyl substituents. We assessed the ability of these lipid analogues to assemble into phase-separated liposomes and studied their morphology, colloidal stability, and in vivo biodistribution in zebrafish embryos. We found that analogues containing unsaturated, medium length (C16-C18) fatty acids were required to obtain colloidally stable, phase-separated liposomes with cell-specific biodistribution patterns. Moreover, we observed that using the pure DOaG isomer, with acyl chains at the sn -1,3 positions, leads to more colloidally stable liposomes than when a mixture of sn -1,2 and sn -1,3 isomers is used. Similarly, we observed that incorporating a DOaG analogue with fatty tails shorter than DSPC, as well as PEGylation, endows liposomes with long term stability while retaining cell-selective biodistribution. Diacylglycerols are known to promote fusion, lipid polymorphism, signaling and protein recruitment on lipid membranes. In this study, we showed that diacylglycerol derivatives can induce phase separation in liposomes, unlocking the potential for cell-specific targeting in vivo . We believe that these findings can be the foundation for future use of diacylglycerols in lipid-based nanomedicines and could lead to the development of novel targeted delivery strategies.

Pickering foams have great potential for applications in aerated foods, but their foaming ability and physical stability are still far from satisfactory. Herein, solid lipid particles (SLNs) were fabricated by using diacylglycerol of varying acyl chain lengths with modification by a protein. The SLNs showed different crystal polymorphisms and air-water interfacial activity. C14-DAG SLN with a contact angle ∼ 79° formed aqueous foam with supreme stability and high plasticity. Whey protein isolate and sodium caseinate (0.1 wt %) considerably enhanced the foamability and interfacial activity of SLNs and promoted the packing of particles at the bubble surface. However, high protein concentration caused foam destruction due to the competitive adsorption effect. β-sheet increased in protein after adsorption and changed the polymorphism and thermodynamic properties of SLN. The foam collapsing behaviors varied in the presence of protein. The results gave insights into fabricating ultrastable aqueous foams by using high-melting DAG particles. The obtained foams demonstrated good temperature sensitivity and plasticity, which showed promising application prospects in the food and cosmetic fields.

In the glycerolysis process for diacylglycerol (DAG) preparation, free lipases suffer from poor stability and the inability to be reused. To address this, a cost-effective immobilized lipase preparation was developed by cross-linking macroporous resin with poly (ethylene glycol) diglycidyl ether (PEGDGE) followed by lipase adsorption. The selected immobilization conditions were identified as pH 7.0, 35 °C, cross-linking agent concentration 2.0%, cross-linking time 4 h, lipase amount 5 mg/g of support, and adsorption time 4 h. Enzymatic properties of the immobilized lipase were analyzed, revealing enhanced pH stability, thermal stability, storage stability, and operational stability post-immobilization. The conditions for immobilized enzyme-catalyzed glycerolysis to produce DAG were selected, demonstrating the broad applicability of the immobilized lipase. The immobilized lipase catalyzed glycerolysis reactions using various oils as substrates, with DAG content in the products ranging between 35 and 45%, demonstrating broad applicability. Additionally, the changes during the repeated use of the immobilized lipase were characterized, showing that mechanical damage, lipase leakage, and alterations in the secondary structure of the lipase protein contributed to the decline in catalytic activity over time. These findings provide valuable insights for the industrial application of lipase.

This study examined the impacts of ultrasonic power (0, 150, 300, 450, 600, and 750 W) and ultrasonic durations (3, 6, 9, 12, and 15 min) on the physicochemical properties and microstructure of diacylglycerol (DAG)-loaded emulsions stabilized with soybean protein isolate (SPI) and sodium alginate (SA). The findings indicated that the smallest particle size, zeta potential, and contact angle for SPI-SA-DAG emulsions were respectively 5.58 μm, -49.85 mV, and 48.65°, achieved at an ultrasonic power of 450 W. The emulsification properties, loss modulus, storage modulus, and apparent viscosity of the emulsions were optimal at this power setting and at a duration of 9 min. Analytical techniques, including confocal laser scanning-, scanning electron-, and atomic force microscopy, revealed that ultrasonication significantly altered emulsion aggregation state, with the surface roughness (Rq) being minimized at 450 W. These results demonstrated that the stability of SPI-SA-DAG emulsions can be effectively enhanced by an appropriate ultrasonic treatment at 450 W for 9 min. This research provides theoretical support for the broad application of sonication techniques in the food industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Currently, the poor whipping capabilities of anhydrous milk fat (AMF) in aerated emulsion products are a major obstacle for their use in beverages like tea and coffee, as well as in cakes and desserts, presenting fresh hurdles for the food industry. In this study, the mechanism of action of diacylglycerols (DAGs) with different carbon chain lengths and degrees of saturation on the partial coalescence of aerated emulsions was systematically investigated from three fundamental perspectives: fat crystallization, air-liquid interface rheology, and fat globule interface properties. The optimized crystallization of long carbon chain length diacylglycerol (LCD) based on stearate enhances interactions between fat globules at the air-liquid interface (with an elastic modulus E' reaching 246.42 mN/m), leading to a significantly reduced interface membrane strength. This promotes fat crystal-membrane interactions during whipping, resulting in a thermally stable foam structure with excellent shaping capability due to enhanced partial coalescence of fat globules. Although Laurate based medium carbon chain length diacylglycerol (MCD) promoted fat crystallization and optimized interface properties, it showed weaker foam properties because it did not adequately encapsulate air bubbles during whipping. Conversely, oleate long carbon chain length diacylglycerol (OCD) proved to be ineffective in facilitating fat crystal-membrane interaction, causing foam to have a subpar appearance. Hence, drawing from the carefully examined fat crystal-membrane interaction findings, a proposed mechanism sheds light on how DAGs impact the whipping abilities of aerated emulsions. This mechanism serves as a blueprint for creating aerated emulsions with superior whipping capabilities and foam systems that are resistant to heat., Competing Interests: Declaration of competing interest The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript., (Copyright © 2024. Published by Elsevier Ltd.)

Diacylglycerol (DAG) has garnered attention for its safe and nutritious qualities, and its utilization in emulsion systems to encapsulate hydrophobic bioactives is anticipated to enhance their bioaccessibility. Thus, this study aimed to evaluate the influence of DAG oil as a carrier on the stability and digestive characteristics of nanostructured lipid carriers (NLCs) containing lycopene (LYC). The results indicated that DAG oil demonstrated superior storage and heating stability in comparison to triacylglycerol (TAG) oil. Furthermore, NLCs formulated with DAG oil exhibited a faster rate of lipolysis (>76.3%) and higher loading capacity (1.48%), resulting in an approximate 11% enhancement in the bioaccessibility of LYC (reaching up to 31.4%). DAG oils show considerable potential for enhancing and prolonging the properties and bioactivity of NLC carriers, thereby boosting bioaccessibility. The incorporation of DAG oil in food systems holds promise for enriching their functionality over traditional TAG oil., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Bing Shao reports financial support was provided by Guangdong Provincial Key R&D Programme. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Phospholipase C (PLC) is a key enzyme that regulates physiological processes via lipid and calcium signaling. Despite advances in protein engineering, no tools are available for direct PLC control. Here, we developed a novel optogenetic tool, light-controlled PLCβ (opto-PLCβ). Opto-PLCβ uses a light-induced dimer module, which directs an engineered PLC to the plasma membrane in a light-dependent manner. Our design includes an autoinhibitory capacity, ensuring stringent control over PLC activity. Opto-PLCβ triggers reversible calcium responses and lipid dynamics in a restricted region, allowing precise spatiotemporal control of PLC signaling. Using our system, we discovered that phospholipase D-mediated phosphatidic acid contributes to diacylglycerol clearance on the plasma membrane. Moreover, we extended its applicability in vivo, demonstrating that opto-PLCβ can enhance amygdala synaptic plasticity and associative fear learning in mice. Thus, opto-PLCβ offers precise spatiotemporal control, enabling comprehensive investigation of PLC-mediated signaling pathways, lipid dynamics, and their physiological consequences in vivo., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Lipid-based drug delivery systems offer the potential to enhance bioavailability, reduce dosing frequency, and improve patient adherence. In aqueous environment, initially dry lipid depots take up water and form liquid crystalline phases. Variation of lipid composition, depot size and hydration-induced phase transitions will plausibly affect the diffusion in and out of the depot. Lipid depots of soybean phosphatidylcholine (SPC) and glycerol dioleate (GDO) mixtures were hydrated for varying time durations in a phosphate-buffered saline (PBS) buffer and then analyzed with Karl Fischer titration, magnetic resonance imaging (MRI) and gravimetrically. Mathematical modeling of the swelling process using diffusion equations, was used to estimate the parameters of diffusion. Both composition of lipid mixture and depot size affect swelling kinetics… The diffusion parameters obtained in Karl Fischer titration and MRI (with temporal and spatial resolution respectively) are in good agreement. Remarkably, the MRI results show a gradient of water content within the depot even after the end of diffusion process. Apparently contradicting the first Fick's law in its classical form, these results find an explanation using the generalized Fick's law that considers the gradient of chemical potential rather than concentration as the driving force of diffusion., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jenni Engstedt reports financial support was provided by Knowledge Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Toward human immunodeficiency virus type-1 (HIV-1) cure, cells latently infected with HIV-1 must be eliminated from people living with HIV-1. We previously developed a protein kinase C (PKC) activator, diacylglycerol (DAG)-lactone derivative 3 , with high HIV-1 latency-reversing activity, based on YSE028 ( 2 ) as a lead compound and found that the activity was correlated with binding affinity for PKC and stability against esterase-mediated hydrolysis. Here, we synthesized new DAG-lactone derivatives not only containing a tertiary ester group or an isoxazole surrogate but also several symmetric alkylidene moieties to improve HIV-1 latency reversing activity. Compound 9a , with a dimethyl group at the α-position of the ester group, exerted twice higher HIV-1 latency reversing activity than compound 3 , and compound 26 , with the isoxazole moiety, was significantly active. In addition, DAG-lactone derivatives with moderate hydrophobicity and potent biostability showed high biological activity.

Torreya grandis wax (TGW), a new nut wax and by-product of refined Torreya grandis oil, lacks sufficient research and application. In this study, the gelling behavior in diacylglycerol (DAG) and chemical compositions of TGW were investigated. Compared with four typical natural waxes, TGW exhibited the lowest critical gelling concentration (C g , 1 %wt) in DAG. The results performed that TGW-DAG oleogels at C g possessed the highest G' LVR and G″, highest critical stress, good thermal stability, moderate viscosity recovery, and osc. yields stress, indicating strong gel. The microstructure and correlation analysis revealed that excellent gelling behaviors of TGW-DAG oleogels were due to the solid three-dimensional network formed by rod-like TGW crystal, and the higher hydrocarbon compound (HC) content and HC/wax ester in TGW. Formulation optimization suggested that oleogel containing 3.2 % TGW and 1.0 % diosgenin (DSG) better mimicked the characteristics of shortening in terms of hardness, adhesiveness, spreadability. The bread prepared with TGW/DSG-DAG oleogel owned uniform and dense pores, the best moisture retention capability, and soft and firm taste, demonstrating that TGW/DSG-DAG oleogel was a good shortening substitute. Therefore, this study provides the systematically fundamental knowledge of TGW and develops DSG-TGW-DAG oleogels as promising shortening substitutions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The process of protein transport across membranes involves a variety of factors and has been extensively investigated. Traditionally, proteinaceous translocons and chaperones have been recognized as crucial factors in this process. However, recent studies have highlighted the significant roles played by lipids and a glycolipid present in biological membranes in membrane protein transport. Membrane lipids can influence transport efficiency by altering the physicochemical properties of membranes. Notably, our studies have revealed that diacylglycerol (DAG) attenuates mobility in the membrane core region, leading to a dramatic suppression of membrane protein integration. Conversely, a glycolipid in Escherichia coli inner membranes, named membrane protein integrase (MPIase), enhances integration not only through the alteration of membrane properties but also via direct interactions with membrane proteins. This review explores the mechanisms of membrane protein integration mediated by membrane lipids, specifically DAG, and MPIase. Our results, along with the employed physicochemical analysis methods such as fluorescence measurements, nuclear magnetic resonance, surface plasmon resonance, and docking simulation, are presented to elucidate these mechanisms., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Mono- and diglycerides play a crucial role in the food industry as multifunctional food additives and emulsifiers. Their importance stems from their unique properties, which allow them to improve the quality, texture, and stability of various food products. Here, results of the kinetic modeling of the mono- and diglycerides synthesis mediated by the lipase Lipozyme® TL 100 L immobilized on the clayey support Spectrogel® type C are reported. The support was characterized by TEM, SEM, and FTIR. Firstly, the influence of pH and lipase load on the immobilization process was analyzed, resulting in an enzymatic activity of 93.2 ± 0.7 U g -1 under optimized conditions (170.9 U g -1 of lipase and pH of 7.1). Afterward, the effects of reaction temperature and concentration of immobilized biocatalyst in the feedstock conversion were evaluated. At optimized parameters, a triglycerides conversion of 97% was obtained at 36.5 °C, 7.9 vol.% of enzyme, a glycerol to feedstock molar ratio of 2:1, and 2 h. The optimized conditions were used to determine the kinetic constants of the elementary reactions involved in the glycerolysis, where a fit superior to 0.99 was achieved between experimental values and predicted data., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Regulation of membrane protein integration involves molecular devices such as Sec-translocons or the insertase YidC. We have identified an integration-promoting factor in the inner membrane of Escherichia coli called membrane protein integrase (MPIase). Structural analysis revealed that, despite its enzyme-like name, MPIase is a glycolipid with a long glycan comprising N-acetyl amino sugars, a pyrophosphate linker, and a diacylglycerol (DAG) anchor. Additionally, we found that DAG, a minor membrane component, blocks spontaneous integration. In this review, we demonstrate how they contribute to Sec-independent membrane protein integration in bacteria using a comprehensive approach including synthetic chemistry and biophysical analyses. DAG blocks unfavorable spontaneous integrations by suppressing mobility in the membrane core, whereas MPIase compensates for this. Moreover, MPIase plays critical roles in capturing a substrate protein to prevent its aggregation, attracting it to the membrane surface, facilitating its insertion into the membrane, and delivering it to other factors. The combination of DAG and MPIase efficiently regulates the integration of membrane proteins.

Diacylglycerols (DAG) of varying chain lengths were synthesized and the acyl migrated samples with different 1,3-DAG/1,2-DAG ratios were obtained. The crystallization profile and surface adsorption differed depending on DAG structure. C12 and C14 DAGs formed small platelet- and needle-like crystals at the oil-air interface which can better reduce surface tension and pack in an ordered lamellar structure in oil. The acyl migrated DAGs with higher ratios of 1,2-DAG showed reduced crystal size and lower oil-air interfacial activity. C14 and C12 DAG oleogels exhibited higher elasticity and whipping ability with crystal shells surrounding bubbles, whereas C16 and C18 DAG oleogels had low elasticity and limited whipping ability due to the formation of aggregated needle-like crystals and loose gel network. Thus, acyl chain length dramatically influences the gelation and foaming behaviors of DAGs whereas the isomers exert little influence. This study provides basis for applying DAG of different structures in food products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Konjac glucomannan (KGM) combined with hydroxypropyl methyl cellulose was used to fabricate diacylglycerol oleogels (DGOs) through the emulsion-templated method, and compared with triacylglycerol oleogels (TGOs). The appearance and microstructure results showed that stable emulsions and oleogels could be formed in the presence of 0.2-0.6 wt% KGM. Higher KGM concentrations resulted in a stronger gel structure in oleogels, whose thixotropic recovery percentages were 50.45-75.20 %. From LF-NMR determination, the higher concentration of KGM presented earlier transverse relaxation (T 2 ) time, and the T 2 parameters of DGOs were higher than that of TGOs. Texture and oil loss analysis indicated that the mechanical strength and oil holding ability of DGOs were slightly lower than those of TGOs. This study demonstrated the advantages of biopolymers as thickening agents for obtaining stable emulsion and oleogels. The specific characteristics of DGOs distinguished from TGOs should be attributed to their different properties (unsaturation, viscosity, polarity, etc.) between the liquid oils., Competing Interests: Declaration of competing interest The authors claim no conflict of interest in this work., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The study aimed to evaluate the effect of ultrasonic pretreatment on the transesterification of lard with glycerol monolaurate (GML) using Lipozyme TL IM to synthesize diacylglycerol (DAG), and the physicochemical properties of lard, GML, ultrasonic-treated diacylglycerol (named U-DAG), purified ultrasonic-treated diacylglycerol obtained by molecular distillation (named P-U-DAG), and without ultrasonic-treated diacylglycerol (named N-U-DAG) were analyzed. The optimized ultrasonic pretreatment conditions were: lard to GML mole ratio 3:1, enzyme dosage 6 %, ultrasonic temperature 80 °C, time 9 min, power 315 W. After ultrasonic pretreatment, the mixtures reacted for 4 h in a water bath at 60 °C, the content of DAG reached 40.59 %. No significant variations were observed between U-DAG and N-U-DAG in fatty acids compositions and iodine value, while P-U-DAG had lower unsaturated fatty acids than U-DAG. Differential scanning calorimetry analysis showed that the melting and crystallization properties of DAGs prepared by ultrasonic pretreatment significantly differed from lard. FTIR spectra noted transesterification reaction from lard and GML with and without ultrasonic pretreatment would not change the structure of lard. However, thermogravimetric analysis proved that N-U-DAG, U-DAG, and P-U-DAG had lower oxidation stability than lard. The higher the content of DAG, the faster the oxidation speed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

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.

Background: Diacylglycerol (DAG)-enriched oil has been attracting attention because of its nutritional benefits and biological functions, although the composition of its various free fatty acids (FFAs) and an unclear relationship between substrate and yield make it difficult to be identified and qualified with respect to its production. In the present study, linoleic acid-enriched diacylglycerol (LA-DAG) was synthesized and enriched from Camellia oil by the esterification process using the combi-lipase Lipozyme TL IM/RM IM system., Results: The relationship between FFA composition and DAG species productivity was revealed. The results showed that heterogeneous FFA with a major constituent (more than 50%) exhibited higher DAG productivity and inhibited triacylglycerol productivity compared to homogeneous constituents. Joint characterization by high-performance liquid chromatography-evaporative light scattering detection, gas chromatography-mass spectrometry and ultra-performance liquid chromatography-heated electrospray ionization-tandem mass spectrometry identified that DAG components contained dilinoleic acid acyl glyceride, linoleyl-oleyl glyceride and dioleic acid acyl glyceride in esterification products. Under the optimum conditions, 60.4% 1,3-DAG and 61.3% LA-DAG in the crude product at 1 h reaction were obtained, and further purified to 81.7% LA-DAG and 94.7% DAG via silica column chromatography., Conclusion: The present study provides a guideline for the identification of DAG species, as well as a structure-guided preparation method of DAG-enriched oils via the cost-effective combi-lipase. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Diacylglycerols (DAGs) are anabolic precursors to membrane lipid and storage triacylglycerol biosynthesis, metabolic intermediates of lipid catabolism, and potent cellular signaling molecules. The different DAG molecular species that accumulate over development or in different tissues reflect the changing aspects of cellular lipid metabolism. Consequently, an accurate determination of DAG molecular species in biological samples is essential to understand various metabolic processes and their diagnostic relevance. However, quantification of DAG molecular species in various biological samples represents a challenging task because of their low abundance, hydrophobicity, and instability. This chapter describes the most common chromatographic (TLC and HPLC) and mass spectrometry (MS) methods used to analyze DAG molecular species. In addition, we directly compared the three methods using DAG obtained by phospholipase C hydrolysis of phosphatidylcholine purified from a Nicotiana benthamiana leaf extract. We conclude that each method identified similar major molecular species, however, the exact levels of those varied mainly due to sensitivity of the technique, differences in sample preparation, and processing. This chapter provides three different methods to analyze DAG molecular species, and the discussion of the benefits and challenges of each technique will aid in choosing the right method for your analysis., (Copyright © 2023 Elsevier Inc. All rights reserved.)

This study developed an alpha-linolenic acid (ALA) supplement with emulsion form using ALA-rich diacylglycerol (ALA-DAG) and ALA-DAG stearin (DAG-SF) as a new source of ALA and emulsifier. Stable, commercial surfactant-free W/O emulsions with 90 wt% oil phase (including DAG-SF and ALA-DAG with 10:90 - 20:80 wt ratio) was fabricated. Microstructure and Raman spectra revealed that the compact crystal networks and high amounts of solid acyl chains were responsible for high emulsion stability. These emulsions exhibited good potential in improving the ALA nutritional status (with ALA release level of 60.49% - 62.98%). Furthermore, the emulsifier-to-oil ratio greatly impacted the emulsion texture (solid-like or liquid-like) and emulsions showed great oxidation stability (2.80 - 3.09 meq/kg lipid of peroxide value at 6th week). The tunable texture and high oxidation stability make this emulsion system useful for a wide range of food products. This developed emulsion system could provide valuable information for other important fatty acids supplement., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Diacylglycerol kinases (DGKs) are important enzymes in molecular membrane biology, as they can lower the concentration of diacylglycerol through phosphorylation while at the same time producing phosphatidic acid. Dysfunction of DGK is linked with multiple diseases including cancer and autoimmune disorders. Currently, the high-resolution structures have not been determined for any of the 10 human DGK paralogs, which has made it difficult to gain a more complete understanding of the enzyme's mechanism of action and regulation. In the present study, we have taken advantage of the significant developments in protein structural prediction technology by artificial intelligence (i.e., Alphafold 2.0), to conduct a comprehensive investigation on the properties of all 10 human DGK paralogs. Structural alignment of the predictions reveals that the C1, catalytic, and accessory domains are conserved in their spatial arrangement relative to each other, across all paralogs. This suggests a critical role played by this domain architecture in DGK function. Moreover, docking studies corroborate the existence of a conserved ATP-binding site between the catalytic and accessory domains. Interestingly, the ATP bound to the interdomain cleft was also found to be in proximity of the conserved glycine-rich motif, which in protein kinases has been suggested to function in ATP binding. Lastly, the spatial arrangement of DGK, with respect to the membrane, reveals that most paralogs possess a more energetically favorable interaction with curved membranes. In conclusion, AlphaFold predictions of human DGKs provide novel insights into the enzyme's structural and functional properties while also paving the way for future experimentation.

Diacylglycerol kinase ε (DGKε), an enzyme of the phosphatidylinositol (PI) cycle, bears a highly conserved hydrophobic N-terminal segment, which was proposed to anchor the enzyme into the membrane. However, the importance of this segment to the DGKε function remains to be determined. To address this question, it is here reported an in silico and in vitro combined research strategy. Capitalizing on the AlphaFold 2.0 predicted structure of human DGKε, it is shown that its hydrophobic N-terminal segment anchors it into the membrane via a transmembrane α-helix. Coarse-grained based elastic network model studies showed that a conformational change in the hydrophobic N-terminal segment determines the proximity between the active site of DGKε and the membrane-water interface, likely regulating its kinase activity. In vitro studies with a purified DGKε construct lacking the hydrophobic N-terminal segment (His-SUMO*-Δ 50 -DGKε) corroborated the role of the N-terminus in regulating DGKε enzymatic properties. The comparison between the enzymatic properties of DGKε and His-SUMO*-Δ 50 -DGKε showed that the conserved N-terminal segment markedly inhibits the enzyme activity and its sensitivity to membrane intrinsic negative curvature, while also playing a role in the modulation of the enzyme by phosphatidylserine. On the other hand, this segment did not strongly affect its diacylglycerol acyl chain specificity, the modulation of the enzyme by membrane morphological changes, or the activation by phosphatidic acid-rich lipid domains. Hence, these results suggest that the conservation of the hydrophobic N-terminal segment of DGKε throughout evolution guaranteed not only membrane anchorage but also an efficient and elegant manner to regulate the rate of the PI cycle.

Four types of pure lipid, namely lauric acid (LA), glycerol monolaurate (MAG), diglycerol laurate (DAG) and triglyceride laurate (TAG) were used to prepare oleofoams. The relationship between crystal profiles and their performance in oleofoams was established. DAG formed small needle-like crystals while MAG formed large flake-like crystals in oleogels, and crystal shells around air bubbles were observed in LA-, MAG- and DAG-based oleofoams. LA and DAG displayed higher over-run whereas DAG-stabilised foam possessed smaller bubbles and higher physical stability due to the presence of small β and β' crystals. Upon heating, DAG and TAG-based foams showed varying extents of oil drainage indicating the crystals were distributed in a different manner. Therefore, DAG was shown to be an excellent gelator in the fabrication of ultra-stable oleofoams. This work extends the lipid varieties with nutritional features and allows a better understanding on the stabilization mechanisms of lauric acid lipids in oleofoams., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Ceramides and diacylglycerols are groups of lipids capable of nucleating and stabilizing ordered lipid domains, structures that have been implicated in a range of biological processes. Previous studies have used fluorescence reporter molecules to explore the influence of ceramide acyl chain structure on sphingolipid-rich ordered phases. Here, we use small-angle neutron scattering (SANS) to examine the ability of ceramides and diacylglycerols to promote lipid domain formation in the well-characterized domain-forming mixture DPPC/DOPC/cholesterol. SANS is a powerful, probe-free technique for interrogating membrane heterogeneity, as it is differentially sensitive to hydrogen's stable isotopes protium and deuterium. Specifically, neutron contrast is generated through selective deuteration of lipid species, thus enabling the detection of nanoscopic domains enriched in deuterated saturated lipids dispersed in a matrix of protiated unsaturated lipids. Using large unilamellar vesicles, we found that upon replacing 10 mol% DPPC with either C16:0 or C18:0 ceramide, or 16:0 diacylglycerol (dag), lipid domains persisted to higher temperatures. However, when DPPC was replaced with short chain (C6:0 or C12:0) or very long chain (C24:0) ceramides, or ceramides with unsaturated acyl chains of any length (C6:1(3), C6:1(5), C18:1, and C24:1), as well as C18:1-dag, lipid domains were destabilized, melting at lower temperatures than those in the DPPC/DOPC/cholesterol system. These results show how ceramide acyl chain length and unsaturation influence lipid domains and have implications for how cell membranes might modify their function through the generation of different ceramide species., (Copyright © 2022. Published by Elsevier B.V.)

Diacylglycerol (DAG) is a versatile lipid whose 1,2-sn-stereoisomer serves both as second messenger in signal transduction pathways that control vital cellular processes, and as metabolic precursor for downstream signaling lipids such as phosphatidic acid. Effector proteins translocate to available DAG pools in the membranes by using conserved homology 1 (C1) domains as DAG-sensing modules. Yet, how C1 domains recognize and capture DAG in the complex environment of a biological membrane has remained unresolved for the 40 years since the discovery of Protein Kinase C (PKC) as the first member of the DAG effector cohort. Herein, we report the high-resolution crystal structures of a C1 domain (C1B from PKCδ) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts and that command intense therapeutic interest. This structural information details the mechanisms of stereospecific recognition of DAG by the C1 domains, the functional properties of the lipid-binding site, and the identities of the key residues required for the recognition and capture of DAG and exogenous agonists. Moreover, the structures of the five C1 domain complexes provide the high-resolution guides for the design of agents that modulate the activities of DAG effector proteins., (© 2022. The Author(s).)

There is a need to better understand lipid metabolism during mosquito ovarian development. Lipids are the major source of energy supporting ovarian follicles development in mosquitoes. In this paper, we describe the complementary use of stable isotope labeling (SIL) and high-resolution mass spectrometry-based tools for the investigation of de novo triglycerides (TG) and diglycerides (DG) during the ovarian previtellogenic (PVG) stage (4-6 days posteclosion) of female adult Aedes aegypti . Liquid chromatography coupled to high-resolution trapped ion mobility spectrometry-parallel accumulation sequential fragmentation-time-of-flight tandem mass spectrometry (LC-TIMS-PASEF-TOF MS/MS) allowed the separation and quantification of nonlabeled and 2 H/ 13 C-labeled TG and DG species. Three SIL strategies were evaluated (H 2 O/ 2 H 2 O with 50:50 and 95:5 mixtures, 13 C-sucrose, and 13 C-glucose). Results showed wide applicability with no signs of lipid ovarian impairment by SIL induced toxicity. The analytical workflow based on LC-TIMS-TOF MS/MS provided high confidence and high reproducibility for lipid DG and TG identification and SIL incorporation based on their separation by retention time (RT), collision cross section (CCS), and accurate m / z . In addition, the SIL fatty acid chain incorporation was evaluated using PASEF MS/MS. The 2 H/ 13 C incorporation into the mosquito diet provided information on how TG lipids are consumed, stored, and recycled during the PVG stage of ovarian development.

PI3Kβ is required for invadopodia-mediated matrix degradation by breast cancer cells. Invadopodia maturation requires GPCR activation of PI3Kβ and its coupling to SHIP2 to produce PI(3,4)P 2 . We now test whether selectivity for PI3Kβ is preserved under conditions of mutational increases in PI3K activity. In breast cancer cells where PI3Kβ is inhibited, short-chain diC8-PIP 3  rescues gelatin degradation in a SHIP2-dependent manner; rescue by diC8-PI(3,4)P 2  is SHIP2-independent. Surprisingly, the expression of either activated PI3Kβ or PI3Kα mutants rescued the effects of PI3Kβ inhibition. In both cases, gelatin degradation was SHIP2-dependent. These data confirm the requirement for PIP 3 conversion to PI(3,4)P 2 for invadopodia function and suggest that selectivity for distinct PI3K isotypes may be obviated by mutational activation of the PI3K pathway., (© 2022 Federation of European Biochemical Societies.)

This study determined the influence of diacylglycerol (DAG) pre-emulsion on the gel properties and microstructure of golden thread surimi gels. DAG emulsion stabilized using sodium caseinate was pre-emulsified through ultrasound. The average particle size of DAG pre-emulsion decreased from 1324.15 nm to 41.19 nm, with notable improvements in apparent viscosity and storage stability. The surimi gels with different amounts (0%, 1%, 3%, 5%, and 7% w/w) of DAG pre-emulsion were prepared under heat induction. The whiteness of the composite gels markedly increased with the incorporation of DAG pre-emulsion. The peak T 22 value of immobilized water, the gel strength, and water-holding capacity increased gradually, but it slightly decreased with the addition of 7% pre-emulsion. The curve of G' and G″ kept climbing as the concentration of pre-emulsion, and the microstructure of the gel network tended to become denser and more orderly. Principal component analysis (PCA) of electronic nose results showed that the surimi gels containing pre-emulsion could be clearly distinguished from the control group. In conclusion, the addition of 5% DAG pre-emulsion to surimi not only improved gel properties to the highest extent but also be compensated for lipid loss during the rinsing of surimi., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

In this study, lipase A from Candida antarctica (CALA) was immobilized onto the macroporous resin NKA-9. Immobilization conditions (pH, time and CALA concentration) were studied, enzymatic activity and immobilization efficiency (IE) up to 968.89 U/g and 53.19% were respectively obtained under optimal conditions (immobilization pH 5.0, time 5 h and CALA concentration at 30 mg/mL). Then, the NKA-9 supported CALA (CALA@NKA-9) samples were used to catalyze glycerolysis in solvent-free system. With 0.25 g of the present CALA@NKA-9 (soybean oil 3.52 g and glycerol 0.184 g) and after 12 h reaction at 50 °C, diacylglycerols (DAG) content up to 64.37% and triacylglycerols (TAG) conversion at 83.33% were obtained. The relationship between temperature and TAG conversion was LnV 0 = 13.9310-6.4212/T for CALA@NKA-9. Meanwhile, the activation energy (Ea) of CALA@NKA-9 was calculated to be 53.39 kJ/mol. In addition, reusability in the glycerolysis reaction was also evaluated, and 57.82% of the initial glycerolysis activity was retained after 9 consecutive applications. Furthermore, the CALA@NKA-9 was also used to catalyze the esterification (esterification of fatty acids with glycerol), however, the present CALA@NKA-9 cannot initiate the esterification. Therefore, the present CALA@NKA-9 is shown to be potential for DAG production through glycerolysis reaction.

Bipolar tetraether lipids (BTL) have been long thought to play a critical role in allowing thermoacidophiles to thrive under extreme conditions. In the present study, we demonstrated that not all BTLs from the thermoacidophilic archaeon Sulfolobus acidocaldarius exhibit the same membrane behaviors. We found that free-standing planar membranes (i.e., black lipid membranes, BLM) made of the polar lipid fraction E (PLFE) isolated from S.   acidocaldarius  formed over a pinhole on a cellulose acetate partition in a dual-chamber Teflon device exhibited remarkable stability showing a virtually constant capacitance (~28 pF) for at least 11 days. PLFE contains exclusively tetraethers. The dominating hydrophobic core of PLFE lipids is glycerol dialky calditol tetraether (GDNT, ~90%), whereas glycerol dialkyl glycerol tetraether (GDGT) is a minor component (~10%). In sharp contrast, BLM made of BTL extracted from microvesicles (Sa-MVs) released from the same cells exhibited a capacitance between 36 and 39 pF lasting for only 8 h before membrane dielectric breakdown. Lipids in Sa-MVs are also exclusively tetraethers; however, the dominating lipid species in Sa-MVs is GDGT (>99%), not GDNT. The remarkable stability of BLM PLFE can be attributed to strong PLFE-PLFE and PLFE-substrate interactions. In addition, we compare voltage-dependent channel activity of calcium-gated potassium channels (MthK) in BLM PLFE to values recorded in BLM Sa-MV . MthK is an ion channel isolated from a methanogenic that has been extensively characterized in diester lipid membranes and has been used as a model for calcium-gated potassium channels. We found that MthK can insert into BLM PLFE and exhibit channel activity, but not in BLM Sa-MV . Additionally, the opening/closing of the MthK in BLM PLFE is detectable at calcium concentrations as low as 0.1 mM; conversely, in diester lipid membranes at such a low calcium concentration, no MthK channel activity is detectable. The differential effect of membrane stability and MthK channel activity between BLM PLFE and BLM Sa-MV may be attributed to their lipid structural differences and thus their abilities to interact with the substrate and membrane protein. Since Sa-MVs that bud off from the plasma membrane are exclusively tetraether lipids but do not contain the main tetraether lipid component GDNT of the plasma membrane, domain segregation must occur in S. acidocaldarius . The implication of this study is that lipid domain formation is existent and functionally essential in all kinds of cells, but domain formation may be even more prevalent and pronounced in hyperthermophiles, as strong domain formation with distinct membrane behaviors is necessary to counteract randomization due to high growth temperatures while BTL in general make archaea cell membranes stable in high temperature and low pH environments whereas different BTL domains play different functional roles.

Pickering water-in-oil (W/O) emulsions were fabricated by using medium-long chain diacylglycerol (MLCD)-based solid lipid nanoparticles (SLNs) and the connection between the characteristics of the SLNs and the colloidal stability of the emulsions was established. Via melt-emulsification and ultrasonication, MLCD-based SLNs with particle sizes of 120-300 nm were obtained with or without other surfactants. The particle size of the SLNs was influenced by the chemical properties of the surfactants, and surfactants decreased the contact angle of SLNs at the oil-water interface. Gelation was observed in SLNs modified by sodium stearoyl lactylate and lecithin, whereas the addition of Tween 20 resulted in a homogeneous SLN solution. The adsorption of surfactants onto SLN surfaces caused the production of higher amounts of α crystals accompanied by delayed crystallization onset which contributed to the reduction of particle size, interfacial tension and oil wetting ability. The W/O emulsions with higher rigidity and physical stability can be obtained by varying surfactant types and by increasing SLN mass ratios to 60%, whereby more SLNs are adsorbed at the droplet surface as a Pickering stabilizer. This study provides useful insights for the development of diacylglycerol-based SLNs and Pickering W/O emulsions which have great potential for food, cosmetic and pharmaceutical applications.

Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One β-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the β-carotene, SQDG and PsbO in formation of the PSII dimer., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mass spectrometry is routinely employed for structure elucidation of molecules. Structural information can be retrieved from intact molecular ions by fragmentation; however, the interpretation of fragment spectra is often hampered by poor understanding of the underlying dissociation mechanisms. For example, neutral headgroup loss from protonated glycerolipids has been postulated to proceed via an intramolecular ring closure but the mechanism and resulting ring size have never been experimentally confirmed. Here we use cryogenic gas-phase infrared (IR) spectroscopy in combination with computational chemistry to unravel the structures of fragment ions and thereby shed light on elusive dissociation mechanisms. Using the example of glycerolipid fragmentation, we study the formation of protonated five-membered dioxolane and six-membered dioxane rings and show that dioxolane rings are predominant throughout different glycerolipid classes and fragmentation channels. For comparison, pure dioxolane and dioxane ions were generated from tailor-made dehydroxyl derivatives inspired by natural 1,2- and 1,3-diacylglycerols and subsequently interrogated using IR spectroscopy. Furthermore, the cyclic structure of an intermediate fragment occurring in the phosphatidylcholine fragmentation pathway was spectroscopically confirmed. Overall, the results contribute substantially to the understanding of glycerolipid fragmentation and showcase the value of vibrational ion spectroscopy to mechanistically elucidate crucial fragmentation pathways in lipidomics.

Due to their special chemical structure, tetraether lipids (TEL) represent essential elements of archaeal membranes, providing these organisms with extraordinary properties. Here we describe the characterization of a newly isolated structural element of the main lipids. The TEL fragment GDNT-β-Glu was isolated from Sulfolobus metallicus and characterized in terms of its chemical structure by NMR- and MS-investigations. The obtained data are dissimilar to analogically derived established structures - in essence, the binding relationships in the polar head group are re-determined and verified. With this work, we provide an important contribution to the structure elucidation of intact TEL also contained in other Sulfolobus strains such as Solfulobus acidocaldarius and Sulfolobus solfataricus., (© 2021 The Authors. Published by Wiley-VCH GmbH.)

DAG-lactones represent useful templates for the design of potent and selective C1 domain ligands for PKC isozymes. The ester moiety at the sn -1 position, a common feature in this template, is relevant for C1 domain interactions, but it represents a labile group susceptible to endogenous esterases. An interesting challenge involves replacing the ester group of these ligands while still maintaining biological activity. Here, we present the synthesis and functional characterization of novel diacylglycerol-lactones containing heterocyclic ring substituents at the sn -1 position. Our results showed that the new compound 10B12 , a DAG-lactone with an isoxazole ring, binds PKCα and PKCε with nanomolar affinity. Remarkably, 10B12 displays preferential selectivity for PKCε translocation in cells and induces a PKCε-dependent reorganization of the actin cytoskeleton into peripheral ruffles in lung cancer cells. We conclude that introducing a stable isoxazole ring as an ester surrogate in DAG-lactones emerges as a novel structural approach to achieve PKC isozyme selectivity.

Oleofoams have emerged as attractive low-calorie aeration systems, but saturated lipids or large amount of surfactants are commonly required. Herein, an innovative strategy was proposed to create oleofoams using medium-long chain diacylglycerol (MLCD) and β-sitosterol (St). The oleofoams prepared using MLCD and St in ratios of 15:5 and 12:8 exhibited smaller bubble size and much higher stability. MLCD crystals formed rigid Pickering shell, whereby air bubbles acted as "active fillers" leading to enhanced rigidity. Both Pickering and network stabilization for the MLCD-St oleofoam provided a steric hindrance against coalescence. The gelators interacted via hydrogen bonding, causing a condensing effect in improving the gel elasticity. The oleofoams and foam-based emulsions exhibited a favorable capacity in controlling volatile release where the maximum headspace concentrations and partition coefficients showed a significantly decrease. Overall, the oleofoams have shown great potential for development of low-calorie foods and delivery systems with enhanced textural and nutritional features., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Sulfoquinovosyldiacylglycerol (SQDG) is a glycolipid ubiquitously found in photosynthetically active organisms. It has attracted much attention in recent years due to its biological activities. Similarly, the increasing demand for vegan and functional foods has led to a growing interest in micronutrients such as sulfolipids and their physiological influence on human health. To study this influence, reference materials are needed for developing new analytical methods and providing enough material for model studies on the biological activity. However, the availability of these materials is limited by the difficulty to isolate and purify sulfolipids from natural sources and the unavailability of chemical standards on the market. Consequently, an alternative synthetic route for the comprehensive preparation of sulfolipids was established. Here, the synthesis of a sulfolipid with two identical saturated fatty acids is described exemplarily. The method opens possibilities for the preparation of a diverse range of interesting derivatives with different saturated and unsaturated fatty acids.

Diacylglycerol kinases catalyze the ATP-dependent phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA). In humans, the alpha isoform (DGKα) has emerged as a potential target in the treatment of cancer due to its anti-tumor and pro-immune responses. However, its mechanism of action at a molecular level is not fully understood. In this work, a systematic investigation of the role played by the membrane in the regulation of the enzymatic properties of human DGKα is presented. By using a cell-free system with purified DGKα and model membranes of variable physical and chemical properties, it is shown that membrane physical properties determine human DGKα substrate acyl chain specificity. In model membranes with a flat morphology; DGKα presents high enzymatic activity, but it is not able to differentiate DAG molecular species. Furthermore, DGKα enzymatic properties are insensitive to membrane intrinsic curvature. However, in the presence of model membranes with altered morphology, specifically the presence of physically curved membrane structures, DGKα bears substrate acyl chain specificity for palmitic acid-containing DAG. The present results identify changes in membrane morphology as one possible mechanism for the depletion of specific pools of DAG as well as the production of specific pools of PA by DGKα, adding an extra layer of regulation on the interconversion of these two potent lipid-signaling molecules. It is proposed that the interplay between membrane physical (shape) and chemical (lipid composition) properties guarantee a fine-tuned signal transduction system dependent on the levels and molecular species of DAG and PA., (© 2021 Federation of American Societies for Experimental Biology.)

Background: Diacylglycerols as a fat substitute in meat products is a growing area of interest. This study was conducted to analyze the digestion rate, digestion extent, and changes in interfacial properties of lard, glycerolized lard (GL), and purified GL (PGL) in an emulsions system by pH-stat in vitro digestion., Results: PGL had significantly higher hydrolysis rate and final digestion extent than lard (P ≤ 0.05) during in vitro digestion. The analysis on diameter variations of lard, GL, and PGL during digestion indicated that the surface- and volume-weighted mean particle diameters of all samples had the same variation trend, but variation degree was different. Concurrently, the ζ-potential analysis of the lard, GL, and PGL during the digestion process showed that the absolute values of the ζ-potentials of the three types of lipids increased at first and subsequently decreased. The microstructure changes results for lard, GL, and PGL showed there was no obvious flocculation, and the particle size of lard throughout the digestion process was larger than that of GL and PGL., Conclusion: This study showed that lard-based diacylglycerols had high digestibility characteristics and could be applied as a functional lipid in meat products to improve human health. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Herein, we prepared 1,3-dipalmitoyl-2-oleoyl glycerol (POP)-rich fats with reduced levels of diacylglycerols (DAGs), adversely affecting the tempering of chocolate, via two-step hexane fractionation of palm stearin. DAG content in the as-prepared fats was lower than that in POP-rich fats obtained by previously reported conventional two-step acetone fractionation. Cocoa butter equivalents (CBEs) were fabricated by blending the as-prepared fats with 1,3-distearoyl-2-oleoyl glycerol (SOS)-rich fats obtained by hexane fractionation of degummed shea butter. POP-rich fats achieved under the best conditions for the fractionation of palm stearin had a significantly lower DAG content (1.6 w/w %) than that in the counterpart (4.6 w/w %) prepared by the previously reported method. The CBEs fabricated by blending the POP- and SOS-rich fats in a weight ratio of 40:60 contained 63.7 w/w % total symmetric monounsaturated triacylglycerols, including 22.0 w/w % POP, 8.6 w/w % palmitoyl-2-oleoyl-3-stearoyl- rac -glycerol, 33.1 w/w % SOS, and 1.3 w/w % DAGs, which was not substantially different from the DAG content in cocoa butter (1.1 w/w %). Based on the solid-fat content results, it was concluded that, when these CBEs were used for chocolate manufacture, they blended with cocoa butter at levels up to 40 w/w %, without distinctively altering the hardness and melting behavior of cocoa butter.

Single-cell analysis is critical to revealing cell-to-cell heterogeneity that would otherwise be lost in ensemble analysis. Detailed lipidome characterization for single cells is still far from mature, especially when considering the highly complex structural diversity of lipids and the limited sample amounts available from a single cell. We report the development of a general strategy enabling single-cell lipidomic analysis with high structural specificity. Cell fixation is applied to retain lipids in the cell during batch treatments prior to single-cell analysis. In addition to tandem mass spectrometry analysis revealing the class and fatty acyl-chain for lipids, batch photochemical derivatization and single-cell droplet treatment are performed to identify the C=C locations and sn-positions of lipids, respectively. Electro-migration combined with droplet-assisted electrospray ionization enables single-cell mass spectrometry analysis with easy operation but high efficiency in sample usage. Four subtypes of human breast cancer cells are correctly classified through quantitative analysis of lipid C=C location or sn-position isomers in ~160 cells. Most importantly, the single-cell deep lipidomics strategy successfully discriminates gefitinib-resistant cells from a population of wild-type human lung cancer cells (HCC827), highlighting its unique capability to promote precision medicine.

Small molecular probes designed for photopharmacology and opto-chemogenetics are rapidly gaining widespread recognition for investigations of transient receptor potential canonical (TRPC) channels. This protocol describes the use of three photoswitchable diacylglycerol analogs-PhoDAG-1, PhoDAG-3, and OptoDArG-for ultrarapid activation and deactivation of native TRPC2 channels in mouse vomeronasal sensory neurons and olfactory type B cells, as well as heterologously expressed human TRPC6 channels. Photoconversion can be achieved in mammalian tissue slices and enables all-optical stimulation and shutoff of TRPC channels. For complete details on the use and execution of this protocol, please refer to Leinders-Zufall et al. (2018)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Munc13-1 is a presynaptic active zone protein that acts as a master regulator of synaptic vesicle priming and neurotransmitter release in the brain. It has been implicated in the pathophysiology of several neurodegenerative diseases. Diacylglycerol and phorbol ester activate Munc13-1 by binding to its C1 domain. The objective of this study is to identify the structural determinants of ligand binding activity of the Munc13-1 C1 domain. Molecular docking suggested that residues Trp-588, Ile-590, and Arg-592 of Munc13-1 are involved in ligand interactions. To elucidate the role of these three residues in ligand binding, we generated W588A, I590A, and R592A mutants in full-length Munc13-1, expressed them as GFP-tagged proteins in HT22 cells, and measured their ligand-induced membrane translocation by confocal microscopy and immunoblotting. The extent of 1,2-dioctanoyl- sn -glycerol (DOG)- and phorbol ester-induced membrane translocation decreased in the following order: wild type > I590A > W588A > R592A and wild type > W588A > I590A > R592A, respectively. To understand the effect of the mutations on ligand binding, we also measured the DOG binding affinity of the isolated wild-type C1 domain and its mutants in membrane-mimicking micelles using nuclear magnetic resonance methods. The DOG binding affinity decreased in the following order: wild type > I590A > R592A. No binding was detected for W588A with DOG in micelles. This study shows that Trp-588, Ile-590, and Arg-592 are essential determinants for the activity of Munc13-1 and the effects of the three residues on the activity are ligand-dependent. This study bears significance for the development of selective modulators of Munc13-1.

Enrichment of mayonnaise using delivery emulsions (DEs) containing 70% fish oil versus neat fish oil was investigated. DEs were produced with combined use of sodium caseinate, diacetyl tartaric acid esters of mono- and diglycerides (DATEM), and/or modified DATEMs with different length (C12 or C14) and covalently attached caffeic acid. Physical and oxidative stability of the mayonnaises were analyzed based on parameters including droplet size, viscosity, peroxide value, volatile compounds, and sensory properties. DEs addition to mayonnaise resulted in larger droplets and lower viscosity compared to neat fish oil. However, zeta potential was higher in mayonnaises with DEs containing DATEMs. Mayonnaise containing DATEM C14 had higher protein surface load leading to a thicker interfacial layer, lower formation of hexanal, (E)-2-hexenal, and (E)-2-heptenal as well as lower rancid odour intensity compared to mayonnaise containing DATEM and free caffeic acid, and thus benefitted from the location of the antioxidant at the interface., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Lipid droplets (LDs) are intracellular organelles responsible for lipid storage, and they emerge from the endoplasmic reticulum (ER) upon the accumulation of neutral lipids, mostly triglycerides (TG), between the two leaflets of the ER membrane. LD biogenesis takes place at ER sites that are marked by the protein seipin, which subsequently recruits additional proteins to catalyze LD formation. Deletion of seipin, however, does not abolish LD biogenesis, and its precise role in controlling LD assembly remains unclear. Here, we use molecular dynamics simulations to investigate the molecular mechanism through which seipin promotes LD formation. We find that seipin clusters TG, as well as its precursor diacylglycerol, inside its unconventional ring-like oligomeric structure and that both its luminal and transmembrane regions contribute to this process. This mechanism is abolished upon mutations of polar residues involved in protein-TG interactions into hydrophobic residues. Our results suggest that seipin remodels the membrane of specific ER sites to prime them for LD biogenesis., Competing Interests: The authors declare no competing interest.

The c subunits of F 0 F 1 -ATP synthase (F 0 c) assemble into a ring structure, following membrane insertion that is dependent on both glycolipid MPIase and protein YidC. We analyzed the insertion and assembly processes of Propionigenium modestum F 0 c (Pm-F 0 c), of which the ring structure is resistant to SDS. Ring assembly of Pm-F 0 c requires P. modestum UncI (Pm-UncI). Ring assembly of in vitro synthesized Pm-F 0 c was observed when both YidC and Pm-UncI were reconstituted into liposomes of Escherichia coli phospholipids. Under the physiological conditions where spontaneous insertion had been blocked by diacylglycerol, MPIase was necessary for Pm-F 0 c insertion allowing the subsequent YidC/Pm-UncI-dependent ring assembly. Thus, we have succeeded in the complete reconstitution of membrane insertion and subsequent ring assembly of Pm-F 0 c., (© 2021 Federation of European Biochemical Societies.)

In this study, α-linolenic acid-enriched diacylglycerols (ALA-DAGs) were prepared via a two-step enzymatic way by combi-lipase using silkworm pupae oils as substrates. Firstly, several factors including temperature, mass ratio of water to oil, pH and enzyme loading were optimized for the hydrolysis of silkworm pupae oil. The maximum fatty acid content (96.51%) was obtained under the conditions: temperature 40 °C, water/oil 3:2 (w/w), pH 7, lipase TL100L loading 400 U/g, lipase PCL loading 30 U/g. Then, ALA was enriched by urea inclusion, with an increased ALA content of 82.50% being obtained. Secondly, the ALA-enriched silkworm pupae DAG oil (SPDO) was prepared by lipase PCL-catalyzed esterification reaction. After molecular distillation, the final SPDO product contained contents of DAGs (97.01%) and ALA (82.50%). This two-step enzymatic way for production of ALA-DAGs was successfully applied in a 100-fold scale-up reaction. Overall, our study provides a promising way for the preparation of ALA-DAGs.