Membrane Lipid Research Articles

Overexpression of the GmPM35 Gene Significantly Enhances Drought Tolerance in Transgenic Arabidopsis and Soybean

Drought stress is one of the major adversity stresses affecting soybean (Glycine max [L.] Merr.) yield. Late embryogenesis abundant protein (LEA protein) is a large family of proteins widely distributed in various types of organisms, and this class of proteins plays an important role in protecting proteins, membrane lipids, and lipids inside the cell. The soybean GmPM35 gene is a member of the LEA_6 subfamily. The expression of the GmPM35 gene was significantly increased after drought stress in soybeans. A subcellular localization assay confirmed that the gene acts on the cell membrane. Against wild-type Arabidopsis thaliana, we found that Arabidopsis lines overexpressing the GmPM35 gene were significantly more drought-tolerant at germination and seedling stages under drought stress. To further investigate the drought tolerance function of this gene in soybeans, nine overexpression lines of the T3 generation soybean GmPM35 gene and two editing lines of the T3 generation soybean GmPM35 gene were obtained by Agrobacterium-mediated method using a wild-type soybean strain (JN28) as a receptor. Germination rate, root length, chlorophyll (CHL) content, Proline (Pro) content, malondialdehyde (MDA) content, superoxide anion (O2−) content, hydrogen peroxide (H2O2) content, (NBT, DAB) staining, and activities of antioxidant enzymes (CAT, SOD, POD), and photosynthetic physiological indexes of the three different types of strains were measured and analyzed before and after drought stress. Combined with the results of rehydration experiments and physiological and biochemical indices, we found that overexpression of the GmPM35 gene protected the activities of antioxidant enzymes under drought stress. The activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were increased by an average of 34.28%, 26.12%, and 30.01%, respectively, in soybean plants overexpressing the GmPM35 gene compared with wild-type soybeans. Under drought stress conditions, soybean plants overexpressing the GmPM35 gene showed an average increase of 76.81% in photosynthesis rate (Pn), 39.8% in transpiration rate (Tr), 126% in stomatal conductance (Gs), 47.71% in intercellular CO2 concentration (Ci), and 26.44% in instantaneous water use efficiency (WUEi). The improvement of these indexes helped to reduce the accumulation of reactive oxygen species (ROS) in the plants. In addition, we found that under drought stress, the MDA content was reduced by an average of 18.8%, and the Pro content was increased by an average of 60.14% in soybean plants overexpressing the GmPM35 gene, and the changes in these indexes indicated that the plants had stronger antioxidant and osmoregulatory capacities in response to drought stress. In summary, this experiment demonstrated that the GmPM35 gene plays an important role in soybean tolerance to drought stress, and by overexpressing the GmPM35 gene, soybean plants can better tolerate drought stress and maintain normal physiological functions.

Pharmacodynamic characterization and evaluation of oxidative stress effects of digitoxigenin derivatives on HeLa cells.

Cancer is a leading cause of death worldwide and its treatment is hampered by the lack of specificity and side effects of current drugs. Cardiotonic steroids (CTS) interact with Na+/K+-ATPase (NKA) and induce antineoplastic effects, but their narrow therapeutic window is key limiting factor. The synthesis of digitoxigenin derivatives with glycosidic unit modifications is a promising approach to develop more selective and effective antitumor agents. This study aimed to compare the pharmacological properties as well as the cytotoxic effects of digitoxigenin-α-L-amiceto-pyranoside and digitoxigenin-α-L-rhamno-pyranoside and to evaluate the mechanism of these derivatives in oxidative conditions in HeLa cells. The rhamnose derivative increased the binding affinity and inhibitory effect of digitoxigenin by approximately 5-15 times, unlike the amicetose derivative. Despite this difference, both compounds similarly increased H2O2 levels, induced membrane lipid peroxidation, and reduced GSH levels and SOD activity at nanomolar concentrations. This study highlights the importance of the sugar moiety in CTS structure for NKA binding and demonstrates that a primary mechanism of cytotoxicity of digitoxigenin derivatives may involve cellular oxidative stress, underscoring their potential as therapeutic agents for cancer treatment.

Effects of aluminum on metabolism of reactive oxygen species and reactive nitrogen species in root tips of different Eucalyptus species

On acidified soil, the growth of Eucalyptus is seriously restricted by aluminum (Al) stress. Therefore, breeding Eucalyptus species with excellent Al tolerance, developing the genetic potential of species, and improving tolerance to Al stress are important for the sustainable development of artificial Eucalyptus forests. By observing the occurrence and distribution of the main reactive oxygen species (ROS) and reactive nitrogen species (RNS) in root tips of Eucalyptus seedlings under Al stress, this study analyzed change in the growth and physiological indexes of Eucalyptus seedlings under Al stress. The antioxidant enzymes activities of the root tips of different Eucalyptus species induced by Al stress resulted in different ROS and RNS contents, ultimately resulting in differing degrees of membrane lipid peroxidation. In addition to suppressions of root relative elongation and root activity, the accumulations of soluble sugar, soluble protein, and proline can be used as indicators of Al sensitivity in Eucalyptus species. This may be an important determinant of the differences in Al tolerance among Eucalyptus species. The accumulation of ROS and RNS in the roots of E. grandis and E. tereticornis resulted in severe oxidative and nitrification stress. The tolerance of E. urophylla and E. urophylla × E. grandis to Al stress was stronger than that of E. grandis and E. tereticornis. Differences in Al toxicity tolerance were related to long-term selection of the original habitat of the species; moreover, the Al tolerance was hereditary. Eucalyptus urophylla × E. grandis had stronger Al tolerance than its parents, which is indicative of heterosis. These results provide theoretical support for the breeding of tree species in areas with acidic soil.

Utilizing physiologies, transcriptomics, and metabolomics to unravel key genes and metabolites of Salvia miltiorrhiza Bge. seedlings in response to drought stress

Drought stress inhibits Salvia miltiorrhiza Bunge (S. miltiorrhiza) seedling growth and yield. Here, we studied the effects of drought stress on the different parts of S. miltiorrhiza seedlings through physiological, transcriptomic, and metabolomics analyses, and identified key genes and metabolites related to drought tolerance. Physiological analysis showed that drought stress increased the accumulation of hydrogen peroxide (H2O2), enhanced the activity of peroxidase (POD), decreased the activity of catalase (CAT) and the contents of chlorophyll b and total chlorophyll, reduced the degree of photosynthesis, enhanced oxidative damage in S. miltiorrhiza seedlings, and inhibited the growth of S. miltiorrhiza plants. Transcriptome analyses revealed 383 genes encoding transcription factors and 80 genes encoding plant hormones as hypothetical regulators of drought resistance in S. miltiorrhiza plants. Moreover, differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs) are involved in a variety of biological processes, such as proline and glycine betaine metabolism, and biosynthesis of tanshinones and phenolic acids. Additionally, it has barely been reported that the AHL gene family may be involved in regulating the neocryptotanshinone biosynthesis. In conclusion, our results suggest that drought stress inhibits S. miltiorrhiza seedling growth by enhancing membrane lipid peroxidation, attenuating the antioxidant system, photosynthesis, and regulating proline and glycine betaine metabolism, transcription factors and plant hormones, and tanshinones and phenolic acid metabolism pathways. This study provides new insights into the complex mechanisms by which S. miltiorrhiza responds to drought stress.

The Function of Two Brassica napus β-Ketoacyl-CoA Synthases on the Fatty Acid Composition

Rapeseed (Brassica napus L.) is one of the four major oilseed crops in the world and is rich in fatty acids. Changes in the fatty acid composition affect the quality of rapeseed. Fatty acids play various roles in plants, but the functions of the genes involved in the fatty acid composition during plant development remain unclear. β-Ketoacyl-CoA synthase (KCS) is a key enzyme involved in the elongation of fatty acids. Various types of fatty acid products are used to build lipid molecules, such as oils, suberin, wax, and membrane lipids. In B. napus, BnaKCSA8 and BnaKCSC3 belong to the KCS family, but their specific functions remain unclear. This study cloned BnaKCSA8 and BnaKCSC3 from Brassica napus L. and analyzed their functions. The gene structures of BnaKCSA8 and BnaKCSC3 were similar and they were localized to the endoplasmic reticulum (ER). In yeast, overexpression of BnaKCSA8 increased the ratios of palmitoleic acid and oleic acid, while BnaKCSC3 decreased the ratios of oleic acid. In Arabidopsis, overexpression of BnaKCSA8 and BnaKCSC3 lead to an increase in the proportion of linoleic acid and a decrease in the erucic acid. In summary, BnaKCSA8 and BnaKCSC3 altered the composition ratios of fatty acids. These findings lay the foundation for an understanding of the role of KCS in the fatty acids in rapeseed, potentially improving its health and nutritional qualities.

Long-term fasting induces a remodelling of fatty acid composition in erythrocyte membranes.

Long-term fasting (LF) activates an adaptative response to switch metabolic fuels from food glucose to lipids stored in adipose tissues. The increase in free fatty acid (FFA) oxidation during fasting triggers health benefits. We questioned if the changes in lipid metabolism during LF could affect lipids in cell membranes in humans. We thus analysed the FA composition in erythrocyte membranes (EM) during 12.6 ± 3.5 days of LF and 1 month after food reintroduction. A total of 98 subjects out of three single-arm interventional studies underwent a medical supervised long-term fasting (12.6 ± 3.5 days) programme. The distribution pattern of 26 FA as well as the HS-Omega-3 Index were assessed in the EM using gas chromatography. Eighteen of 26 FA showed significant changes. Within the group of saturated FA, myristic (14:0) and stearic acid (18:0) decreased while palmitic (16:0) and arachid acid (20:0) increased. While most monounsaturated FA increased, trans fatty acids decreased or remained unchanged. Within the polyunsaturated FA, arachidonic (20:4n6) and docosahexaenoic (22:6n3) acid increased, while linoleic (18:2n6), alpha-linolenic (18:3n3) and eicosapentaenoic acid (20:5n3) decreased. Consequently, the HS-Omega-3 Index increased. 11 out of the 18 FA with significant changes returned to baseline levels 1 month afterwards. Levels of linoleic and alpha-linolenic acid increased over baseline levels. Long-term fasting triggers changes in the FA composition of EM.

α-Synuclein interaction with POPC/POPS vesicles.

We have investigated the adsorption of the amyloid-forming protein α-Synuclein (αSyn) onto small unilamellar vesicles composed of a mixture of zwitterionic POPC and anionic POPS lipids. αSyn monomers adsorb onto the anionic lipid vesicles where they adopt an α-helical secondary structure. The degree of adsorption depends on the fraction of anionic lipid in the mixed lipid membrane, but one needs to consider the electrostatic shift of the serine pKa with increasing fraction of POPS. The vesicles with adsorbed αSyn monomers are kinetically stable. However, after fibrils have been formed, here triggered by the addition of a small concentration of pre-formed fibrils (seeds), we observed that the average vesicle size increased by approximately a factor of two. This increase in the vesicle size can be explained by vesicle fusion taking place during the fibril formation process.

Zahabba Patch: Innovation of Gingival Mucoadhesion Patch Combination of Ginger (Zingiber Officinale) and Black Cubit (Nigella Sativa) as Gingivitis Treatment

Background The prevalence of periodontal disease in Indonesia reaches 74.1%. Treatment of gingivitis by administering NSAIDs has a side effect of gastritis. Anti-inflammatory and analgesic derived from natural ingredients are known to cause minimal side effects on the patient's body. The active compounds gingerol in ginger (Zingiber officinale) and quercetin in black cumin (Nigella sativa) are known to function as good anti-inflammatory and analgesic agents. Objectives This study aims to analyze the effectiveness of gingerol in ginger extract and qurcetin in black cumin as an anti-inflammatory and analgesic packaged in a mucoadhesion patch as a treatment for gingivitis. Material and Methods Preparations of gingerol and quercetin were obtained by biocomputation using the PyRx program, the Pymol program, the RCSB PDB website, the Lipinski rule of five page, the PDBSum website, and also the pkCSM website. Physicochemical, pharmacokinetic, toxicity, and molecular docking tests were carried out to determine the biocompatibility of the active ingredients used. Results In the physicochemical tests, the compounds glycerol and quercetin can penetrate somatic cell membranes, have good solubility in water, can dissolve easily in the blood, are easily distributed in the body, and can maintain their position and bond strength with the target macro protein. The results of the AMES toxicity, Hepatotoxicity and skin sensitization tests showed negative values for both gingerol and quercetin. In the molecular docking test, the gingerol and quercetin compounds have a tendency to form bonds with COX-1 compared to arachidonic acid without having to increase the dose so that they become competitors to the native ligand. Conclusion The active compounds gingerol in ginger extract (Zingiber officinale) and quercetin in black cumin extract (Nigella sativa) contained in Zahabba patch are good anti-inflammatory and analgesic by inhibiting the synthesis of pro-inflammatory cytokines such as PGI2, PGE2, and TXA2 by being competitive inhibitor of the enzyme phospholipase A2 against membrane lipids and the enzyme cyclooxygenase-1 (COX-1) against arachidonic acid.

Patterns of variations in lipid molecular profile during larval development of red king crab, Paralithodes camtschaticus, and Japanese mitten crab, Eriocheir Japonica

The red king crab, Paralithodes camtschaticus, and the Japanese mitten crab, Eriocheir japonica, are the major commercially valuable species. In addition to their high nutritional value, these crabs are used as objects of ecological research. To extend our knowledge of crustacean biochemistry and provide a more comprehensive model of lipidomic patterns during embryonic and larval development of these crab species, we studied the dynamics of molecular species profiles of reserve lipids such as triacylglycerols (TG) and membrane lipids such as glycerophospholipids (PL). A complete disappearance of TG was observed in zoea IV larvae of E. japonica and zoea III larvae of P. camtschaticus. The appearance of TG at older stages of larval development was accompanied by considerable changes in TG composition. The dynamics of PL with major polyunsaturated fatty acids (PUFA) (20:5n-3, 22:6n-3, and 20:4n-3) during the larval development was species-specific. The obtained results indicate different demands for PUFA in P. camtschaticus and E. japonica, which can be taken into account when selecting optimum diets. The lipidomic approach allows identifying new patterns of lipid changes during crab embryonic development, which may be useful for improvement of aquaculture techniques.

Cadmium translocation combined with metabolomics analysis revealed potential mechanisms of MT@MSN-CS and GSH@MSN-CS in reducing cadmium accumulation in rice (Oryza sativa L.) grains.

Applying nano-delivery systems for phytohormones via foliar application has proven effective in reducing grain cadmium (Cd) levels in crops. However, the mechanisms underlying this reduction remain inadequately understood. This study integrated the determination of leaf photosynthetic parameters, Cd translocation analysis, and metabolomics to elucidate the effects of reduced glutathione (GSH) and melatonin (MT), delivered with or without chitosan-encapsulated mesoporous silica nanoparticles (MSN-CS), on grain Cd levels in rice. Our findings revealed that the foliar application of MT@MSN-CS significantly outperformed MT alone in reducing grain Cd levels and enhancing leaf photosynthesis under Cd stress. Conversely, GSH@MSN-CS showed comparable effects to GSH alone. Foliar-applied GSH@MSN-CS and MT@MSN-CS both decreased the Cd transport coefficients from panicle nodes to brown rice by 26.2-53.3%, with MT@MSN-CS demonstrating superior efficiency in reducing Cd concentrations across roots, stems, leaves, panicle nodes, and grains. Metabolomic analysis revealed substantial shifts in rice metabolite profiles following GSH@MSN-CS and MT@MSN-CS treatments. Foliar application of MT@MSN-CS or GSH@MSN-CS may rapidly and effectively activate the primary antioxidant defense system and alleviate membrane lipid peroxidation in rice grown on low-to-moderately Cd-contaminated soils by upregulating amino acid metabolism. The secondary defense mechanism, phenylpropanoid biosynthesis, was reprogrammed to reduce energy expenditure and decrease Cd translocation.

Mitochondrial Porin Is Required for Versatile Biocontrol Trait-Involved Biological Processes in a Filamentous Insect Pathogenic Fungus.

The mitochondrial voltage-dependent anion channel (VDAC) is the major channel in the mitochondrial outer membrane for metabolites and ions. VDACs also regulate a variety of biological processes, which vary in the number of VDAC isoforms across different eukaryotes. However, little is known about VDAC-mediated biocontrol traits in biocontrol fungi. Here, the only VDAC isoform (BbOmm1) in the filamentous insect pathogenic fungus Beauveria bassiana was characterized, which was crucial for maintenance of mitochondrial homeostasis and function and important biocontrol traits. Besides serious impairment of fungal growth, conidiation, and germination, inactivation of BbOmm1 led to increased sensitivity/tolerance to oxidative and osmotic stresses and production of oosporein and other secondary metabolites, which corresponded to the mitochondrial damage, downregulation of membrane lipid and cell wall homeostasis-involved genes, and upregulation of detoxification genes and biosynthesis gene clusters of those metabolites. These results enrich our understanding of VDAC-mediated biocontrol traits in insect fungal pathogens.

Molecular dynamics at immune synapse lipid rafts influence the cytolytic behavior of CAR T cells.

Chimeric antigen receptor T cells (CART) targeting CD19 through CD28.ζ signaling induce rapid lysis of leukemic blasts, contrasting with persistent tumor control exhibited by 4-1BB.ζ-CART. We reasoned that molecular dynamics at the CART immune synapse (CARIS) could explain differences in their tumor rejection kinetics. We observed that CD28.ζ-CART engaged in brief highly lethal CARIS and mastered serial killing, whereas 4-1BB.ζ-CART formed lengthy CARIS and relied on robust expansion and cooperative killing. We analyzed CARIS membrane lipid rafts (mLRs) and found that, upon tumor engagement, CD28.ζ-CAR molecules rapidly but transiently translocated into mLRs, mobilizing the microtubular organizing center and lytic granules to the CARIS. This enabled fast CART recovery and sensitivity to low target site density. In contrast, gradual accumulation of 4-1BB.ζ-CAR and LFA-1 molecules at mLRs built mechanically tonic CARIS mediating chronic Fas ligand-based killing. The differences in CD28.ζ- and 4-1BB.ζ-CARIS dynamics explain the distinct cytolytic behavior of CART and can guide engineering of more adaptive effective cellular products.

Characterization of Key Lipid Components in the Cell Membrane of Freeze-Drying Resistant Lacticaseibacillus paracasei Strains Using Nontargeted Lipidomics.

Lactic acid bacteria (LAB) are usually freeze-dried into powder for transportation and storage, with the bacterial membrane playing a crucial role in this process. However, different strains exhibit different levels of freeze-drying resistance in their cell membranes. In this study, Lacticaseibacillus paracasei (L. paracasei) strains 1F20, K56, and J5, demonstrating survival rates of 59.51, 25.86, and 4.05% after freeze-drying, respectively, were selected. The membrane structure and composition of these strains were subsequently analyzed. Bacterial live/dead staining results indicated that strain 1F20 maintained the highest membrane integrity after drying. Nontargeted lipidomics analysis revealed six differential lipid species that differed in membrane lipid compositions. KEGG functional enrichment analysis revealed 13 significantly different pathways, with glycerophospholipid metabolism being the most critical. This study explored the membrane composition of L. paracasei at the cellular level and identified key lipid species associated with freeze-drying resistance, providing a reference for screening highly resistant strains.

Autoantibodies against phosphatidylserine and DNA during canine Dirofilaria immitis infection.

Heartworm infection caused by Dirofilaria immitis induces a devastating disease that greatly affects the global canine population. The mechanism leading to heartworm pathology has been attributed to be mostly by mechanical damage of the worm to the dog´s vascular system and immune-mediated, but the latter processes are not completely understood. Autoantibodies targeting host molecules such as lipids and nucleic acids have been described with pathological roles during malaria and COVID-19 and mediating anemia and thrombocytopenia. We hypothesized that autoantibodies could be present and have a pathological role during canine heartworm disease caused by D. immitis. In this study, we analyzed the levels of autoantibodies (IgM and IgG) against membrane lipid phosphatidylserine (PS) and DNA in the serum of 169 canine samples based on D. immitis infection. First, our results found significant levels of anti-PS IgM and IgG autoantibodies that were associated with D. immitis-positive when compared to D. immitis-negative samples. Second, we found that autoantibodies, particularly anti-PS, are correlated with hematological parameters such as low platelet count suggesting an association with pathologies such as thrombocytopenia. Altogether, these findings elucidate the understudied presence and pathological role of autoantibodies during canine heartworm disease by D. immitis with implications as biomarkers of disease.

Toxic Effects of Butanol in the Plane of the Cell Membrane.

Solvent toxicity limits n-butanol fermentation titer, increasing the cost and energy consumption for subsequent separation processes and making biobased production more expensive and energy-intensive than petrochemical approaches. Amphiphilic solvents such as n-butanol partition into the cell membrane of fermenting microorganisms, thinning the transverse structure, and eventually causing a loss of membrane potential and cell death. In this work, we demonstrate the deleterious effects of n-butanol partitioning upon the lateral dimension of the membrane structure, called membrane domains or lipid rafts. Lipid rafts are regions of the cell membrane enriched with certain lipids, providing a reservoir of high melting temperature lipids and a platform for membrane protein partitioning and oligomerization. Neutron scattering experiments and molecular dynamics simulations revealed that n-butanol increased the size of the lipid domains in a model membrane system. The data showed that n-butanol partitions more into the disordered lipid regions than into the raft-like phase, leading to a differential thinning of these coexisting phases in the plane of the membrane and increasing the hydrophobic mismatch. The resulting increase in line tension at the interface favors domain coalescence to minimize the ratio of the interfacial length to domain area. A detailed computational investigation of the lipid domain interface identifies the boundary as a site of membrane disorder and thinning due to an accumulation of n-butanol. Solvent-induced changes to domain morphology and membrane instability at the domain interface are unrecognized modes of solvent-induced stress to fermenting microbes, representing targets for new solvent tolerance strategies to increase the n-butanol titer.

The influence of phosphoinositide lipids in the molecular biology of membrane proteins: recent insights from simulations.

The phosphoinositide family of membrane lipids play diverse and critical roles in eukaryotic molecular biology. Much of this biological activity derives from interactions of phosphoinositide lipids with integral and peripheral membrane proteins, leading to modulation of protein structure, function, and cellular distribution. Since the discovery of phosphoinositides in the 1940s, combined molecular biology, biophysical, and structural approaches have made enormous progress in untangling this vast and diverse cellular network of interactions. More recently, in silico approaches such as molecular dynamics simulations have proven to be an asset in prospectively identifying, characterising, explaining the structural basis of these interactions, and in the best cases providing atomic level testable hypotheses on how such interactions control the function of a given membrane protein. This review details a number of recent seminal discoveries in phosphoinositide biology, enabled by advanced biomolecular simulation, and its integration with molecular biology, biophysical, and structural biology approaches. The results of the simulation studies agree well with experimental work, and in a number of notable cases have arrived at the key conclusion several years in advance of the experimental structures. Condensed title:Simulations of phosphoinositides and membrane proteins SUMMARY: Hedger and Yen review developments in simulations of phosphoinositides and membrane proteins.

Triacylglycerol mobilization underpins mitochondrial stress recovery.

Mitochondria are central to myriad biochemical processes, and thus even their moderate impairment could have drastic cellular consequences if not rectified. Here, to explore cellular strategies for surmounting mitochondrial stress, we conducted a series of chemical and genetic perturbations to Saccharomyces cerevisiae and analysed the cellular responses using deep multiomic mass spectrometry profiling. We discovered that mobilization of lipid droplet triacylglycerol stores was necessary for strains to mount a successful recovery response. In particular, acyl chains from these stores were liberated by triacylglycerol lipases and used to fuel biosynthesis of the quintessential mitochondrial membrane lipid cardiolipin to support new mitochondrial biogenesis. We demonstrate that a comparable recovery pathway exists in mammalian cells, which fail to recover from doxycycline treatment when lacking the ATGL lipase. Collectively, our work reveals a key component of mitochondrial stress recovery and offers a rich resource for further exploration of the broad cellular responses to mitochondrial dysfunction.

Pharmacological reduction of lipid hydroperoxides as a potential modulator of sarcopenia.

We previously reported that elevated expression of phospholipid hydroperoxide glutathione peroxidase 4, an enzyme that regulates membrane lipid hydroperoxides, can mitigate sarcopenia in mice. However, it is still unknown whether a pharmacological intervention designed to modulate lipid hydroperoxides might be an effective strategy to reduce sarcopenia in aged mice. Here we asked whether a newly developed compound, CMD-35647 (CMD), can reduce muscle atrophy induced by sciatic nerve transection. We treated mice daily with vehicle or CMD (15mg/kg, i.p. injection) starting 1day prior to denervation. CMD treatment reduced hydroperoxide generation and blunted muscle atrophy by over 17% in denervated muscle. To test whether CMD can reduce ageing-induced muscle atrophy and weakness, we treated mice with either vehicle or CMD (15mg/kg, i.p. injection) 3days per week for 8months, starting at 18months of age until 26months of age. We measured muscle mass, functional status of neuromuscular junctions, muscle contractile function and mitochondrial function in control and CMD-treated 26-month-old female mice. Treatment with CMD conferred protection against muscle atrophy in both tibialis anterior and extensor digitorum longus that was associated with maintenance of fibre size of MHC 2b and 2x fibres. Mitochondrial respiration was also protected in CMD-treated mice. We also found that muscle force generation was protected with CMD treatment despite denervation in ∼25% of the muscle fibres. Overall, this study shows that pharmacological interventions designed to reduce lipid hydroperoxides might be effective for preventing sarcopenia. KEY POINTS: Sarcopenia in aged mice is associated with muscle loss, contractile dysfunction, denervation, and reduced mitochondrial respiration. CMD-35647 is a pharmocological compound that can neutralize lipid hydroperoxides. 8 month treatment of CMD-35647 mitigated muscle atrophy in tibialis anterior and extensor digitorum longus. 8 month treatment of CMD-35647 improved muscle function in aged mice independent of the neuromuscular junction. Aged mice treated with CMD-35647 had greater respiration in red gastrocnemius muscle when compared to vehicle treated mice.

Halogenated Cholesterol Alters the Phase Behavior of Ternary Lipid Membranes.

Eukaryotic plasma membranes exhibit nanoscale lateral lipid heterogeneity, a feature that is thought to be central to their function. Studying these heterogeneities is challenging since few biophysical methods are capable of detecting domains at submicron length scales. We recently showed that cryogenic electron microscopy (cryo-EM) can directly image nanoscale liquid-liquid phase separation in extruded liposomes due to its ability to resolve the intrinsic thickness and electron density differences of ordered and disordered phases. However, the intensity contrast between these phases is poor compared with conventional fluorescence microscopy and is thus both a limiting factor and a focal point for optimization. Because the fundamental source of intensity contrast is the spatial variation in electron density within the bilayer, lipid modifications aimed at selectively increasing the electron density of one phase might enhance the ability to resolve coexisting phases. To this end, we investigated model membrane mixtures of DPPC/DOPC/cholesterol in which one hydrogen of cholesterol's C19 methyl group was replaced by an electron-rich halogen atom (either bromine or iodine). We characterized the phase behavior as a function of composition and temperature using fluorescence microscopy, Förster resonance energy transfer, and cryo-EM. Our data suggest that halogenated cholesterol variants distribute approximately evenly between liquid-ordered and liquid-disordered phases and are thus ineffective at enhancing the intensity difference between them. Furthermore, replacing more than half of the native cholesterol with halogenated cholesterol variants dramatically reduces the size of the membrane domains. Our results reinforce how small changes in the sterol structure can have a large impact on the lateral organization of membrane lipids.

Tracking the Geometric and Positional Isomerization of Lipid C═C Bonds in the Bacterial Stress Responses by Mass Spectrometry.

The position and configuration of the C═C bond have a significant impact on the spatial conformation of unsaturated lipids, which subsequently affects their biological functions. Double bond isomerization of lipids is an important mechanism of bacterial stress response, but its in-depth mechanistic study still lacks effective analytical tools. Here, we developed a visible-light-activated dual-pathway reaction system that enables simultaneous [2 + 2] cycloaddition and catalytic cis-trans isomerization of the C═C bond of unsaturated lipids via directly excited anthraquinone radicals. Density functional theory calculations revealed the oxygen radical addition transition state and the addition-elimination isomerization mechanism of the reaction. A full-dimensional resolution method for C═C bond position and configuration was developed based on the bifunctional reaction and liquid chromatography-mass spectrometry. This method was then applied to the study of bacterial environmental stress response mechanisms. The C═C bond cis-trans and positional isomerization patterns of Pseudomonas membrane lipids under temperature stress were discovered, and the effect of temperature stress on fatty acid biosynthesis was also revealed. This study not only provides an effective tool and key information for the study of bacterial stress response mechanisms, but also enriches the toolbox of visible light chemical reactions.