Incorporation Of Lipids Research Articles

Lipid Curvature and Fluidity Influence Lipid Incorporation Disparities in Nanodiscs.

Nanodiscs have become a popular membrane mimetic system offering a well-defined bilayer environment to stabilize membrane proteins for in vitro analyses using a range of analytical methods; however, lipid compositions common to their deployment are simplistic and often fail to model native membrane complexity. Furthermore, there has been a general lack of rigorous analytical and biophysical characterization of nanodiscs comprising more than one lipid. To address these challenges, we coupled a nanodisc formation and purification workflow with targeted LC-MS/MS analysis to quantify lipids in nanodiscs made with different compositions. We screened lipids with a variety of headgroups and acyl chains and found that lipids did not always incorporate into nanodiscs at expected levels. Disparities in lipid incorporation were found to increase upon the addition of lipids known to induce curvature or rigidity to the membrane. Additionally, we found that adding just one additional type of lipid to nanodiscs changes the particle diameter and dispersity compared to nanodiscs containing a single lipid. We also formed and characterized nanodiscs using a complex starting composition inspired by the endoplasmic reticulum membrane and observed native-like cholesterol dynamics that modulated the lipid fluidity in the model bilayer system. Taken together, this work serves as a foundation for understanding nonstoichiometric lipid incorporation into nanodiscs and provides a basis for more thorough nanodisc characterization and quality control, which is critical to ensure multilipid nanodiscs synthesized accurately model the biological system of interest, enabling robust characterization of how the lipid landscape affects membrane protein structure and activity.

Relevance of Lipoprotein Composition in Endothelial Dysfunction and the Development of Hypertension

Endothelial dysfunction and chronic inflammation are determining factors in the development and progression of chronic degenerative diseases, such as hypertension and atherosclerosis. Among the shared pathophysiological characteristics of these two diseases is a metabolic disorder of lipids and lipoproteins. Therefore, the contents and quality of the lipids and proteins of lipoproteins become the targets of therapeutic objective. One of the stages of lipoprotein formation occurs through the incorporation of dietary lipids by enterocytes into the chylomicrons. Consequently, the composition, structure, and especially the properties of lipoproteins could be modified through the intake of bioactive compounds. The objective of this review is to describe the roles of the different lipid and protein components of lipoproteins and their receptors in endothelial dysfunction and the development of hypertension. In addition, we review the use of some non-pharmacological treatments that could improve endothelial function and/or prevent endothelial damage. The reviewed information contributes to the understanding of lipoproteins as vehicles of regulatory factors involved in the modulation of inflammatory and hemostatic processes, the attenuation of oxidative stress, and the neutralization of toxins, rather than only cholesterol and phospholipid transporters. For this review, a bibliographic search was carried out in different online metabases.

Incorporation of ionizable lipids into the outer shell of lipid-coated calcium phosphate nanoparticles boosts cellular mRNA delivery.

Messenger RNA is a highly promising biotherapeutic modality with great potential in preventive and therapeutic vaccination, and in the modulation of cellular function through transient expression of therapeutic proteins. However, for cellular delivery, mRNA requires packaging into delivery vehicles that mediate uptake and also shield the mRNA against degradation. Lipid-coated calcium phosphate (LCP) nanoparticles encapsulate the mRNA in a calcium phosphate core, which is coated by a bilayer of structural lipids, positively charged lipids and pegylated lipid to mediate cellular uptake and achieve colloidal stabilization. Here, we show that such nanoparticles using positively charged lipids achieve cellular uptake but only poor cytosolic mRNA delivery. However, mRNA release could be greatly enhanced through incorporation of ionizable lipids into the outer leaflet of the lipid bilayer. We optimized the composition and molar ratios of ionizable lipids, positive lipid, cholesterol, and polyethylene glycol (PEG) and evaluated the potency of the formulations for the cellular delivery of mRNA. Whereas in lipid nanoparticles, the ionizable lipid has a main role in the complexation of the mRNA, our study provides a new paradigm for the employment of ionizable cationic lipids in nanocarriers other than lipid nanoparticles (LNPs) to boost the endosomal release of nucleic acids.

Advances in the study of LNPs for mRNA delivery and clinical applications.

Messenger ribonucleic acid (mRNA) was discovered in 1961 as an intermediary for transferring genetic information from DNA to ribosomes for protein synthesis. The COVID-19 pandemic brought worldwide attention to mRNA vaccines. The emergency use authorization of two COVID-19 mRNA vaccines, BNT162b2 and mRNA-1273, were major achievements in the history of vaccine development. Lipid nanoparticles (LNPs), one of the most superior non-viral delivery vectors available, have made many exciting advances in clinical translation as part of the COVID-19 vaccine and therefore has the potential to accelerate the clinical translation of many gene drugs. In addition, due to these small size, biocompatibility and excellent biodegradability, LNPs can efficiently deliver nucleic acids into cells, which is particularly important for current mRNA therapeutic regimens. LNPs are composed cationic or pH-dependent ionizable lipid bilayer, polyethylene glycol (PEG), phospholipids, and cholesterol, represents an advanced system for the delivery of mRNA vaccines. Furthermore, optimization of these four components constituting the LNPs have demonstrated enhanced vaccine efficacy and diminished adverse effects. The incorporation of biodegradable lipids enhance the biocompatibility of LNPs, thereby improving its potential as an efficacious therapeutic approach for a wide range of challenging and intricate diseases, encompassing infectious diseases, liver disorders, cancer, cardiovascular diseases, cerebrovascular conditions, among others. Consequently, this review aims to furnish the scientific community with the most up-to-date information regarding mRNA vaccines and LNP delivery systems.

Impact of annealing and incorporation of vegetable oils on physicochemical and rheological properties of wheat starch

This study investigated the impact of annealing treatment and lipids (vegetable oils, such as palm, olive, and grapeseed oils) on the physicochemical and rheological properties of wheat starch. Annealing of wheat starch (WSANN45, WSANN55) under different temperatures (45 °C and 55 °C) and with added vegetable oil (WS-OilANN45, WS-OilANN55) were compared with untreated wheat starch (WS). Annealing at 45 °C resulted in slight changes in the physicochemical properties of starch. However, annealing at 55 °C significantly decreased the relative crystallinity, pasting viscosity, and swelling power. WS-OilANN45 showed a higher ΔH (dissociation peak) than WSANN45, indicating successful lipid incorporation, whereas WS-OilANN55 showed no significant difference from WSANN55, suggesting that lipid integration was not achieved. Rheological tests showed that WS-OilANN45 slightly reduced the shear-thinning behavior and viscoelastic properties of starch. The introduction of oils affected the swelling and pasting properties, weakened the gel network, and significantly reduced the gel hardness. This approach offers a potential method that uses food-grade oils and annealing to modify starch and alter its rheological and physical properties while retaining its native granular characteristics.

An Outline of a Simple, Interpretable Epigenetic Composite Score for Mortality Prediction for Accelerated Underwriting.

In principle, it is generally accepted that DNA methylation measures can be used to predict mortality. However, as of yet, no epigenetic metric has been successfully incorporated into underwriting procedures. In part, this failure results from the relative incompatibility of many DNA methylation measures with conventional underwriting practices. To test the ability of previously established epigenetic markers of smoking, drinking and diabetes to standard lipid-based approaches for predicting mortality. We constructed a series of Cox proportional hazards models for mortality using clinical data and DNA methylation data from 4 previously described loci from the Framingham Heart Study. The incorporation of vital signs, standard lipid and diabetes laboratory assessments to a base model consisting of age and sex only modestly increased prediction of mortality from 0.732 to 0.741 area under the curve (AUC). However, the addition of epigenetic marker information for smoking and drinking to the base model markedly increased prediction (AUC=0.787) while the addition of epigenetic marker for diabetes increased prediction even further (AUC=0.792). These results demonstrate the potential of simple interpretable, epigenetic models to predict mortality in a manner compatible with standard underwriting procedures. Potentially, this epigenetic approach using rapid methylation sensitive digital PCR procedures that can utilize saliva or whole blood DNA would increase prediction power even further while facilitating more accurate accelerated underwriting assessments of mortality.

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles.

Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of Gram-negative bacteria limits the efficacy of many existing antibiotics and adds complexity to antibiotic development by limiting transport of antibiotics to the bacterial cytosol. New methods to mimic this barrier would enable high-throughput studies for antibiotic development. In this study, we introduce an innovative approach to modify outer membrane vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, to generate planar supported lipid bilayer membranes. Our method first involves the incorporation of synthetic lipids into OMVs using a rapid freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when in contact with SiO2 surfaces to form a planar outer membrane supported bilayer (OM-SB). We assessed the formation of OM-Hybrids using dynamic light scattering and a fluorescence quenching assay. To analyze the formation of OM-SBs from OM-Hybrids we used quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP). Additionally, we conducted assays to detect surface-associated DNA and proteins on OM-SBs. The interaction of an antimicrobial peptide, polymyxin B, with the OM-SBs was also assessed. These findings emphasize the capability of our platform to produce planar surfaces of bacterial outer membranes, which in turn, could function as a valuable tool for streamlining the development of antibiotics.

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles.

Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of gram-negative bacteria limits the efficacy of many existing antibiotics and adds complexity to antibiotic development by limiting transport of antibiotics to the bacterial cytosol. New methods to mimic this barrier would enable high-throughput studies for antibiotic development. In this study, we introduce an innovative approach to modify outer membrane vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, to generate planar supported lipid bilayer membranes. Our method first involves the incorporation of synthetic lipids into OMVs using a rapid freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when in contact with SiO2 surfaces to form a planar outer membrane supported bilayer (OM-SB). We assessed the formation of OM-Hybrids using dynamic light scattering and a fluorescence quenching assay. To analyze the formation of OM-SBs from OM-Hybrids we used quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP). Additionally, we conducted assays to detect surface-associated DNA and proteins on OM-SBs. The interaction of an antimicrobial peptide, polymyxin B, with the OM-SBs was also assessed. These findings emphasize the capability of our platform to produce planar surfaces of bacterial outer membranes, which in turn, could function as a valuable tool for streamlining the development of antibiotics.

The brominated flame retardant hexabromocyclododecane causes systemic changes in polyunsaturated fatty acid incorporation in mouse lipids.

Brominated flame retardants are used in many household products to reduce flammability, but often leach into the surrounding environment over time. Hexabromocyclododecane (HBCD) is a brominated flame retardant detected in human blood across the world. HBCD exposure can result in neurological problems and altered lipid metabolism, but to date, the two remain unlinked. As lipids constitute ∼50% of brain dry weight, lipid metabolism plays a critical role in neuronal function and homeostasis. To determine the effect of HBCD exposure on brain lipid metabolism, young adult male C57BL/6 mice were exposed to 1 mg/kg HBCD every 3 d for 28 d. Major lipid classes were found to change across brain regions, including membrane glycerolipids such as phosphatidylcholine and phosphatidylethanolamine, and sphingolipids such as hexosylceramide. In addition, saturated, monounsaturated, and polyunsaturated fatty acids were enriched within brain lipid species. To understand the source of the brain lipidomic alterations, the blood and liver lipidomes and the cecal microbiome were evaluated. The liver and blood demonstrated changes amongst multiple lipid classes, including triacylglycerol suppression, as well as altered esterified fatty acid content. Significant alterations were also detected in the cecal microbiome, with decreases in the Firmicutes to Bacteriodetes ratio, changes in beta diversity, and pathway alterations associated with metabolic pathways and amino acid biosynthesis. These data demonstrate that HBCD can induce lipidomic alterations across brain regions and organs and support a potential role of the microbiome in these alterations.

Preparation and characterization of a nanostructured lipid carrier for phenylethyl resorcinol

Phenylethyl resorcinol (PR) demonstrates inhibitory effects on multiple targets in the melanin synthesis pathway, resulting in a strong whitening effect. However, challenges such as limited solubility in water and susceptibility to oxidation and discoloration restrict its practical application in the cosmetics industry. In order to enhance stability and performance characteristics, a whitening nanostructured lipid carrier (NLC) was synthesized through high-pressure homogenization. This method entailed the incorporation of solid lipids, a liquid lipid, and a compound emulsifier, with deionized water fulfilling the roles of solid phase, liquid phase, and water phase, respectively. The NLC's particle size, Zeta potential, stability, encapsulation efficiency, and other parameters were assessed using techniques such as particle sizer, stability analyzer, and HPLC. The results showed that the NLC for phenylethyl resorcinol prepared by using the optimal formula (7.50 % solid lipids, 3.00 % ethylhexyl palmitate, and 2.00 % Tween 80 and soybean lecithin) has an encapsulation efficiency of 87.11 %, a particle size of 157.2 ± 0.70 nm, a kinetic instability of less than 1.2, and a greatly improved stability, thereby successfully solving the problems of unstable storage and poor solubility of phenylethyl resorcinol.

Ionizable lipid nanoparticles for RAS protease delivery to inhibit cancer cell proliferation

Mutations in RAS, a family of proteins found in all human cells, drive a third of cancers, including many pancreatic, colorectal, and lung cancers. However, there is a lack of clinical therapies that can effectively prevent RAS from causing tumor growth. Recently, a protease was engineered that specifically degrades active RAS, offering a promising new tool for treating these cancers. However, like many other intracellularly acting protein-based therapies, this protease requires a delivery vector to reach its site of action within the cell. In this study, we explored the incorporation of cationic lipids into ionizable lipid nanoparticles (LNPs) to develop a RAS protease delivery platform capable of inhibiting cancer cell proliferation in vitro and in vivo. A library of 13 LNPs encapsulating RAS protease was designed, and each formulation was evaluated for in vitro delivery efficiency and toxicity. A subset of four top-performing LNP formulations was identified and further evaluated for their impact on cancer cell proliferation in human colorectal cancer cells with mutated KRAS in vitro and in vivo, as well as their in vivo biodistribution and toxicity. In vivo, both the concentration of cationic lipid and type of cargo influenced LNP and cargo distribution. All lead candidate LNPs showed RAS protease functionality in vitro, and the top-performing formulation achieved effective intracellular RAS protease delivery in vivo, decreasing cancer cell proliferation in an in vivo xenograft model and significantly reducing tumor growth and size. Overall, this work demonstrates the use of LNPs as an effective delivery platform for RAS proteases, which could potentially be utilized for cancer therapies.

Long-term glucocorticoid infusion impairs epididymal adipocyte metabolism and maturation and affects miR-150–5p actions

The most common form of hypercortisolism is iatrogenic Cushing's syndrome. Lipodystrophy and metabolic disorders can result from the use of exogenous glucocorticoids (GC). Adipocytes play an important role in the production of circulating exosomal microRNAs, and knockdown of Dicer promotes lipodystrophy. The aim of this study is to investigate the effect of GCs on epididymal fat and to assess their influence on circulating microRNAs associated with fat turnover. The data indicate that despite the reduction in adipocyte volume due to increased lipolysis and apoptosis, there is no difference in tissue mass, suggesting that epididymal fat pad, related to animal size, is not affected by GC treatment. Although high concentrations of GC have no direct effect on epididymal microRNA-150–5p expression, GC can induce epididymal adipocyte uptake of microRNA-150–5p, which regulates transcription factor Ppar gamma during adipocyte maturation. In addition, GC treatment increased lipolysis and decreased glucose-derived lipid and glycerol incorporation. In conclusion, the similar control and GC epididymal fat mass results from increased dense fibrogenic tissue and decreased adipocyte volume induced by the lipolytic effect of GC. These findings demonstrate the complexity of epididymal fat. They also highlight how this disease alters fat distribution. This study is the first in a series published by our laboratory showing the detailed mechanism of adipocyte turnover in this disease.

Antioxidant capacity and lipid composition of Brachionus plicatilis and Artemia enriched with a mixture of different post-processing formats of Navicula salinicola and Isochrysis galbana and lipid emulsions

Live prey used by the aquaculture industry are usually poor in some essential nutrients including long chain polyunsaturated fatty acids (LC-PUFA) and must be enriched to improve their nutritional value prior to larval feeding. Standard enrichment protocols are commonly based on lipid emulsions, being associated to a high oxidative stress condition. The combination of microalgae and lipid emulsion can palliate this situation, where the oxidative stress can be partially compensated by the antioxidant compounds present in microalgae. The maintenance of living microalgae in culture facilities is laborious, and the produced biomass may present fluctuating properties, leading to a serious bottleneck in the cultivation of live prey. Hence, substitutes for live microalgae including pastes or dried formats are receiving increasing research attention due to its nutritional stability, longer shelf-life and easy handling. In this study four different microalgae formats combined with a lipid emulsion are tested as enrichment products for Brachionus plicatilis and Artemia. Thus, fresh, frozen and spray-dried Navicula salinicola (NFRE, NFRO and NSD, respectively), and spray-dried Isochrysis galbana (ISD) were mixed with a commercial oil concentrate (IncromegaTM) or a marine lecithin (LC 60®), and added for 5 h to the rotifer or Artemia culture media. The antioxidant capacity of the microalgae extracts and the live prey activity of antioxidant enzymes, peroxides index (PxI) and thiobarbituric acid reactive substances (TBARS) were evaluated. The lipid profile of microalgae formats and enriched live preys was also determined. Ethyl acetate extract was the most antioxidant active extract of all microalgae formats. In addition, overall, I. galbana seems to be better than any N. salinicola format for a more effective protection against oxidative stress and for live prey lipid enrichment. Both rotifer and Artemia cultured with the mixture of I. galbana and the lipid emulsion generally showed higher DHA/EPA and EPA/ARA ratios. Moreover, the combination of the microalgae with LC 60® lipid emulsion highly favored Artemia´s polar lipid and DHA incorporation. Among microalgae products, both spray-dried formats better enhanced live prey n-3 LC-PUFA content. Our results highlight the great potential of new microalgae-derived products to improve effectiveness of current live prey lipid enrichment protocols used in aquaculture.

Effect of oil–water colloidal states in liquid feeds on extrudability and textural attributes of high-moisture meat alternatives

Lipid incorporation into high-moisture meat alternatives by extrusion processing and its effect on extrudability, visual appearance, textural and structural properties is highly dependent on the colloidal state of the oil phase.

Reusing a residue from the juice industry: extraction of oil from passion fruit seeds and its incorporation into liposomes

AbstractBACKGROUNDPassion fruit seeds are a residue of the pulp and juice production industry and have the potential to be used for the extraction of vegetable oil contained in them, which is rich in bioactive compounds. This work evaluated the extraction of oil using organic solvents (mixtures of hexane–ethanol–water and hexane–methanol–water) and the use of ethanol as an alternative to methanol, which is used in traditional methodologies. Several solvent mixtures were investigated to determine the condition that provides the highest oil yield. Subsequently, liposomes were produced using the ethanol injection method and the influence of oil addition on the physical–chemical characteristics of the nanoparticulated system was evaluated.RESULTS AND CONCLUSIONThe results showed that the highest oil yield was obtained using the mixture of hexane, ethanol and water, indicating the possibility of replacing methanol with a less toxic solvent. In addition, good results were obtained for the incorporation of lipids with passion fruit oil into liposomes, with the best conditions obtained being for lipid concentrations of 4.64 and 6.96 mmol L−1 with 10% and 17% of oil content, respectively. © 2023 Society of Chemical Industry (SCI).

Novel bioactive lipids enhanced HDL-mediated cholesterol efflux from macrophages through the ABCA1 receptor pathway

High-density lipoprotein (HDL) has traditionally been acknowledged as "good cholesterol" owing to its significant association with a decreased risk of atherosclerosis. This association is primarily attributed to HDL's direct involvement in cholesterol efflux capacity, which plays a pivotal role in reverse cholesterol transport. A novel active compound from Nannochloropsis microalgae termed lyso-DGTS, a lipid that contains EPA fatty acids, was previously isolated and found to increase paraoxonase 1 activity and enhance HDL-mediated cholesterol efflux and HDL-induced endothelial nitric oxide release. Here, the effect of different lyso-DGTS derivatives and analogs on HDL-mediated cholesterol efflux from macrophages was examined, and the mechanism was explored. Structure–activity relationships were established to characterize the essential lipid moieties responsible for HDL-mediated cholesterol efflux from macrophages. Lyso-DGTS, 1-carboxy-N-N-N-trimethyl-3-oleamidopropan-1-aminium, and lyso-platelet-activating factor increased HDL-mediated cholesterol efflux from macrophages dose-dependently, mainly via the ABCA1-mediated cholesterol efflux pathway. The effect of lyso-DGTS derivatives and analogs on the surface polarity of HDL was examined using the Laurdan generalized polarization (GP) assay. A reverse Pearson linear regression was obtained between Laurdan GP values and HDL-mediated cholesterol efflux. Because the incorporation of bioactive lipids into the surface phospholipid layer of HDL leads to a decrease in Laurdan GP, these bioactive lipids may induce lower phospholipid ordering and greater free space on the HDL particle surface, thereby enhancing apolipoprotein A1 binding to the ABCA1 receptor and improving ABCA1 cholesterol-mediated efflux. Our findings suggest a beneficial effect of lyso-DGTS and its bioactive lipid derivatives on increasing HDL-mediated cholesterol efflux activity from macrophages, which may impact atherosclerosis attenuation.

Comparison of the effect of various sources of saturated fatty acids on infant follow-on formulas oxidative stability and nutritional profile.

Fortification of infant follow-on formulas (IFF) with docosahexaenoic acid (DHA), which is prone to lipid oxidation, is required by European regulation. This study aimed to identify lipid formulation parameters that improve the nutritional profile and oxidative stability of IFF. Model IFF were formulated using different lipid and emulsifier sources, including refined (POM) or unrefined red palm oil (RPOM), coconut oil (COM), dairy fat (DFOM), soy lecithin, and dairy phospholipids (DPL). After an accelerated storage, RPOM and DFOM with DPL had improved oxidative stability compared to other IFF. Specifically, they had a peroxide value twice lower than POM and 20% less loss of tocopherols for DFOM-DPL. This higher stability was mainly explained by the presence of compounds such as carotenoids in RPOM and sphingomyelin in DFOM-DPL very likely acting synergistically with tocopherols. Incorporation of dairy lipids and carotenoids into DHA-enriched IFF compositions seems promising to enhance their stability and nutritional quality.

Decline in Cardiolipin in Hematopoietic Stem Cell during Aging Alters Their Regenerative Potential

Hematopoietic Stem Cells (HSCs) are known for their regenerative potential. This property allowed the use of HSC in bone marrow transplantation to treat hematological disorders such as leukemia. However, aging influences these characteristics of HSCs. The major hallmarks of aging are limited self-renewal and reconstitution capacity resulting in HSC exhaustion in aged individuals. Mitochondrial metabolism and activity are active drivers of HSC fate decisions and data from our lab shows that mitochondria in aged HSCs have increased sphericity with a polarized mitochondrial network and a lower mitochondrial membrane potential. An important aspect of mitochondrial functions is the lipid composition of their membranes. A lipid trafficking assay showed an atypical pattern of lipid incorporation by mitochondria in aged HSCs suggesting that mitochondrial lipids become abnormal upon aging. The overall content of Cardiolipin (CL) which is a signature mitochondrial lipid, found exclusively in the inner mitochondrial membrane was also reduced during aging. This was done with flow cytometry using a CL marker NAO (Non-Acridine Orange). Tafazzin encoded by the gene TAZ is crucial for remodeling CL into its mature form which is required for maintaining mitochondrial functions. Mutations in the TAZ gene have been known to cause a reduction in the synthesis of mature CL which is often associated with HSCs functional defect. Western Blot analysis showed a considerable decrease in Taz protein expression in aged stem and progenitor cells (HSPC) compared to young HSPC. To understand the effect of reduced Taz expression on HSC functions, we used a doxycycline inducible, sh-RNA mediated TAZ knock-down (KD) mouse model. Bone marrow analysis indicates that the frequency of HSC and multipotent progenitors decreases significantly in 6-month-old TAZ KD mice compared to wild type (WT) mice. Our data shows that following 5FU induction TAZ KD mice have a significant reduction in the multipotent progenitor and HSC population on day 10 and day 21 post 5FU, indicating TAZ KD decrease the regenerative potential of HSCs under stress conditions. Further pointing towards the significance of cardiolipin in HSC regeneration. TAZ KD mice also have abnormal peripheral blood recovery post 5FU mainly affecting the platelets. We next examined the repopulation potential of HSCs isolated from TAZ KD mice using serial competitive transplantation assays. While primary transplanted mice of TAZ KD HSCs exhibited similar donor-cell chimerism compared to mice transplanted with WT HSCs, about 50-60%, secondary recipient of TAZ KD cells had significantly lower donor-cell chimerism than secondary recipients of WT cells. Further, donor-derived HSC frequency in the bone marrow of recipients of TAZ KD HSCs 24 weeks following engraftment was also significantly lower than recipients of WT HSCs. Hence, Taz-deficiency causes a defect in HSC self-renewal under regenerative conditions. We next evaluated whether restoring cardiolipin levels in mid-aged HSCs could rescue their functions. For this, mid-aged HSC were incubated for 3 days under conditions that maintain HSC functions in vitro with Alcar (Acetyl-L-Carnitine), which promotes CL production. In lipid incorporation assay, Alcar supplementation restored mitochondrial membrane potential, mitochondrial organization and lipid incorporation of mid-aged HSCs. In competitive transplant assay, while mid-aged WT HSC treated with vehicle gave rise to a myeloid-bias graft, Alcar supplemented mid-aged WT HSC gave rise to a more balanced graft with increased ratio lymphoid to myeloid cells in the peripheral blood 16 weeks following transplantation, albeit the rescue was partial. This study indicates that HSC mitochondrial membrane lipids become abnormal with aging which causes a decline in HSC function and highlights the critical role of mitochondrial cardiolipin in HSC functions. It implies that metabolite supplementation is a viable option for restoring age-related HSC functional defects.

Enhancement of Anti-inflammatory Effects of Synthetic High-Density Lipoproteins by Incorporation of Anionic Lipids.

Phosphatidylserine (PS) is an anionic phospholipid component in endogenous high-density lipoprotein (HDL). With the intrinsic anti-inflammatory effects of PS and the correlation between PS content and HDL functions, it was hypothesized that incorporating PS would enhance the therapeutic effects of HDL mimetic particles. To test this hypothesis, a series of synthetic high-density lipoproteins (sHDLs) were prepared with an apolipoprotein A-I (ApoA-1) mimetic peptide, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-glycero-3-phospho-l-serine (POPS). Incorporating PS was found to improve the particle stability of sHDLs. Moreover, increasing the PS content in sHDLs enhanced the anti-inflammatory effects on lipopolysaccharide-activated macrophages and endothelial cells. The incorporation of PS had no negative impact on cholesterol efflux capacity, in vivo cholesterol mobilization, and did not affect the pharmacokinetic profiles of sHDLs. Such results suggest the therapeutic potential of PS-containing sHDLs for inflammation resolution in atherosclerosis and other inflammatory diseases.

Shake It Off! Acoustic Manipulation of Lipid Vesicles for Mass Spectrometric Analysis.

Analyzing lipid assemblies, including liposomes and extracellular vesicles (EVs), is challenging due to their size, diverse composition, and tendency to aggregate. Such vesicles form with a simple phospholipid bilayer membrane, and they play important roles in drug discovery and delivery. The use of mass spectrometry (MS) allows for broad analysis of lipids from different classes; however, their release from the higher order structural aggregates is typically achieved by chemical means. Mechanical disruption by high frequency surface acoustic waves (SAW) is presented as an appealing alternative to preparing lipid vesicles for MS sampling. In this work, SAWs used to disrupt liposomes allow for the direct analysis of their constituent lipids by employing SAW nebulization with corona discharge (CD) ionization. We explore the effects of duration, frequency, and incorporation of nonpolar lipids, including cholesterol, on the SAW's ability to disrupt the liposome. We also report on the successful MS analysis of liposome-derived lipids along with cytochrome C in solution, thus demonstrating applications to aqueous samples and native MS conditions.