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Severe fever with thrombocytopenia syndrome virus (SFTSV), recently named as Dabie bandavirus, belongs to the family Phenuiviridae of the order Bunyavirales, is a newly-identified bunyavirus with a case fatality rate of up to 30%, posing a serious threat to public health. Lipid rafts on plasm membranes are important for the entry of enveloped viruses; however, the role of lipid rafts in bunyavirus entry remains unclear. In this study, we found that methyl-beta-cyclodextrin (MβCD), a drug that disrupts cholesterol in lipid rafts of cell membranes, inhibits SFTSV infection. Additionally, there is a back-complementary effect of SFTSV infection upon the addition of cholesterol. Moreover, the concentration of SFTSV particles in lipid rafts during entry directly indicated the role of lipid rafts as a gateway, whereas MβCD could inhibit SFTSV entry by affecting the structure of lipid rafts. In an in vivo study, MβCD also reduced the susceptibility of mice to SFTSV infection. Our results suggest that SFTSV can interact with Talin1 proteins on lipid rafts to enter host cells by endocytosis of lipid rafts and reveal the potential therapeutic value of MβCD for SFTSV infection., 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 B.V.)

Bioactive metabolites play a crucial role in shaping interactions among diverse organisms. In this study, we identified cyclo(Pro-Tyr), a metabolite produced by Bacillus velezensis, as a potent inhibitor of Botrytis cinerea and Caenorhabditis elegans, two potential cohabitant eukaryotic organisms. Based on our investigation, cyclo(Pro-Tyr) disrupts plasma membrane polarization, induces oxidative stress and increases membrane fluidity, which compromises fungal membrane integrity. These cytological and physiological changes induced by cyclo(Pro-Tyr) may be triggered by the destabilization of membrane microdomains containing the [H + ]ATPase Pma1. In response to cyclo(Pro-Tyr) stress, fungal cells activate a transcriptomic and metabolomic response, which primarily involves lipid metabolism and Reactive Oxygen Species (ROS) detoxification, to mitigate membrane damage. This similar response occurs in the nematode C. elegans, indicating that cyclo(Pro-Tyr) targets eukaryotic cellular membranes., (© 2024. The Author(s).)

Chemotherapy including platinum-based drugs are a possible strategy to enhance the immune response in advanced melanoma patients who are resistant to immune checkpoint blockade (ICB) therapy. However, the immune-boosting effects of these drugs are a subject of controversy, and their impact on the tumor microenvironment are poorly understood. In this study, we discovered that lipid peroxidation (LPO) promotes the formation of lipid rafts in the membrane, which mediated by Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) impairs the sensitivity of melanoma cells to platinum-based drugs. This reduction primarily occurs through the inhibition of immunogenic ferroptosis and pyroptosis by reducing cell membrane pore formation. By disrupting ACSL4-mediaged lipid rafts via the removal of membrane cholesterol, we promoted immunogenic cell death, transformed the immunosuppressive environment, and improved the antitumor effectiveness of platinum-based drugs and immune response. This disruption also helped reverse the decrease in CD8 + T cells while maintaining their ability to secrete cytokines. Our results reveal that ACSL4-dependent LPO is a key regulator of lipid rafts formation and antitumor immunity, and that disrupting lipid rafts has the potential to enhance platinum-based drug-induced immunogenic ferroptosis and pyroptosis in melanoma. This novel strategy may augment the antitumor immunity of platinum-based therapy and further complement ICB therapy., (© 2024. The Author(s).)

β2-Adrenoceptors (β2-ARs) are the most abundant subtype of adrenergic receptors in skeletal muscles. Their activation via a stabilization of postsynaptic architecture has beneficial effects in certain models of neuromuscular disorders. However, the ability of β2-ARs to regulate neuromuscular transmission at the presynaptic level is poorly understood. Using electrophysiological recordings and fluorescent FM dyes, we found that β2-AR activation with fenoterol enhanced an involvement of synaptic vesicles in exocytosis and neurotransmitter release during intense activity at the neuromuscular junctions of mouse diaphragm. This was accompanied by an improvement of contractile responses to phrenic nerve stimulation (but not direct stimulation of the muscle fibers) at moderate-to-high frequencies. β2-ARs mainly reside in lipid microdomains enriched with cholesterol and sphingomyelin. The latter is hydrolyzed by sphingomyelinases, whose upregulation occurs in many conditions characterized by muscle atrophy and sympathetic nerve hyperactivity. Sphingomyelinase treatment reversed the effects of β2-AR agonist on the neurotransmitter release and synaptic vesicle recruitment to the exocytosis during intense activity. Inhibition of G i protein with pertussis toxin completely prevented the sphingomyelinase-mediated inversion in the β2-AR agonist action. Note that lipid raft disrupting enzyme cholesterol oxidase had the same effect on β2-AR agonist-mediated changes in neurotransmission as sphingomyelinase. Thus, β2-AR agonist fenoterol augmented recruitment and release of synaptic vesicles during intense activity in the diaphragm neuromuscular junctions. Sphingomyelin hydrolysis inversed the effects of β2-AR agonist on neurotransmission probably via switching to G i protein-dependent signaling. This phenomenon may reflect a dependence of the β2-AR signaling on lipid raft integrity in the neuromuscular junctions., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Aims: Despite aggressive treatment, the recurrence of glioma is an inevitable occurrence, leading to unsatisfactory clinical outcomes. A plausible explanation for this phenomenon is the phenotypic alterations that glioma cells undergo aggressive therapies, such as TMZ-therapy. However, the underlying mechanisms behind these changes are not well understood., Methods: The TMZ chemotherapy resistance model was employed to assess the expression of intercellular adhesion molecule-1 (ICAM1) in both in vitro and in vivo settings. The potential role of ICAM1 in regulating TMZ chemotherapy resistance was investigated through knockout and overexpression techniques. Furthermore, the mechanism underlying ICAM1-mediated TMZ chemotherapy resistance was examined using diverse molecular biological methods, and the lipid raft protein was subsequently isolated to investigate the cellular subcomponents where ICAM1 operates., Results: Acquired TMZ resistant (TMZ-R) glioma models heightened production of intercellular adhesion molecule-1 (ICAM1) in TMZ-R glioma cells. Additionally, we observed a significant suppression of TMZ-R glioma proliferation upon inhibition of ICAM1, which was attributed to the enhanced intracellular accumulation of TMZ. Our findings provide evidence supporting the role of ICAM1, a proinflammatory marker, in promoting the expression of ABCB1 on the cell membrane of TMZ-resistant cells. We have elucidated the mechanistic pathway by which ICAM1 modulates phosphorylated moesin, leading to an increase in ABCB1 expression on the membrane. Furthermore, our research has revealed that the regulation of moesin by ICAM1 was instrumental in facilitating the assembly of ABCB1 exclusively on the lipid raft of the membrane., Conclusions: Our findings suggest that ICAM1 is an important mediator in TMZ-resistant gliomas and targeting ICAM1 may provide a new strategy for enhancing the efficacy of TMZ therapy against glioma., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rong Hu reports administrative support, article publishing charges, equipment, drugs, or supplies, statistical analysis, and writing assistance were provided by National Natural Science Foundation of China. Qing Wang reports administrative support, article publishing charges, equipment, drugs, or supplies, statistical analysis, travel, and writing assistance were provided by Natural Science Foundation of Jiangsu Province., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Euphorbia lathyris L. (EL) is a traditional poisonous herbal medicine used to treat dropsy, ascites, amenorrhea, anuria and constipation. Processing to reduce toxicity of EL is essential for its safe and effective application. However, there is little known regarding the molecular mechanism of reducing toxicity after EL processing. This research aimed to screen the differential markers for EL and PEL, explore the differential mechanisms of inflammatory injury induced by EL and processed EL (PEL) to expound the mechanism of alleviating toxicity after EL processing. The results showed that 15 potential biomarkers, mainly belonging to diterpenoids, were screened to distinguish EL from PEL. EL promoted the expressions of TLR4, NLRP3, NF-κB p65, IL-1β and TNF-α, increased lipid rafts abundance and promoted TLR4 positioning to lipid rafts. Meanwhile, EL decreased LXRα and ABCA1 expression, and reduced cholesterol efflux. In contrast to EL, the effects of PEL on these indicators were markedly weakened. In addition, Euphorbia factors L 1 , L 2 , and L 3 affected LXRα, ABCA1, TLR4, NLRP3, NF-κB p65, TNF-α and IL-1β expression, influenced cholesterol efflux and lipid rafts abundance, and interfered with the colocalization of TLR4 and lipid rafts. The inflammatory injury caused by processed EL was significantly weaker than that caused by crude EL, and reduction of Euphorbia factors L 1 , L 2 , and L 3 as well as attenuation of inflammatory injury participated in processing-based detoxification of EL. Our results provide valuable insights into the attenuated mechanism of EL processing and will guide future research on the processing mechanism of toxic traditional Chinese medicine., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest regarding the publication of this paper., (Copyright © 2023. Published by Elsevier B.V.)

Autophagy dysfunction has been closely related with pathogenesis of many neurodegenerative diseases and therefore represents a potential therapeutic target. Extracellular vesicles (EVs) may act as potent anti-inflammatory agents and also modulators of autophagy in target cells. However, the molecular mechanisms by which EVs modulate autophagy flux in human microglia remain largely unexplored. In the present study, we investigated the effects of EVs derived from human oral mucosa stem cells on the autophagy in human microglia. We demonstrate that EVs promoted autophagy and autophagic flux in human microglia and that this process was dependent on the integrity of lipid rafts. Lipopolysaccharide (LPS) also activated autophagy, but combined treatment with EVs and LPS suppressed autophagy response, indicating interference between these signaling pathways. Blockage of Toll-like receptor 4 (TLR4) with anti-TLR4 antibody suppressed EV-induced autophagy. Furthermore, inhibition of the EV-associated heat shock protein (HSP70) chaperone which is one of the endogenous ligands of the TLR4 also suppressed EV-induced lipid raft formation and autophagy. Pre-treatment of microglia with a selective inhibitor of αvβ3/αvβ5 integrins cilengitide inhibited EV-induced autophagy. Finally, blockage of purinergic P2X4 receptor (P2X4R) with selective inhibitor 5-BDBD also suppressed EV-induced autophagy. In conclusion, we demonstrate that EVs activate autophagy in human microglia through interaction with HSP70/TLR4, αVβ3/αVβ5, and P2X4R signaling pathways and that these effects depend on the integrity of lipid rafts. Our findings could be used to develop new therapeutic strategies targeting disease-associated microglia., (© 2024 Federation of European Biochemical Societies.)

Despite successfully utilizing anesthetics for over 150 years, the mechanism of action remains relatively unknown. Recent studies have shown promising results, but due to the complex interactions between anesthetics and their targets, there remains a clear need for further mechanistic research. We know that lipophilicity is directly connected to anesthetic potency since lipid solubility relates to anesthetic partition into the membrane. However, clinically relevant concentrations of anesthetics do not significantly affect lipid bilayers but continue to influence various molecular targets. Lipid rafts are derived from liquid-ordered phases of the plasma membrane that contain increased concentrations of cholesterol and sphingomyelin and act as staging platforms for membrane proteins, including ion channels. Although anesthetics do not perturb membranes at clinically relevant concentrations, they have recently been shown to target lipid rafts. In this review, we summarize current research on how different types of anesthetics-local, inhalational, and intravenous-bind and affect both lipid rafts and voltage-gated sodium channels, one of their major targets, and how those effects synergize to cause anesthesia and analgesia. Local anesthetics block voltage-gated sodium channel pores while also disrupting lipid packing in ordered membranes. Inhalational anesthetics bind to the channel pore and the voltage-sensing domain while causing an increase in the number, size, and diameter of lipid rafts. Intravenous anesthetics bind to the channel primarily at the voltage-sensing domain and the selectivity filter, while causing lipid raft perturbation. These changes in lipid nanodomain structure possibly give proteins access to substrates that have translocated as a result of these structural alterations, resulting in lipid-driven anesthesia. Overall, anesthetics can impact channel activity either through direct interaction with the channel, indirectly through the lipid raft, or both. Together, these result in decreased sodium ion flux into the cell, disrupting action potentials and producing anesthetic effects. However, more research is needed to elucidate the indirect mechanisms associated with channel disruption through the lipid raft, as not much is known about anionic lipid products and their influence over voltage-gated sodium channels. Anesthetics' effect on S-palmitoylation, a promising mechanism for direct and indirect influence over voltage-gated sodium channels, is another auspicious avenue of research. Understanding the mechanisms of different types of anesthetics will allow anesthesiologists greater flexibility and more specificity when treating patients., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 International Anesthesia Research Society.)

Background: Premature infants may suffer from high levels of bilirubin that could lead to neurotoxicity. Bilirubin has been shown to decrease L1-mediated ERK1/2 signaling, L1 phosphorylation, and L1 tyrosine 1176 dephosphorylation. Furthermore, bilirubin redistributes L1 into lipid rafts (LR) and decreases L1-mediated neurite outgrowth. We demonstrate that choline supplementation improves L1 function and signaling in the presence of bilirubin., Methods: Cerebellar granule neurons (CGN) were cultured with and without supplemental choline, and the effects on L1 signaling and function were measured in the presence of bilirubin. L1 activation of ERK1/2, L1 phosphorylation and dephosphorylation were measured. L1 distribution in LR was quantified and neurite outgrowth of CGN was determined., Results: Forty µM choline significantly reduced the effect of bilirubin on L1 activation of ERK1/2 by 220% (p = 0.04), and increased L1 triggered changes in tyrosine phosphorylation /dephosphorylation of L1 by 34% (p = 0.026) and 35% (p = 0.02) respectively. Choline ameliorated the redistribution of L1 in lipid rafts by 38% (p = 0.02) and increased L1-mediated mean neurite length by 11% (p = 0.04)., Conclusion: Choline pretreatment of CGN significantly reduced the disruption of L1 function by bilirubin. The supplementation of pregnant women and preterm infants with choline may increase infant resilience to the effects of bilirubin., Impact: This article establishes choline as an intervention for the neurotoxic effects of bilirubin on lipid rafts. This article provides clear evidence toward establishing one intervention for bilirubin neurotoxicity, where little is understood. This article paves the way for future investigation into the mechanism of the ameliorative effect of choline on bilirubin neurotoxicity., (© 2023. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Background: Docosahexaenoic acid (DHA) controls the biophysical organization of plasma membrane sphingolipid/cholesterol-enriched lipid rafts to exert anti-inflammatory effects, particularly in lymphocytes. However, the impact of DHA on the spatial arrangement of alveolar macrophage lipid rafts and inflammation is unknown., Objectives: The primary objective was to determine how DHA controls lipid raft organization and function of alveolar macrophages. As proof-of-concept, we also investigated DHA's anti-inflammatory effects on select pulmonary inflammatory markers with a murine influenza model., Methods: MH-S cells, an alveolar macrophage line, were treated with 50 μM DHA or vehicle control and were used to study plasma membrane molecular organization with fluorescence-based methods. Biomimetic membranes and coarse grain molecular dynamic (MD) simulations were employed to investigate how DHA mechanistically controls lipid raft size. qRT-PCR, mass spectrometry, and ELISAs were used to quantify downstream inflammatory signaling transcripts, oxylipins, and cytokines, respectively. Lungs from DHA-fed influenza-infected mice were analyzed for specific inflammatory markers., Results: DHA increased the size of lipid rafts while decreasing the molecular packing of the MH-S plasma membrane. Adding a DHA-containing phospholipid to a biomimetic lipid raft-containing membrane led to condensing, which was reversed with the removal of cholesterol. MD simulations revealed DHA nucleated lipid rafts by driving cholesterol and sphingomyelin into rafts. Downstream of the plasma membrane, DHA lowered the concentration of select inflammatory transcripts, oxylipins, and IL-6 secretion. DHA lowered pulmonary Il6 and Tnf-α mRNA expression and increased anti-inflammatory oxylipins of influenza-infected mice., Conclusions: The data suggest a model in which the localization of DHA acyl chains to nonrafts is driving sphingomyelin and cholesterol molecules into larger lipid rafts, which may serve as a trigger to impede signaling and lower inflammation. These findings also identify alveolar macrophages as a target of DHA and underscore the anti-inflammatory properties of DHA for lung inflammation., (Copyright © 2024 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Protopanaxadiol (PPD), which has a molecular structure similar to cholesterol, is a potent anticancer agent that has been proposed to target the lipid membrane for the pharmacological effects. However, the underlying mechanism by which PPD modulates the cell membrane leading to cancer cell death is not be fully understood. In this work, we used single cell infrared spectroscopy, scanning electron microscopy and confocal microscopy to investigate the effects of PPD on human hepatocellular carcinoma (HepG2) cells, focusing on the change in membrane structure. We found that PPD significantly reduced the number of membrane tubules over the course of treatment. Interestingly, the addition of PPD could promote the formation of lipid raft-like domains (PPD rafts) and even restore the domain disruption caused by methyl-beta-cyclodextrin depletion of membrane cholesterol. In addition, PPD pre-treatment may increase the induction effect of FasL, which impairs cell viability, although it does not appear to be beneficial for Fas clustering in the PPD rafts. Collectively, these results highlight a non-classical mechanism by which PPD induces HepG2 apoptosis by directly affecting the physical properties of the cell membrane, providing a novel insight into understanding membrane-targeted therapy., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the plasma membrane. We have already described that cholesterol and sphingomyelin depletion, or inhibition of sphingolipid biosynthesis decreased the TRPM8 but not the TRPM3 channel opening on cultured sensory neurons. We aimed to test the effects of lipid raft disruptors on channel activation on TRPM3- and TRPM8-expressing HEK293T cells in vitro, as well as their potential analgesic actions in TRPM3 and TRPM8 channel activation involving acute pain models in mice. CHO cell viability was examined after lipid raft disruptor treatments and their effects on channel activation on channel expressing HEK293T cells by measurement of cytoplasmic Ca 2+ concentration were monitored. The effects of treatments were investigated in Pregnenolone-Sulphate-CIM-0216-evoked and icilin-induced acute nocifensive pain models in mice. Cholesterol depletion decreased CHO cell viability. Sphingomyelinase and methyl-beta-cyclodextrin reduced the duration of icilin-evoked nocifensive behavior, while lipid raft disruptors did not inhibit the activity of recombinant TRPM3 and TRPM8. We conclude that depletion of sphingomyelin or cholesterol from rafts can modulate the function of native TRPM8 receptors. Furthermore, sphingolipid cleavage provided superiority over cholesterol depletion, and this method can open novel possibilities in the management of different pain conditions.

Coronavirus disease 2019 (COVID-19) is currently a worldwide emergency caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In observational clinical studies, statins have been identified as beneficial to hospitalized patients with COVID-19. However, experimental evidence of underlying statins protection against SARS-CoV-2 remains elusive. Here we reported for the first-time experimental evidence of the protective effects of simvastatin treatment both in vitro and in vivo . We found that treatment with simvastatin significantly reduced the viral replication and lung damage in vivo , delaying SARS-CoV-2-associated physiopathology and mortality in the K18-hACE2-transgenic mice model. Moreover, simvastatin also downregulated the inflammation triggered by SARS-CoV-2 infection in pulmonary tissue and in human neutrophils, peripheral blood monocytes, and lung epithelial Calu-3 cells in vitro , showing its potential to modulate the inflammatory response both at the site of infection and systemically. Additionally, we also observed that simvastatin affected the course of SARS-CoV-2 infection through displacing ACE2 on cell membrane lipid rafts. In conclusion, our results show that simvastatin exhibits early protective effects on SARS-CoV-2 infection by inhibiting virus cell entry and inflammatory cytokine production, through mechanisms at least in part dependent on lipid rafts disruption., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Teixeira, Temerozo, Pereira-Dutra, Ferreira, Mattos, Gonçalves, Sacramento, Palhinha, Cunha-Fernandes, Dias, Soares, Barreto, Cesar-Silva, Fintelman-Rodrigues, Pão, de Freitas, Reis, Hottz, Bozza, Bou-Habib, Saraiva, de Almeida, Viola, Souza and Bozza.)

Lipid rafts, also known as microdomains, are important components of cell membranes and are enriched in cholesterol, glycophospholipids and receptors. They are involved in various essential cellular processes, including endocytosis, exocytosis and cellular signaling. Receptors are concentrated at lipid rafts, through which cellular signaling can be transmitted. Pathogens exploit these signaling mechanisms to enter cells, proliferate and egress. However, lipid rafts also play an important role in initiating antimicrobial responses by sensing pathogens via clustered pathogen-sensing receptors and triggering downstream signaling events such as programmed cell death or cytokine production for pathogen clearance. In this review, we discuss how both host and pathogens use lipid rafts and associated proteins in an arms race to survive. Special attention is given to the involvement of the major vault protein, the main constituent of a ribonucleoprotein complex, which is enriched in lipid rafts upon infection with vaccinia virus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kulkarni, Wiemer and Chang.)

The increase in Aβ 1-42 is a neurotoxic effect induced by aluminum which can lead to impairment of learning and memory, but its mechanism has yet to be fully elucidated. Studies have shown that APP palmitoylation is appears to be involved in the production process of Aβ 1-42 . Here, we investigated whether APP palmitoylation is related to the increase in Aβ caused by aluminum and its specific mechanism of action. In this study, APP palmitoylation was studied in the setting of aluminum-induced increases in Aβ 1-42 from two perspectives: whole animal experiments and in vitro cell experiments. First, the learning and memory of rats were impaired and the number of rat cortical neurons was decreased after staining with aluminum. Second, the expression of palmitoyl APP, APP in lipid rafts and palmitoyl acyltransferase zDHHC7 both in rat cerebral cortex and PC12 cells increased with the production of Aβ 1-42 induced by aluminum in a dose-dependent manner. Finally, the intervention with the palmitoylation inhibitors 2-BP and siRNA zDHHC7 in PC12 cells reduced levels of palmitoyl APP, the expression of APP in lipid rafts and the content of Aβ 1-42 induced by aluminum to a certain extent. Our results indicate that increased APP palmitoylation levels may be related to the increase in Aβ 1-42 caused by aluminum, and the mechanism may involve APP palmitoylation promoting the accumulation of APP protein on lipid rafts and the cleavage of APP by BACE1 in amyloidogenic pathway. The increase in expression of zDHHC7 may be one of the reasons for the increase in levels of APP palmitoylation caused by aluminum., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The lipid raft-resident protein, MAL2, has been implicated as contributing to the pathogenesis of several malignancies, including breast cancer, but the underlying mechanism for its effects on tumorigenesis is unknown. Here, we show that MAL2-mediated lipid raft formation leads to HER2 plasma membrane retention and enhanced HER2 signaling in breast cancer cells. We demonstrate physical interactions between HER2 and MAL2 in lipid rafts using proximity ligation assays. Super-resolution structured illumination microscopy imaging displays the structural organization of the HER2/Ezrin/NHERF1/PMCA2 protein complex. Formation of this protein complex maintains low intracellular calcium concentrations in the vicinity of the plasma membrane. HER2/MAL2 protein interactions in lipid rafts are enhanced in trastuzumab-resistant breast cancer cells. Our findings suggest that MAL2 is crucial for lipid raft formation, HER2 signaling, and HER2 membrane stability in breast cancer cells, suggesting MAL2 as a potential therapeutic target., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Platelet cytosolic cyclic adenosine monophosphate (cAMP) levels are balanced by synthesis, degradation, and efflux. Efflux can occur via multidrug resistant protein-4 (MRP4; ABCC4) present on dense granule and/or plasma membranes. As lipid rafts have been shown to interfere on cAMP homeostasis, we evaluated the relationships between the distribution and activity of MRP4 in lipid rafts and cAMP efflux., Methods: Platelet activation and cAMP homeostasis were analyzed in human and wild-type or MRP4-deleted mouse platelets in the presence of methyl-β-cyclodextrin (MßCD) to disrupt lipid rafts, and of activators of the cAMP signalling pathways. Human platelet MRP4 and effector proteins of the cAMP pathway were analyzed by immunoblots in lipid rafts isolated by differential centrifugation., Results: MßCD dose dependently inhibited human and mouse platelet aggregation without affecting per se cAMP levels. An additive inhibitory effect existed between the adenylate cyclase (AC) activator forskolin and MßCD that was accompanied by an overincrease of cAMP, and which was significantly enhanced upon MRP4 deletion. Finally, an efflux of cAMP out of resting platelets incubated with prostaglandin E1 (PGE 1 ) was observed that was partly dependent on MRP4. Lipid rafts contained a small fraction (≈15%) of MRP4 and most of the inhibitory G-protein Gi, whereas Gs protein, AC3, and phosphodiesterases PDE2 and PDE3A were all present as only trace amounts., Conclusion: Our results are in favour of part of MRP4 present at the platelet surface, including in lipid rafts. Lipid raft integrity is necessary for cAMP signalling regulation, although MRP4 and most players of cAMP homeostasis are essentially located outside rafts., Competing Interests: None declared., (Thieme. All rights reserved.)

Objective: The aim of the study was to show the effect that two naturally occurring compounds, a cyclodextrin and hydroxytyrosol, can have on the entry of SARS-CoV-2 into human cells., Materials and Methods: The PubMed database was searched to retrieve studies published from 2000 to 2020, satisfying the inclusion criteria. The search keywords were: SARS-CoV, SARS-CoV-2, coronavirus, lipid raft, endocytosis, hydroxytyrosol, cyclodextrin. Modeling of alpha-cyclodextrin and hydroxytyrosol were done using UCSF Chimera 1.14., Results: The search results indicated that cyclodextrins can reduce the efficiency of viral endocytosis and that hydroxytyrosol has antiviral properties. Bioinformatic docking studies showed that alpha-cyclodextrin and hydroxytyrosol, alone or in combination, interact with the viral spike protein and its host cell receptor ACE2, thereby potentially influencing the endocytosis process., Conclusions: Hydroxytyrosol and alpha-cyclodextrin can be useful against the spread of SARS-CoV-2.

The activity of local anesthetics (LAs) has been attributed to the inhibition of ion channels, causing anesthesia. However, there is a growing body of research showing that LAs act on a wide range of receptors and channel proteins far beyond simple analgesia. The current concept of ligand recognition may no longer explain the multitude of protein targets influenced by LAs. We hypothesize that LAs can cause anesthesia without directly binding to the receptor proteins just by changing the physical properties of the lipid bilayer surrounding these proteins and ion channels based on LAs' amphiphilicity. It is possible that LAs act in one of the following ways: They 1) dissolve raft-like membrane microdomains, 2) impede nerve impulse propagation by lowering the lipid phase transition temperature, or 3) modulate the lateral pressure profile of the lipid bilayer. This could also explain the numerous additional effects of LAs besides anesthesia. Furthermore, the concepts of membrane-mediated activity and binding to ion channels do not have to exclude each other. If we were to consider LA as the middle part of a continuum between unspecific membrane-mediated activity on one end and highly specific ligand binding on the other end, we could describe LA as the link between the unspecific action of general anesthetics and toxins with their highly specific receptor binding. This comprehensive membrane-mediated model offers a fresh perspective to clinical and pharmaceutical research and therapeutic applications of local anesthetics. SIGNIFICANCE STATEMENT: Local anesthetics, according to the World Health Organization, belong to the most important drugs available to mankind. Their rediscovery as therapeutics and not only anesthetics marks a milestone in global pain therapy. The membrane-mediated mechanism of action proposed in this review can explain their puzzling variety of target proteins and their thus far inexplicable therapeutic effects. The new concept presented here places LAs on a continuum of structures and molecular mechanisms in between small general anesthetics and the more complex molecular toxins., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Lipid rafts (LRs), cholesterol-enriched microdomains on cell membranes, are increasingly viewed as signalling platforms governing critical facets of cancer progression. The phenotype of cancer stem-like cells (CSCs) presents significant hurdles for successful cancer treatment, and the expression of several CSC markers is associated with LR integrity. However, LR implications in CSCs remain unclear., Methods: This study evaluated the biological and molecular functions of LRs in colorectal cancer (CRC) by using an LR-disrupting alkylphospholipid (APL) drug, miltefosine. The mechanistic role of miltefosine in CSC inhibition was examined through normal or tumour intestinal mouse organoid, human CRC cell, CRC xenograft and miltefosine treatment gene expression profile analyses., Results: Miltefosine suppresses CSC populations and their self-renewal activities in CRC cells, a CSC-targeting effect leading to irreversible disruption of tumour-initiating potential in vivo. Mechanistically, miltefosine reduced the expression of a set of genes, leading to stem cell death. Among them, miltefosine transcriptionally inhibited checkpoint kinase 1 (CHEK1), indicating that LR integrity is essential for CHEK1 expression regulation. In isolated CD44 high CSCs, we found that CSCs exhibited stronger therapy resistance than non-CSC counterparts by preventing cell death through CHEK1-mediated cell cycle checkpoints. However, inhibition of the LR/CHEK1 axis by miltefosine released cell cycle checkpoints, forcing CSCs to enter inappropriate mitosis with accumulated DNA damage and resulting in catastrophic cell death., Conclusion: Our findings underscore the therapeutic potential of LR-targeting APLs for CRC treatment that overcomes the therapy-resistant phenotype of CSCs, highlighting the importance of the LR/CHEK1 axis as a novel mechanism of APLs., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Equinatoxin II (EqtII) and Fragaceatoxin C (FraC) are pore-forming toxins (PFTs) from the actinoporin family that have enhanced membrane affinity in the presence of sphingomyelin (SM) and phase coexistence in the membrane. However, little is known about the effect of these proteins on the nanoscopic properties of membrane domains. Here, we used combined confocal microscopy and force mapping by atomic force microscopy to study the effect of EqtII and FraC on the organization of phase-separated phosphatidylcholine/SM/cholesterol membranes. To this aim, we developed a fast, high-throughput processing tool to correlate structural and nano-mechanical information from force mapping. We found that both proteins changed the lipid domain shape. Strikingly, they induced a reduction in the domain area and circularity, suggesting a decrease in the line tension due to a lipid phase height mismatch, which correlated with proteins binding to the domain interfaces. Moreover, force mapping suggested that the proteins affected the mechanical properties at the edge, but not in the bulk, of the domains. This effect could not be revealed by ensemble force spectroscopy measurements supporting the suitability of force mapping to study local membrane topographical and mechanical alterations by membranotropic proteins.

Agents that induce immunogenic cell death (ICD) alter the cellular localization of calreticulin (CRT), causing it to become cell surface-exposed within the plasma membrane lipid raft microdomain [cell surface-exposed CRT (ectoCRT)] where it serves as a damage associated-molecular pattern that elicits an antitumor immune response. We have identified the sphingolipid metabolic pathway as an integral component of the process of ectoCRT exposure. Inhibition of the sphingosine kinases (SphKs) enhances mitoxantrone-induced production of hallmarks of ICD, including ectoCRT production, with an absolute mean difference of 40 MFI (95% CI: 19-62; P = 0.0014) and 1.3-fold increase of ATP secretion with an absolute mean difference of 87 RLU (95% CI: 55-120; P < 0.0001). Mechanistically, sphingosine kinase inhibition increases mitoxantrone-induced accumulation of ceramide species, including C16:0 ceramide 2.8-fold with an absolute mean difference of 1.390 pmol/nmol Pi (95% CI: 0.798-1.983; P = 0.0023). We further examined the localization of ectoCRT to the lipid raft microdomain and demonstrate that ectoCRT forms disulfide-bridged dimers. Together, our findings suggest that ceramide accumulation impinges on the homeostatic function of the endoplasmic reticulum to induce ectoCRT exposure and that structural alterations of ectoCRT may underlie its immunogenicity. Our findings further suggest that inhibition of the SphKs may represent a means to enhance the therapeutic immunogenic efficacy of ICD-inducing agents while reducing overt toxicity/immunosuppressive effects by allowing for the modification of dosing regimens or directly lowering the dosages of ICD-inducing agents employed in therapeutic regimens. SIGNIFICANCE STATEMENT: This study demonstrates that inhibition of sphingosine kinase enhances the mitoxantrone-induced cell surface exposure of a dimeric form of the normally endoplasmic reticulum resident chaperone calreticulin as part of the process of a unique form of regulated cell death termed immunogenic cell death. Importantly, inhibition of sphingosine kinase may represent a means to enhance the therapeutic efficacy of immunogenic cell death-inducing agents, such as mitoxantrone, while reducing their overt toxicity and immunosuppressive effects, leading to better therapeutic outcomes for patients., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Termination of antidepressant therapy often has negative consequences. Although symptoms of antidepressant withdrawal are widely recognized, the molecular processes that underlie them are not well characterized. We show that certain aspects of G α s signaling remain suppressed after antidepressant withdrawal, even after others have reverted to baseline. Antidepressant treatment causes translocation of G α s protein from lipid rafts to nonraft membrane regions. This results in augmented G α s signaling, including facilitated activation of adenylyl cyclase and increased cAMP accumulation. Using CC6 or SK-N-SH cells and a lipid raft-localized cAMP sensor, we show that G α s signaling is reduced in lipid rafts, even while signaling is enhanced elsewhere in the cell. These signaling changes mirror the changes in G α s localization observed after antidepressant treatment. Furthermore, we show that suppression of G α s signaling in lipid rafts persists at least 24 hours after cessation of antidepressant treatment. G α s localization was quantified after membrane isolation and sequential detergent extraction. We show that suppression of lipid raft G α s signaling persists for an extended time period after antidepressant withdrawal, whereas increased nonraft membrane G α s signaling reverts partially or fully upon cessation of antidepressant treatment. Translocation of G α s out of lipid rafts is also persistent. These events may reflect cellular adaptations to antidepressant treatment that contribute to antidepressant discontinuation syndromes and may aid in the discovery of new treatments and strategies to mitigate the symptoms of depression and antidepressant withdrawal. SIGNIFICANCE STATEMENT: This work explores, for the first time, the effects of antidepressants on G α s signaling after drug withdrawal. This provides novel insight into the cellular and molecular processes affected by antidepressant drugs and their persistence after discontinuation of treatment., (U.S. Government work not protected by U.S. copyright.)

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy- N -methyl- N , N -bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.

Hereditary sensory neuropathy type 1 (HSN1) is caused by mutations in the SPTLC1 or SPTLC2 sub-units of the enzyme serine palmitoyltransferase, resulting in the production of toxic 1-deoxysphingolipid bases (DSBs). We used induced pluripotent stem cells (iPSCs) from patients with HSN1 to determine whether endogenous DSBs are neurotoxic, patho-mechanisms of toxicity and response to therapy. HSN1 iPSC-derived sensory neurons (iPSCdSNs) endogenously produce neurotoxic DSBs. Complex gangliosides, which are essential for membrane micro-domains and signaling, are reduced, and neurotrophin signaling is impaired, resulting in reduced neurite outgrowth. In HSN1 myelinating cocultures, we find a major disruption of nodal complex proteins after 8 weeks, which leads to complete myelin breakdown after 6 months. HSN1 iPSC models have, therefore, revealed that SPTLC1 mutation alters lipid metabolism, impairs the formation of complex gangliosides, and reduces axon and myelin stability. Many of these changes are prevented by l-serine supplementation, supporting its use as a rational therapy., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Aims: Plasma hyperlipidemia is a protective factor in amyotrophic lateral sclerosis (ALS) while cholesterol-lowering drugs aggravate the pathology. We hypothesize that this phenomenon can be linked with membrane lipid alterations in the neuromuscular junctions (NMJs) occurring before motor neuron loss., Methods: Neurotransmitter release in parallel with lipid membrane properties in diaphragm NMJs of SOD1G93A (mSOD) mice at nine weeks of age (pre-onset stage) were assessed., Key Findings: Despite on slight changes in spontaneous and evoked quantum release of acetylcholine, extracellular levels of choline at resting conditions, an indicator of non-quantum release, were significantly increased in mSOD mice. The use of lipid-sensitive fluorescent probes points to lipid raft disruption in the NMJs of mSOD mice. However, content of cholesterol, a key raft component was unchanged implying another pathway responsible for the loss of raft integrity. In the mSOD mice we found marked increase in levels of raft-destabilizing lipid ceramide. This was accompanied by enhanced ability to uptake of exogenous ceramide in NMJs. Acute and chronic administration of 25-hydroxycholesterol, whose levels increase due to hypercholesterolemia, recovered early alterations in membrane properties. Furthermore, chronic treatment with 25-hydroxycholesterol prevented increase in ceramide and extracellular choline levels as well as suppressed lipid peroxidation of NMJ membranes and fragmentation of end plates., Significance: Thus, lipid raft disruption likely due to ceramide accumulation could be early event in ALS which may trigger neuromuscular abnormalities. Cholesterol derivative 25-hydroxycholesterol may serve as a molecule restoring the membrane and functional properties of NMJs at the early stage., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: The mechanism of bilirubin neurotoxicity is poorly understood. We hypothesize that bilirubin inhibits the function of lipid rafts (LR), microdomains of the plasma membrane critical for signal transduction. To test this hypothesis, we measured the effect of free bilirubin (Bf) between 7.6 and 122.5 nM on LR-dependent functions of L1 cell adhesion molecule (L1)., Methods: Cerebellar granule neurons (CGN) were plated on poly-L-lysine overnight, and neurite length was determined after 1 h treatment with L1 alone or L1 and bilirubin. L1 activation of ERK1/2 was measured in CGN in the presence or absence of bilirubin. The effect of bilirubin on L1 distribution in LR was quantitated, and the localization of bilirubin to LR was determined., Results: The addition of bilirubin to CGN treated with L1 significantly decreased neurite length compared to L1 alone. L1 activation of ERK1/2 was inhibited by bilirubin. Bilirubin redistributed L1 into LR. Bilirubin was associated only with LR-containing fractions of a sucrose density gradient., Conclusion: Bf significantly inhibits LR-dependent functions of L1 and are found only associated with LR, suggesting one mechanism by which bilirubin may exert neurotoxicity is through the dysfunction of protein-LR interactions., Impact: This article establishes lipid rafts as a target for the neurotoxic effects of bilirubin. This article provides clear evidence toward establishing one mechanism of bilirubin neurotoxicity, where little is understood. This article paves the way for future investigation into lipid raft dependent functions, and its role in neurodevelopmental outcome.

Objective: Polydatin has been used in the treatment of various inflammatory diseases. However, its role in the regulation of neuroinflammation has not been reported. In this study, we designed to investigate the anti-inflammatory effects of polydatin in LPS-stimulated BV2 microglia cells., Methods: Inflammatory mediators TNF-α, IL-1β, NO, and PGE2 production were measured by ELISA. The protein of signaling pathways were detected by western blot analysis., Results: The results showed that polydatin significantly ameliorated the production of TNF-α, IL-1β, NO, and PGE2 up-regulated by LPS. Polydatin also blocked LPS-induced NF-κB activation. In addition, PI3K and AKT, the up-stream molecules of NF-κB signaling pathway, were inhibited by the treatment of polydatin. Also, we found the formation of lipid rafts was inhibited by polydatin through attenuating the cholesterol content. Finally, polydatin was found to increase the expression of ABCA1 and ABCG1., Conclusion: In conclusion, the results of the present study suggested that polydatin exhibited its anti-inflammatory effects in BV2 cells through disrupting lipid rafts, which subsequently inhibiting PI3K/AKT signaling pathway.

In uremic patients, high-density lipoprotein (HDL) loses its anti-inflammatory features and can even become pro-inflammatory due to an altered protein composition. In chronic kidney disease (CKD), impaired functions of polymorphonuclear leukocytes (PMNLs) contribute to inflammation and an increased risk of cardiovascular disease. This study investigated the effect of HDL from CKD and hemodialysis (HD) patients on the CD14 expression on PMNLs. HDL was isolated using a one-step density gradient centrifugation. Isolation of PMNLs was carried out by discontinuous Ficoll-Hypaque density gradient centrifugation. CD14 surface expression was quantified by flow cytometry. The activity of the small GTPase Rac1 was determined by means of an activation pull-down assay. HDL increased the CD14 surface expression on PMNLs. This effect was more pronounced for HDL isolated from uremic patients. The acute phase protein serum amyloid A (SAA) caused higher CD14 expression, while SAA as part of an HDL particle did not. Lipid raft disruption with methyl-β-cyclodextrin led to a reduced CD14 expression in the absence and presence of HDL. HDL from healthy subjects but not from HD patients decreased the activity of Rac1. Considering the known anti-inflammatory effects of HDL, the finding that even HDL from healthy subjects increased the CD14 expression was unexpected. The pathophysiological relevance of this result needs further investigation.

Neuronal regeneration in the injured central nervous system is hampered by multiple extracellular proteins. These proteins exert their inhibitory action through interactions with receptors that are located in cholesterol rich compartments of the membrane termed lipid rafts. Here we show that cholesterol-synthesis inhibition prevents the association of the Neogenin receptor with lipid rafts. Furthermore, we show that cholesterol-synthesis inhibition enhances axonal growth both on inhibitory -myelin and -RGMa substrates. Following optic nerve injury, lowering cholesterol synthesis with both drugs and siRNA-strategies allows for robust axonal regeneration and promotes neuronal survival. Cholesterol inhibition also enhanced photoreceptor survival in a model of Retinitis Pigmentosa. Our data reveal that Lovastatin leads to several opposing effects on regenerating axons: cholesterol synthesis inhibition promotes regeneration whereas altered prenylation impairs regeneration. We also show that the lactone prodrug form of lovastatin has differing effects on regeneration when compared to the ring-open hydroxy-acid form. Thus the association of cell surface receptors with lipid rafts contributes to axonal regeneration inhibition, and blocking cholesterol synthesis provides a potential therapeutic approach to promote neuronal regeneration and survival in the diseased Central Nervous System. SIGNIFICANCE STATEMENT: Statins have been intensively used to treat high levels of cholesterol in humans. However, the effect of cholesterol inhibition in both the healthy and the diseased brain remains controversial. In particular, it is unclear whether cholesterol inhibition with statins can promote regeneration and survival following injuries. Here we show that late stage cholesterol inhibition promotes robust axonal regeneration following optic nerve injury. We identified distinct mechanisms of action for activated vs non-activated Lovastatin that may account for discrepancies found in the literature. We show that late stage cholesterol synthesis inhibition alters Neogenin association with lipid rafts, thereby i) neutralizing the inhibitory function of its ligand and ii) offering a novel opportunity to promote CNS regeneration and survival following injuries., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lactoferrin (Lf) is a bioactive milk-derived protein with remarkable wide-spectrum antifungal activity. To deepen our understanding of the molecular mechanisms underlying Lf cytotoxicity, the role of plasma membrane ergosterol- and sphingolipid-rich lipid rafts and their association with the proton pump Pma1p was explored. Pma1p was previously identified as a Lf-binding protein. Results showed that bovine Lf (bLf) perturbs ergosterol-rich lipid rafts organization by inducing intracellular accumulation of ergosterol. Using yeast mutant strains lacking lipid rafts-associated proteins or enzymes involved in the synthesis of ergosterol and sphingolipids, we found that perturbations in the composition of these membrane domains increase resistance to bLf-induced yeast cell death. Also, when Pma1p-lipid rafts association is compromised in the Pma1-10 mutant and in the absence of the Pma1p-binding protein Ast1p, the bLf killing activity is impaired. Altogether, results showed that the perturbation of lipid rafts and the inhibition of both Pma1p and V-ATPase activities mediate the antifungal activity of bLf. Since it is suggested that the combination of conventional antifungals with lipid rafts-disrupting compounds is a powerful antifungal approach, our data will help to pave the way for the use of bLf alone or in combination for the treatment/eradication of clinically and agronomically relevant yeast pathogens/fungi., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Human kidney epithelial cells are supposed to be directly involved in the pathogenesis of the hemolytic-uremic syndrome (HUS) caused by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC). The characterization of the major and minor Stx-binding glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), respectively, of primary human renal cortical epithelial cells (pHRCEpiCs) revealed GSLs with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Using detergent-resistant membranes (DRMs) and non-DRMs, Gb3Cer and Gb4Cer prevailed in the DRM fractions, suggesting their association with microdomains in the liquid-ordered membrane phase. A preference of Gb3Cer and Gb4Cer endowed with C24:0 fatty acid accompanied by minor monounsaturated C24:1-harboring counterparts was observed in DRMs, whereas the C24:1 fatty acid increased in relation to the saturated equivalents in non-DRMs. A shift of the dominant phospholipid phosphatidylcholine with saturated fatty acids in the DRM to unsaturated species in the non-DRM fractions correlated with the GSL distribution. Cytotoxicity assays gave a moderate susceptibility of pHRCEpiCs to the Stx1a and Stx2a subtypes when compared to highly sensitive Vero-B4 cells. The results indicate that presence of Stx-binding GSLs per se and preferred occurrence in microdomains do not necessarily lead to a high cellular susceptibility towards Stx.

The objective of the current study was first to characterize lipid raft microdomains isolated as detergent-resistant membranes (DRM) from mammary gland tissue, and second to determine how dietary fatty acids (FA) such as conjugated linoleic acid (CLA), 19:1 cyclo, and long-chain n-3 polyunsaturated FA affect lipid raft markers of mammary cells, and to finally establish relationships between these markers and lactation performance in dairy cows. Eight Holstein cows were used in a replicated 4 × 4 Latin square design with periods of 28 d. For the first 14 d, cows received daily an abomasal infusion of (1) 406 g of a saturated FA supplement (112 g of 16:0 + 230 g of 18:0) used as a control; (2) 36 g of a CLA supplement (13.9 g of trans-10,cis-12 18:2) + 370 g of saturated FA; (3) 7 g of Sterculia fetida oil (3.1 g of 19:1 cyclo, STO) + 399 g of saturated FA; or (4) 406 g of fish oil (55.2 g of cis-5,cis-8,cis-11,cis-14,cis-17 20:5 + 59.3 g of cis-4,cis-7,cis-10,cis-13,cis-16,cis-19 22:6, FO). Mammary biopsies were harvested on d 14 of each infusion period and were followed by a 14-d washout interval. Cholera toxin subunit B, which specifically binds to ganglioside M-1 (GM-1), a lipid raft marker, was used to assess its distribution in DRM. Infusions of CLA, STO, and FO were individually compared with the control, and significance was declared at P ≤ 0.05. Milk fat yield was decreased with CLA and FO, but was not affected by STO. Milk lactose yield was decreased with CLA and STO, but was not affected by FO. Mammary tissue shows a strong GM-1-signal enrichment in isolated DRM from mammary gland tissue. Caveolin (CAV) and flotillin (FLOT) are 2 proteins considered as lipid raft markers and they are present in DRM from mammary gland tissue. Distributions of GM-1, CAV-1, and FLOT-1 showed an effect of treatments determined by their subcellular distributions in sucrose gradient fractions. Regardless of treatments, data showed positive relationships between the yield of milk fat, protein, and lactose, and the abundance GM-1 in DRM fraction. Milk protein yield was positively correlated with relative proportion of FLOT-1 in the soluble fraction, whereas lactose yield was positively correlated with relative proportion of CAV-1 in the DRM fractions. Infusion of CLA decreased mRNA abundance of CAV-1, FLOT-1, and FLOT-2. Regardless of treatments, a positive relationship was observed between fat yield and mRNA abundance of FLOT-2. In conclusion, although limited to a few markers, results of the current experiment raised potential links between variation in specific biologically active component of raft microdomains in bovine mammary gland and lactation performances in dairy cows., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The binding of vascular endothelial growth factor A (VEGF) to VEGF receptor-2 (VEGFR-2) stimulates angiogenic signaling. Lipid rafts are cholesterol-dense regions of the plasma membrane that serve as an organizational platform for biomolecules. Although VEGFR2 has been shown to colocalize with lipid rafts to regulate its activation, the effect of lipid rafts on non-activated VEGFR2 has not been explored. Here, we characterized the involvement of lipid rafts in modulating the stability of non-activated VEGFR2 in endothelial cells using raft disrupting agents: methyl-β-cyclodextrin, sphingomyelinase and simvastatin. Disrupting lipid rafts selectively decreased the levels of non-activated VEGFR2 as a result of increased lysosomal degradation. The decreased expression of VEGFR2 translated to reduced VEGF-activation of the extracellular signal-regulated protein kinases (ERK). Overall, our results indicate that lipid rafts stabilize VEGFR2 and its associated signal transduction activities required for angiogenesis. Thus, modulation of lipid rafts may provide a means to regulate the sensitivity of endothelial cells to VEGF stimulation. Indeed, the ability of simvastatin to down regulate VEGFR2 and inhibit VEGF activity suggest a potential mechanism underlying the observation that this drug improves outcomes in the treatment of certain cancers.

Lipid rafts are critical cell membrane lipid platforms enriched in sphingolipid and cholesterol content involved in diverse cellular processes. They have been proposed to influence membrane properties and to accommodate receptors within themselves by facilitating their interaction with ligands. Over the past decade, technical advances have improved our understanding of lipid rafts as bioactive structures. In this review, we will cover the more recent findings about cholesterol, sphingolipids and lipid rafts located in cellular and nuclear membranes in cancer. Collectively, the data provide insights on the role of lipid rafts as biomolecular targets in cancer with good perspectives for the development of innovative therapeutic strategies.

The urokinase (uPA) receptor (uPAR) plays a key role in cell migration. We previously showed that uPAR-negative HEK-293 cells efficiently migrate toward serum but, after uPAR ectopic expression, migrate only in a uPAR-dependent manner. In fact, migration of uPAR-transfected HEK-293 (uPAR-293) cells is impaired by anti-uPAR antibodies, without recovery of the uPAR-independent migration mechanisms formerly active. Prostate carcinoma PC3 cells, which express high endogenous uPAR levels, migrated only through a uPAR-dependent mechanism; in fact, the silencing of uPAR expression inhibited their migration. We hypothesize a crucial role of the uPAR glycosyl-phosphatidyl-inositol (GPI) tail, which promotes uPAR partitioning to lipid rafts, in uPAR-controlled cell migration. Here, we show that removal of the uPAR GPI-tail, or lipid rafts disruption by methyl-beta-cyclodextrin impairs migration of PC3 cells, incapable of uPAR-independent migration, whereas it restores uPAR-independent migration in uPAR-293 cells. We then show that, in PC3 cells, both uPAR signaling partners, β1 integrins and receptors for formylated peptides (FPRs), partly associate with lipid rafts. Inhibition of their interaction with uPAR impairs this association and impairs cell migration. Interestingly, blocking uPAR association with FPRs also impairs β1 integrin partitioning to lipid rafts, whereas blocking its association with β1 integrins has no effect on FPRs partitioning. On these bases, we propose that uPAR controls cell migration by connecting β1 integrins and FPRs and, through its GPI tail, by driving them into lipid rafts, thus promoting pro-migratory signals. uPAR-mediated partitioning of integrins to lipid rafts is strictly dependent on uPAR association with FPRs.

We hypothesized that the antimicrobial peptide cathelicidin has a physiological role in regulating gut inflammatory homeostasis. We determined that cathelicidin synergizes with LPS to facilitate its internalization and signaling via endosomic TLR4 in colonic epithelium, evoking synthesis of the human neutrophil chemoattractant, CXCL8 (or murine homolog, CXCL1). Interaction of cathelicidin with LPS in the control of CXCL8/CXCL1 synthesis was assessed in human colon epithelial cells, murine colonoids and cathelicidin-null mice ( Camp -/- ). Mechanistically, human cathelicidin (LL-37), as an extracellular complex with LPS, interacted with lipid raft-associated GM1 gangliosides to internalize and activate intracellular TLR4. Two signaling pathways converged on CXCL8/CXCL1 production: (1) a p38MAPK-dependent pathway regulated by Src-EGFR kinases; and, (2) a p38MAPK-independent, NF-κB-dependent pathway, regulated by MEK1/2-MAPK. Increased cathelicidin-dependent CXCL8 secretion in the colonic mucosa activated human blood-derived neutrophils. These cathelicidin effects occurred in vitro at concentrations well below those needed for microbicidal function. The important immunomodulatory role of cathelicidins was evident in cathelicidin-null/ Camp -/- mice, which had diminished colonic CXCL1 secretion, decreased neutrophil recruitment-activation and reduced bacterial clearance when challenged with the colitis-inducing murine pathogen, Citrobacter rodentium . We conclude that in addition to its known microbicidal action, cathelicidin has a unique pathogen-sensing role, facilitating LPS-mediated intestinal responses, including the production of CXCL8/CXCL1 that would contribute to an integrated tissue response to recruit neutrophils during colitis.

Background: The liver is the central organ for cholesterol homoeostasis, and its dysfunction might cause liver pathological alterations including hepatocellular carcinomas (HCCs). 3β-hydroxysteroid-Δ24 reductase (DHCR24), a crucial enzyme of cholesterol biosynthetic pathway, is involved in lipid rafts formation. Genkwadaphnin (GD) is a daphnane diterpene isolated from the flower buds of Daphne genkwa Siebold et Zuccarini (Thymelaeaceae)., Methods: We evaluated in vitro and in vivo effect of GD using HCC cells and BALB/c nude mice. Microarray assays were used to identify the differential genes by GD. DHCR24 expression and activity, cholesterol level, lipid rafts structure and the role of DHCR24 in human HCC specimens were tested by various molecular biology techniques., Results: High expression of DHCR24 in human HCC specimens was correlated with poor clinical outcome. Interfering DHCR24 altered growth and migration of HCC cells. GD inhibited growth and metastasis of HCC cells both in vivo and in vitro. GD suppressed DHCR24 expression and activity, as well as DHCR24-mediated cholesterol biosynthesis and lipid rafts formation, then further inhibited HCC cell invasion and migration., Conclusions: Our data suggest that DHCR24-mediated cholesterol metabolism might be an effective therapeutic strategy in HCC, and natural product GD might be a promising agent for HCC therapy.

Safe and efficient drugs to combat the current COVID-19 pandemic are urgently needed. In this context, we have analyzed the anti-coronavirus potential of the natural product glycyrrhizic acid (GLR), a drug used to treat liver diseases (including viral hepatitis) and specific cutaneous inflammation (such as atopic dermatitis) in some countries. The properties of GLR and its primary active metabolite glycyrrhetinic acid are presented and discussed. GLR has shown activities against different viruses, including SARS-associated Human and animal coronaviruses. GLR is a non-hemolytic saponin and a potent immuno-active anti-inflammatory agent which displays both cytoplasmic and membrane effects. At the membrane level, GLR induces cholesterol-dependent disorganization of lipid rafts which are important for the entry of coronavirus into cells. At the intracellular and circulating levels, GLR can trap the high mobility group box 1 protein and thus blocks the alarmin functions of HMGB1. We used molecular docking to characterize further and discuss both the cholesterol- and HMG box-binding functions of GLR. The membrane and cytoplasmic effects of GLR, coupled with its long-established medical use as a relatively safe drug, make GLR a good candidate to be tested against the SARS-CoV-2 coronavirus, alone and in combination with other drugs. The rational supporting combinations with (hydroxy)chloroquine and tenofovir (two drugs active against SARS-CoV-2) is also discussed. Based on this analysis, we conclude that GLR should be further considered and rapidly evaluated for the treatment of patients with COVID-19., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome., (Copyright © 2020 Elsevier Inc. All rights reserved.)

COVID-19 is a global pandemic currently in an acute phase of rapid expansion. While public health measures remain the most effective protection strategy at this stage, when the peak passes, it will leave in its wake important health problems. Historically, very few viruses have ever been eradicated. Instead, the virus may persist in communities causing recurrent local outbreaks of the acute infection as well as several chronic diseases that may arise from the presence of a "suppressed" virus or as a consequence of the initial exposure. An ideal solution would be an anti-viral medication that (i) targets multiple stages of the viral lifecycle, (ii) is insensitive to frequent changes of viral phenotype due to mutagenesis, (iii) has broad spectrum, (iv) is safe and (v) also targets co-morbidities of the infection. In this Perspective we discuss a therapeutic approach that owns these attributes, namely "lipid raft therapy." Lipid raft therapy is an approach aimed at reducing the abundance and structural modifications of host lipid rafts or at targeted delivery of therapeutics to the rafts. Lipid rafts are the sites of the initial binding, activation, internalization and cell-to-cell transmission of SARS-CoV-2. They also are key regulators of immune and inflammatory responses, dysregulation of which is characteristic to COVID-19 infection. Lipid raft therapy was successful in targeting many viral infections and inflammatory disorders, and can potentially be highly effective for treatment of COVID-19., (Copyright © 2020 Sviridov, Miller, Ballout, Remaley and Bukrinsky.)

Pharmacologic intervention to affect the membrane lipid homeostasis of lipid rafts is a potent therapeutic strategy for cancer. Here we showed that gallic acid (GA) caused the complex formation of inactive Ras-related C3 botulinum toxin substrate 1 (Rac1)-phospho (p)-casein kinase 2 α (CK2α) (Tyr 255) in human tongue squamous carcinoma (TSC) cells, which disturbed the lipid raft membrane-targeting of phosphatidylinositol 3-kinase (PI3K)-Rac1-protein kinase B (Akt) signal molecules by inducing the association of p110α-free p85α with unphosphorylated phosphatase tensin homolog deleted on chromosome 10 (PTEN) in lipid rafts. The effects on induction of inactive Rac1-p-CK2α (Tyr 255) complex formation and attenuation of p-Akt (Ser 473), GTP-Rac1, glucose transporter-1 (GLUT-1) lipid raft membrane-targeting, and cell invasive activity by GA were counteracted either by CK2α short hairpin RNA or cellular-Src (c-Src) inhibitor PP1. PP1 treatment, GLUT-1 or constitutively active Rac1 ectopic-expression blocked GA-induced decreases in cellular glucose, sphingolipid and cholesterol of lipid raft membranes, p85α-p110α-GTP-Rac1 complexes, glucosylceramide synthase activity and increase in ceramide and p110α-free p85α-PTEN complex levels of lipid raft membranes, which reversed the inhibition on matrix metalloproteinase (MMP)-2/-9-mediated cell invasion induced by GA. Using transient ectopic expression of nuclear factor-kappa B (NF-κB) p65, MMP-2/-9 promoter-driven luciferase, and NF-κB-dependent luciferase reporter genes and NF-κB specific inhibitors or Rac1 specific inhibitor NSC23766, we confirmed that an attenuation of Rac1 activity by GA confers inhibition of NF-κB-mediated MMP-2/-9 expression and cell invasion. In conclusion, GA-induced c-Src activation is a key inductive event for the formation of inactive Rac1-p-CK2α (Tyr 255) complexes, which disturbed lipid raft compartment of PI3K and PTEN molecules by impairing Akt-regulated GLUT-1-mediated sphingolipid synthesis, and finally resulting in inhibition of TSC cell invasion.

Retinoblastoma (RB) is the most common childhood malignant intraocular tumor. The clinical efficacy of vincristine (VCR) in the treatment of RB is severely limited by drug resistance. Here, we found that CD24, a GPI-anchored protein, was overexpressed in human RB tissues and RB cell lines, and was associated with the sensitivity of RB cells in response to VCR therapy. We demonstrated that CD24 plays a critical role in impairing RB sensitivity to VCR via regulating autophagy. Mechanistically, CD24 recruits PTEN to the lipid raft domain and regulates the PTEN/AKT/mTORC1 pathway to activate autophagy. Lipid raft localization was essential for CD24 recruitment function. Collectively, our findings revealed a novel role of CD24 in regulating RB sensitivity to VCR and showed that CD24 is a potential target for improving chemotherapeutic sensitivity and RB patient outcomes., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

The antioxidant vitamin E is a commonly used vitamin supplement. Although the multi-billion dollar vitamin and nutritional supplement industry encourages the use of vitamin E, there is very little evidence supporting its actual health benefits. Moreover, vitamin E is now marketed as a lipid raft destabilizing anti-cancer agent, in addition to its antioxidant behaviour. Here, we studied the influence of vitamin E and some of its vitamers on membrane raft stability using phase separating unilamellar lipid vesicles in conjunction with small-angle scattering techniques and fluorescence microscopy. We find that lipid phase behaviour remains unperturbed well beyond physiological concentrations of vitamin E (up to a mole fraction of 0.10). Our results are consistent with a proposed line active role of vitamin E at the domain boundary. We discuss the implications of these findings as they pertain to lipid raft modification in native membranes, and propose a new hypothesis for the antioxidant mechanism of vitamin E., 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 © 2020 Elsevier B.V. All rights reserved.)

10-gingerol is a major phenolic lipid found in the rhizomes of ginger (Zingiber officinale). Being amphiphilic in nature, phenolic lipids have the ability to incorporate into cell membranes and modulate membrane properties. The purpose of the present study was to evaluate the effects of 10-gingerol on lipid raft/membrane raft modulation in radio-resistant triple negative breast cancer (MDA-MB-231/IR) cells. The effects of 10-gingerol on MDA-MB-231/IR cells' proliferation, clonogenic growth, migration, and invasion were assayed using MTT, colony formation, cell migration, and invasion assays, respectively. Sucrose density gradient centrifugation was used to extract lipid rafts. Western blotting and immunofluorescence were employed to assess the effects of 10-gingerol on lipid raft/membrane raft modulation and lipid rafts-associated PI3K/Akt signaling. Cholesterol measurements were carried out using a commercially available kit. 10-gingerol suppressed the proliferation, migration, invasion, and induced apoptosis through targeting the PI3K/Akt signaling pathway in MDA-MB-231/IR cells. Moreover, 10-gingerol was found to modulate the lipid rafts of MDA-MB-231/IR cells and attenuate the key PI3K/Akt signaling components in lipid rafts. The cholesterol content of the lipid rafts and rafts-resident Akt signaling were also affected by exposure to 10-gingerol. The results of the present study highlight rafts-associated PI3K/Akt signaling as a new target of 10-gingerol in MDA-MB-231/IR cells, thus rationalizing a new rafts-mediated treatment approach for radio-resistant triple negative breast cancer cells., Competing Interests: The authors declare no conflict of interest

Acute lung injury (ALI) is a kind of lung serious disease which leads to the damage of alveolar epithelial cells and capillary endothelial. Lipopolysaccharide (LPS) is one of the common factors inducing ALI. The previous study has reported that the anti-inflammatory effect of peiminine, but little is known about its effect on the ALI induced by LPS. The aim of this study is to investigate the therapeutic effect of peiminine on LPS-induced acute lung injury and potential mechanisms. Mice were given LPS through nasal cavity to establish ALI model, and then the peiminine (1, 3, or 5 mg/kg) was injected into the mice as the experimental group. In the present study, we would measure the W/D ratio, activity of MPO, the histopathological changes, and the levels of cytokines. The results showed that peiminine could reduce the W/D ratio and the MPO activity significantly. Furthermore, the histopathological changes and the expression of TNF-α, IL-1β, and IL-6 were inhibited after the peiminine treatment. In vitro, peiminine significantly inhibited LPS-induced IL-8 production in A549 lung epithelial cells. Meanwhile, the activity of NF-κB signaling pathway was suppressed obviously by peiminine with the western blot analysis. Also, peiminine significantly attenuated LPS-induced AKT and PI3K phosphorylation. In addition, peiminine was found to disrupt lipid rafts formation by attenuating the cholesterol content. In conclusion, peiminine could attenuate LPS-induced ALI in mice and it may become a new approach to treat ALI.

Mycobacterium abscessus is an emerging multidrug-resistant bacterial pathogen causing severe lung infections in cystic fibrosis patients. A remarkable trait of this mycobacterial species is its ability to form morphologically smooth (S) and rough (R) colonies. The S-to-R transition is caused by the loss of glycopeptidolipids (GPLs) in the outer layer of the cell envelope and correlates with an increase in cording and virulence. Despite the physiological and medical importance of this morphological transition, whether it involves changes in cell surface properties remains unknown. Herein, we combine recently developed quantitative imaging (QI) atomic force microscopy (AFM) with hydrophobic tips to quantitatively map the surface structure and hydrophobicity of M. abscessus at high spatiotemporal resolution, and to assess how these properties are modulated by the S-to-R transition and by treatment with an inhibitor of the mycolic acid transporter MmpL3. We discover that loss of GPLs leads to major modifications in surface hydrophobicity, without any apparent change in cell surface ultrastructure. While R bacilli are homogeneously hydrophobic, S bacilli feature unusual variations of nanoscale hydrophobic properties. These previously undescribed cell surface nanodomains are likely to play critical roles in bacterial adhesion, aggregation, phenotypic heterogeneity and transmission, and in turn in virulence and pathogenicity. Our study also suggests that MmpL3 inhibitors show promise in nanomedicine as chemotherapeutic agents to interfere with the highly hydrophobic nature of the mycobacterial cell wall. The advantages of QI-AFM with hydrophobic tips are the ability to map chemical and structural properties simultaneously and at high resolution, applicable to a wide range of biosystems.

Alkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, erufosine (EPC3) is a promising drug for the treatment of several types of tumors. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy and analytical chemistry approaches (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy, generalized polarization imaging, as well as thin layer and gas chromatography) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid-lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 could be linked to its effects on fundamental biophysical properties of lipid membranes, as well as on lipid metabolism in cancer cells.

Colorectal cancer (CRC) is one of the most common cancers worldwide. Epidemiological studies indicate that consumption of fruits and vegetables containing procyanidins is associated with lower CRC risk. This study investigated the capacity of two dimeric procyanidins composed of epicatechin gallate (ECG) or epigallocatechin gallate (EGCG) isolated from persimmons, to inhibit CRC cell growth and promote apoptosis, characterizing the underlying mechanisms. ECG and EGCG dimers reduced the growth of five human CRC cell lines in a concentration (10-60 μM)- and time (24-72 h)-dependent manner, with a 72 h-IC 50 value in Caco-2 cells of 10 and 30 μM, respectively. ECG and EGCG dimers inhibited Caco-2 cell proliferation by arresting the cell cycle in G 2 /M phase and by inducing apoptosis via the mitochondrial pathway. In addition, ECG and EGCG dimers inhibited cell migration, invasion, and adhesion, decreasing the activity of matrix metalloproteinases (MMP-2/9). Mechanistically, ECG and EGCG dimers inhibited the activation of lipid raft-associated epidermal growth factor (EGF) receptor (EGFR), without affecting its localization at lipid rafts. In particular, ECG and EGCG dimers reduced EGFR phosphorylation at Tyr 1068 residue, prevented EGFR dimerization and activation upon stimulation, and induced EGFR internalization both in the absence and presence of EGF. Furthermore, ECG and EGCG dimers increased EGFR phosphorylation at Tyr 1045 residue, providing a docking site for ubiquitin ligase c-Cbl and induced EGFR degradation by the proteasome. Downstream of EGFR, ECG and EGCG dimers inhibited the activation of the MEK/ERK1/2 and PI3K/AKT signaling pathways, downregulating proteins involved in the modulation of cell survival. In conclusion, ECG and EGCG dimers reduced CRC cell growth by inhibiting EGFR activation at multiple steps, including the disruption of lipid rafts integrity and promoting EGFR degradation. These results shed light on a potential molecular mechanism on how procyanidins-rich diets may lower CRC risk., 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 © 2020 Elsevier Inc. All rights reserved.)

Background/aim: We previously showed that oxaliplatin induces necrotic-like cell death in hepatocarcinomas, and combination with ursodexoycholic acid (UDCA) significantly shifts the necrotic-like death to apoptosis. Since cell death mode is crucial on inflammatory responses and chemotherapeutic efficacy, the mechanism underlying determination of cell death mode by UDCA was investigated in this study., Materials and Methods: Apoptosis or necrosis was determined by apoptotic body formation, caspase-8 activity, LDH release and PI inclusion. The involvement of lipid rafts and death receptors was examined by rafts fractionation, confocal microscopy and gene silencing assays., Results: UDCA combination enhanced recruitment of death receptors and adaptors into cholesterol-enriched lipid rafts, and induced a stronger raft clustering. Lipid raft disruption decreased the UDCA/oxaliplatin-mediated apoptosis and increased necrotic-like death., Conclusion: UDCA promotes lipid raft localization of multiple death receptors, thereby contributing to a shift of cell death mode from oxaliplatin-induced necrotic death to apoptosis in HepG2 cells., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)