Membrane Concentration Research Articles

Alterations in The Functional and Structural Properties of Erythrocytes Due to Alcohol Intoxication; Correction of Disorders

Alcohol abuse is among the leading reasons for disability and accounts for at least 5% of the global health burden. This study aims to define changes in the functional and structural properties of erythrocytes in chronic ethanol intoxication and to design methods for correction of the identified disorders. The experiments has been conducted on 93 healthy sexually mature and newborn Wistar rats. Acute and chronic alcohol intoxication has been designed by forced intragastric administration of 20% ethanol solution at a dose of 3 ml/kg (2.92 g/kg) on a daily basis for 5, 30 and 60 days. From the newborn animals we obtained allogeneic hepatocytes and their cultural fluid, from which proteins were isolated. The proteins of allogeneic hepatocyte and the combined administration of immunomodulatory, hepatoprotective and antioxidant agents were used to correct disorders in chronic intoxication. The membranes of circulating erythrocytes were obtained by the method of Dodge; electrophoretic separation of membrane proteins was carried out according to the method of Laemli. The lipid content in the erythrocyte membrane was determined by liquid thin-layer chromatography. It has been elucidated that in presence of chronic alcohol intoxication there is a change in the content of the membrane proteins in circulating erythrocytes, which are responsible for flexibility and shaping, intrinsic cellular metabolism, stabilization and structure formation, and a disruption in the lipids representativeness and ratio that form the basis for the lipid framework of the plasmalemma and play the major role in arrangement of protein macromolecules and the normal metabolism of red blood cells as well. When analyzing the corrective activity of the proteins of allogeneic hepatocytes and the combination Glutoxim + Mexidol + Heptral, we have established their high efficiency towards the disorders in the functional and structural properties of circulating erythrocytes caused by chronic alcohol intoxication.

Syntaxin interacts with arachidonic acid to prevent diabetes mellitus

Syntaxin regulates pancreatic β cell mass and participates in insulin secretion by regulating insulin exocytosis. In addition, syntaxin 4 reduces IFNγ and TNF-α signaling via NF-ĸB in islet β-cells that facilitates plasma glucose sensing and appropriate insulin secretion. Arachidonic acid (AA) has potent anti-inflammatory actions and prevents the cytotoxic actions of alloxan and streptozotocin (STZ) against pancreatic β cells and thus, prevents the development of type 1 diabetes mellitus (induced by alloxan and STZ) and by virtue of its anti-inflammatory actions protects against the development of type 2 diabetes mellitus (DM) induced by STZ in experimental animals that are models of type 1 and type 2 DM in humans. AA has been shown to interact with syntaxin and thus, potentiate exocytosis. AA enhances cell membrane fluidity, increases the expression of GLUT and insulin receptors, and brings about its anti-inflammatory actions at least in part by enhancing the formation of its metabolite lipoxin A4 (LXA4). Prostaglandin E2 (PGE2), the pro-inflammatory metabolite of AA, activates ventromedial hypothalamus (VMH) neurons of the hypothalamus and inhibits insulin secretion leading to reduced glucose tolerance and decreases insulin sensitivity in the skeletal muscle and liver. This adverse action of PGE2 on insulin release and action can be attributed to its (PGE2) pro-inflammatory action and inhibitory action on vagal tone (vagus nerve and its principal neurotransmitter acetylcholine has potent anti-inflammatory actions). High fat diet fed animals have hypothalamic inflammation due to chronic elevation of PGE2. Patients with type 2 DM show low plasma concentrations of AA and LXA4 and elevated levels of PGE2. Administration of AA enhances LXA4 formation without altering or reducing PGE2 levels and thus, tilts the balance more towards anti-inflammatory events. These results suggest that administration of AA is useful in the prevention and management of DM by enhancing the action of syntaxin, increasing cell membrane fluidity, and reducing VMH inflammation. Docosahexaenoic acid (DHA) has actions like AA: it increases cell membrane fluidity; has anti-inflammatory actions by enhancing the formation of its anti-inflammatory metabolites resolvins, protectins and maresins; interacts with syntaxin and enhance exocytosis in general and of insulin. But the DHA content of cell membrane is lower compared to AA and its content in brain is significant. Hence, it is likely DHA is important in neurotransmitters secretion and regulating hypothalamic inflammation. It is likely that a combination of AA and DHA can prevent DM.

Rapid macrovoid characterization in membranes prepared via nonsolvent-induced phase separation: A comparison between 2D and 3D techniques

Optimizing the performance of asymmetric membranes prepared via nonsolvent-induced phase separation (NIPS) requires a quantitative understanding of how processing variables influence membrane morphology. Presently, the most useful structural quantification techniques require 3D visualization of the membrane structure and are best suited for studies seeking detailed information on small datasets. This study proposes and validates a rapid and accurate technique for quantifying macroporosity (i.e., Dm), a simple descriptor of sublayer macrovoid content in asymmetric membranes. Dm values measured from segmented cross-sectional imaging performed via X-ray computed tomography (XCT) and scanning electron microscopy (SEM) are presented and compared for three asymmetric membranes prepared from commercial polymers. Importantly, analyses of 3D XCT membrane reconstructions reveal that Dm is described by a single, centralized mean, which demonstrates that macrovoid content is spatially homogenous. Thus, Dm can be approximated from limited sampling of the 2D cross-sectional membrane structure via SEM. A proposed 2D SEM sampling method provides Dm estimates within ±6% of corresponding 3D XCT values with 30 independent measurements for the three membranes. Further sensitivity is achieved using complementary descriptors such as macrovoid count density (i.e., Cm). This technique is thus a useful tool for characterizing macroporosity from a broad selection of membrane samples.

Novel Preclinical Study of Galloylquinic Acid Compounds from Copaifera lucens with Potent Antifungal Activity against Vaginal Candidiasis Induced in a Murine Model via Multitarget Modes of Action.

ABSTRACTVaginal candidiasis is a medical condition characterized by the overgrowth of Candida spp. in the vaginal cavity with complex recurrent pathogenicity as well as tolerance to antifungal therapy and hence is awaiting more safe and effective treatments. This work aimed to assess the potential antifungal activity of galloylquinic acid compounds (GQAs) from Copaifera lucens leaves against vaginal Candida albicans. The antifungal susceptibility test was performed against 20 isolates of multidrug-resistant (MDR) C. albicans using agar diffusion and broth microdilution assays. The results showed that GQAs exhibited strong antagonistic activity against the test isolates, with inhibition zone diameters ranging from 26 to 38 mm and low MICs (1 to 16 μg/mL) as well as minimum fungicidal concentrations (2 to 32 μg/mL). The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay confirmed the safety of GQAs against the Vero cell line, showing a 50% inhibitory concentration (IC50) of 168.17 mg/mL. A marked difference in the growth pattern of the treated and untreated pathogens was also observed, where a concentration-dependent reduction in the growth rate occurred. Moreover, a pronounced fungicidal effect was demonstrated 6 h after treatment with 1× the minimum fungicidal concentration (MFC), as evidenced by time-kill assays, where the number of survivors was decreased a 6-fold. GQAs effectively inhibited and eradicated about 80% of C. albicans biofilm at 6 μg/mL and 32 μg/mL, respectively. Interestingly, GQAs disturbed the fungal membrane integrity, induced cell lysis, and reduced the virulence factors (proteinase and phospholipase) as well as the catalase activity. Moreover, the ergosterol content in the plasma membrane decreased in a concentration-dependent manner. Additionally, the altered mitochondrial membrane potential was associated with an increased release of cytochrome c from mitochondria to the cytosol, suggesting the initiation of early apoptosis in GQA-treated cells. Transcriptional analysis revealed that all test genes encoding virulence traits, including SAP1, PLB1, LIP1, HWP1, and ALS1, were markedly downregulated in GQA-treated cells compared to the control. The in vivo murine model of vaginal candidiasis further confirmed the therapeutic activity of GQAs (4 mg/kg of body weight) against C. albicans. This work comprehensively evaluated the antifungal, antivirulence, and antibiofilm activities of GQAs against C. albicans isolates using in vitro and in vivo models, providing molecular-level insights into the antifungal mechanism of action and experimental evidence that supports the potential use of GQAs for the treatment of vaginal candidiasis.IMPORTANCE Our work presents a new perspective on the potential use of GQAs as safe and highly effective phytochemicals against MDR C. albicans. This microorganism colonizes the human vaginal epithelium, causing vaginal candidiasis, a condition characterized by recurrent pathogenicity and tolerance to traditional antifungal therapy. Based on the results of in vitro tests, our study reports GQAs antifungal modes of action. These compounds exhibited an anticandidal effect by deactivating the fungal hydrolytic enzymes, reducing ergosterol content in the plasma membrane, altering the potential of the mitochondrial membrane, and inducing apoptosis. Additionally, GQAs showed high activity in eradicating the biofilm formed by the fungus via the downregulation of HWP1, ALS, SAP, PLB, and LIP genes, which are constitutively expressed in the biofilm. In an in vivo murine model of vaginal candidiasis, GQAs further demonstrated strong evidence of their effectiveness as an antifungal therapy. In this regard, our findings provide novel insights into the potential therapeutic use of these phytoactive molecules for vaginal candidiasis treatment.

Lysosome exocytosis is required for mitosis in mammalian cells

Mitosis, the accurate segregation of duplicated genetic material into what will become two new daughter cells, is accompanied by extensive membrane remodelling and membrane trafficking activities. Early in mitosis, adherent cells partially detach from the substratum, round up and their surface area decreases. This likely results from an endocytic uptake of plasma membrane material. As cells enter cytokinesis they re-adhere, flatten and exhibit an associated increase in surface area. The identity of the membrane donor for this phase of mitosis remains unclear. In this paper we demonstrate how lysosomes dynamically redistribute during mitosis and exocytose. Antagonism of lysosomal exocytosis by pharmacological and genetic approaches causes mitosis failure in a significant proportion of cells. We speculate that either lysosomal membrane or luminal content release, possibly both, are therefore required for normal mitosis progression. These findings are important as they reveal a new process required for successful cell division.

Fertility Impairment after Trekking at High Altitude: A Proof of Mechanisms on Redox and Metabolic Seminal Changes.

Many authors described negative but reversible effects of high-altitude hypoxic exposure on animal and human fertility in terms of sperm concentration, function, and biochemical alterations. The aim of this study was to evaluate the acute and chronic effects of high-altitude exposure on classical sperm parameters, redox status, and membrane composition in a group of travellers. Five healthy Italian males, all lowlanders not accustomed to the altitude, were evaluated after 19 days-trekking through low, moderate, and high altitudes in the Himalayas. Sperm samples were collected before (Pre), 10 days after (Post), and 70 days after the end of the expedition (Follow-up). Sperm concentration, cholesterol and oxysterol membrane content, and redox status were measured. Hypoxic trek led to a significant reduction in sperm concentration (p < 0.001, η2p = 0.91), with a reduction from Pre to Post (71.33 ± 38.81 to 60.65 ± 34.63 × 106/mL) and a further reduction at Follow-up (to 37.13 ± 39.17 × 106/mL). The seminal volume was significantly affected by the hypoxic trek (p = 0.001, η2p = 0.75) with a significant reduction from Pre to Post (2.86 ± 0.75 to 1.68 ± 0.49 mL) and with partial recovery at Follow-up (to 2.46 ± 0.45 mL). Moreover, subjects had an increase in ROS production (+86%), and a decrease in antioxidant capacity (−37%) in the Post period with partial recovery at Follow-up. These results integrated the hormonal response on thyroid function, hypothalamus–pituitary–gonadal axis, and the prolactin/cortisol pathways previously reported. An uncontrolled ROS production, rather than a compromised antioxidant activity, was likely the cause of impaired sperm quality. The reduction in fertility status observed in this study may lie in an evolutionary Darwinian explanation, i.e., limiting reproduction due to the “adaptive disadvantage” offered by the combined stressors of high-altitude hypoxia and daily physical exercise.

The milk fat globule size governs a physiological switch for biofilm formation by Bacillus subtilis.

Milk lipids are organized in the form of milk fat globules (MFG), ranging in size from 0. 1 to 15 μm. The MFG size is closely associated with the composition of fatty acids, polar lipids, sphingolipids, cholesterol and the content of the MFG membrane (MFGM). Also, the MFGM integral proteins and glycoconjugates differ in composition and structure between different MFG size groups. These compositional differences may modulate the functionality of the MFG and its interaction with microbial cells. We report that small (2.3 μm) MFG facilitates the growth of the Gram-positive bacterium Bacillus subtilis whereas induction of biofilm formation was found in the presence of large (7.0 μm) MFG. Attempting to distinguish between the role played by the size from that played by the composition of the MFG, we compared phospholipid composition between treatments. We found that adjusting the phosphatidylethanolamine (PE) level to the concentration found in the small MFG, increased growth but suppressed biofilm formation in the presence of large MFG. The same normalization protocol for phosphatidylinositol (PI) or sphingomyeline (SM) did not exert a similar effect, suggesting a specific role for PE in regulating bacteria proliferation. We suggest that the content of MFGM, affected by MFG size, governs the ability of B. subtilis to utilize lipids from milk fat. This process might affect the bacterial decision-making toward biofilm formation or growth.

Transcriptome Analysis Reveals Contrasting Plant Responses of Sorghum bicolor upon Colonization by Two Formae Speciales of Sporisorium reilianum

The biotrophic fungus Sporisorium reilianum exists in two host-adapted formae speciales that cause head smut in maize (S. reilianum f. sp. zeae; SRZ) and sorghum (S. reilianum f. sp. reilianum; SRS). In sorghum, the spread of SRZ is limited to the leaves. To understand the plant responses to each forma specialis, we determined the transcriptome of sorghum leaves inoculated either with SRS or SRZ. Fungal inoculation led to gene expression rather than suppression in sorghum. SRZ induced a much greater number of genes than SRS. Each forma specialis induced a distinct set of plant genes. The SRZ-induced genes were involved in plant defense mainly at the plasma membrane and were associated with the Molecular Function Gene Ontology terms chitin binding, abscisic acid binding, protein phosphatase inhibitor activity, terpene synthase activity, chitinase activity, transmembrane transporter activity and signaling receptor activity. Specifically, we found an upregulation of the genes involved in phospholipid degradation and sphingolipid biosynthesis, suggesting that the lipid content of the plant plasma membrane may contribute to preventing the systemic spread of SRZ. In contrast, the colonization of sorghum with SRS increased the expression of the genes involved in the detoxification of cellular oxidants and in the unfolded protein response at the endoplasmic reticulum, as well as of the genes modifying the cuticle wax and lipid composition through the generation of alkanes and phytosterols. These results identified plant compartments that may have a function in resistance against SRZ (plasma membrane) and susceptibility towards SRS (endoplasmic reticulum) that need more attention in the future.

Synthesis and Characterization of Ceramide-Containing Liposomes as Membrane Models for Different T Cell Subpopulations

A fine balance of regulatory (Treg) and conventional CD4+ T cells (Tconv) is required to prevent harmful immune responses, while at the same time ensuring the development of protective immunity against pathogens. As for many cellular processes, sphingolipid metabolism also crucially modulates the Treg/Tconv balance. However, our understanding of how sphingolipid metabolism is involved in T cell biology is still evolving and a better characterization of the tools at hand is required to advance the field. Therefore, we established a reductionist liposomal membrane model system to imitate the plasma membrane of mouse Treg and Tconv with regards to their ceramide content. We found that the capacity of membranes to incorporate externally added azide-functionalized ceramide positively correlated with the ceramide content of the liposomes. Moreover, we studied the impact of the different liposomal preparations on primary mouse splenocytes in vitro. The addition of liposomes to resting, but not activated, splenocytes maintained viability with liposomes containing high amounts of C16-ceramide being most efficient. Our data thus suggest that differences in ceramide post-incorporation into Treg and Tconv reflect differences in the ceramide content of cellular membranes.

Changes in hydration of liposome membranes exposed to nanosecond electric pulses detected by wide-field Coherent anti-Stokes Raman microspectroscopy

Electropulsation has become a powerful technological platform for electromanipulation of cells and tissues for various medical and biotechnological applications, but the molecular changes that underlay the very first initiation step of this process have not been experimentally observed.Here, we endowed a wide-field Coherent anti-Stokes Raman Scattering platform with an ad-hoc electromagnetic exposure device and we demonstrated, using artificial lipid vesicles (i.e. liposomes), that electropulsation is initiated by the increase of interstitial water content in liposome membranes. A pulse-dependent accumulation of the interstitial water molecules is observed in the membranes and a plausible mechanism supported by a computational electrochemical model is presented and discussed.

SERINC5-Mediated Restriction of HIV-1 Infectivity Correlates with Resistance to Cholesterol Extraction but Not with Lipid Order of Viral Membrane.

Serine incorporator 5 (SER5) is a protein that upon incorporation into virions inhibits HIV-1 infectivity by interfering with the ability of the Env glycoprotein to promote viral fusion. The mechanisms by which SER5 antagonizes HIV-1 fusion are not well understood. A recent study of SER5’s structure revealed a lipid-binding pocket, suggesting the ability to sequester lipids. This finding, along with the well-documented modulation of HIV-1 infectivity by viral lipids, especially cholesterol, prompted our examination of SER5′s effect on the general lipid order of the HIV-1 membrane. Pseudoviruses bearing the SER5-sensitive HXB2-Env and containing SER5 or SER2, a control protein that lacks antiviral activity, were analyzed using two distinct lipid-order probes. We show that SER5 incorporation does not noticeably affect the lipid order of pseudoviruses. Although viral cholesterol extraction reduces HIV-1 infectivity, SER5+ viruses are less sensitive to cholesterol extraction than the control samples. In contrast, the virus’ sensitivity to cholesterol oxidation was not affected by SER5 incorporation. The hydrolytic release of sphingomyelin-sequestered cholesterol had a minimal impact on the apparent resistance to cholesterol extraction. Based on these results, we propose that a subpopulation of more stable Env glycoproteins responsible for the residual infectivity of SER5+ viruses is less sensitive to the cholesterol content of the viral membrane.

Characterization and molecular mechanism of the thermal-oxidative gradient aging behavior in asphalt films

To investigate the aging differences of asphalt films in the vertical aggregate direction, a three-layers model of oxygen-asphalt-aggregate was generated to explore the molecular mechanism of gradient aging. Moreover, the asphalt-aggregate specimens aged for 10, 20, and 40 h were layered for three parts. Fourier transforms infrared spectroscopy and thin layer chromatography-flame ionization detector tests verified the simulative findings. Eventually, the moduli of the asphalt pellicles were tested using nanoindentation test. Results denote that the oxygen’s diffusion and distribution of four components are more sensitive to pressure. The aging grade and component content of the asphalt membranes at miscellaneous thicknesses have significant differences. The combined effects of component volatilization, oxidation reactions and component migration lead to mutates in the modulus gradient of the asphalt films. The carbonyl content difference between the top and bottom of the three binders are approximately 0.22, −0.13, and − 0.27, respectively. Meanwhile, the maximum difference in modulus of binders and mastics are about 2.7 and 320.2 MPa.

Preparation of Alumina-Sphere-Supported Potassium Chabazite Zeolite Membrane with Excellent Potassium Extraction Performance at Room Temperature

In this paper, a potassium chabazite (KCHA) zeolite membrane was prepared by coating KCHA zeolite on the surface of a porous alumina sphere. The performance of the KCHA zeolite membrane in extracting potassium from seawater and sea bittern at room temperature was studied in detail. The XRD results show that the prepared KCHA zeolite was a KCHA membrane. The EDS test indicated that the potassium content of the KCHA zeolite membrane reached a value of 18.33 wt.%. The morphology of the KCHA zeolite grown on the surface of the alumina sphere was similar to a sphere, and it had good symmetry. The potassium ion-exchange capacities of the KCHA zeolite membrane reached 32 mg/g in seawater and 77 mg/g in sea bittern at room temperature. Ion exchange between the ammonium ions and potassium ions in the KCHA zeolite membrane could be completed in a short time at room temperature. The KCHA zeolite membrane was proven to have good reusability in seawater and sea bittern. The selective ion-exchange mechanism of the KCHA zeolite membrane was controlled by a specific K+ ion memory.

Diagnosing Improper Membrane Water Content in Proton Exchange Membrane Fuel Cell Using Two-Dimensional Convolutional Neural Network

In existing proton exchange membrane fuel cell (PEMFC) applications, improper membrane water management will cause PEMFC performance decay, which restricts the reliability and durability of PEMFC systems. Therefore, diagnosing improper water content in the PEMFC membrane is the key to taking appropriate mitigations to guarantee its operating safety. This paper proposes a novel approach for diagnosing improper PEMFC water content using a two-dimensional convolutional neural network (2D-CNN). In the analysis, the collected PEMFC voltage signal is transformed into 2D image data, which is then used to train the 2D-CNN. Data enhancement and pre-processing techniques are applied to PEMFC voltage data before the training. Results demonstrate that with the trained model, the diagnostic accuracy for PEMFC membrane improper water content can reach 97.5%. Moreover, by comparing it with a one-dimensional convolutional neural network (1D-CNN), the noise robustness of the proposed method can be better highlighted. Furthermore, t-distributed Stochastic Neighbor Embedding (t-SNE) is used to visualize the feature separability with different methods. With the findings, the effectiveness of using 2D-CNN for diagnosing PEMFC membrane improper water content is explored.

Preparation of AQS/GO/PVDF redox mediator membrane and the decolorization of azo dyes

AQS/PVDF and AQS/GO/PVDF redox mediator membranes were prepared with poly (vinylidene fluoride) (PVDF) as the raw supporting material of membrane, anthraquinone-2-sulfonic acid (AQS) as the redox mediator, and graphene oxide (GO) as an additive by an immersion precipitation method. The AQS content in the redox mediator membrane was optimized with the formation performance of AQS/PVDF membrane. With the optimal casting solution for AQS/PVDF membrane, GO was introduced as an additive to form AQS/GO/PVDF membrane. The effects of the redox mediator membranes on the bacterial decolorization of dye wastewaters were investigated. The results suggest that both AQS/PVDF and AQS/GO/PVDF membranes can promote the bacterial decolorization of dyes and the introduction of GO improves the decolorization rate more significantly because it can accelerate the electron transfer. Therefore, the conductivity of GO and the electron shuttle of AQS show a synergetic effect on accelerating the bacterial decolorization of dyes.

Membrane elasticity modulated by cholesterol in model of porcine eye lens-lipid membrane

Experimental evidence shows that the eye lens loses its elasticity dramatically with age. It has also been reported that the cholesterol (Chol) content in the eye lens fiber cell plasma membrane increases significantly with age. High Chol content leads to the formation of cholesterol bilayer domains (CBDs) in the lens membrane. The role of high Chol associated with lens elasticity is unclear. The purpose of this research is to investigate the membrane elasticity of the model of porcine lens-lipid (MPLL) membrane with increasing Chol content to elucidate the role of high Chol in lens membrane elasticity. In this study, we used atomic force microscopy (AFM) to study the mechanical properties (breakthrough force and area compressibility modulus (KA)) of the MPLL membrane with increasing Chol content where KA is the measure of membrane elasticity. We varied Chol concentration in Chol/MPLL membrane from 0 to ∼71 mol%. Supported Chol/MPLL membranes were prepared by fusion of small unilamellar vesicles (SUVs) on top of a flat mica surface. SUVs of the Chol/MPLL lipid mixture were prepared with the rapid solvent exchange method followed by probe-tip sonication. For the Chol/MPLL mixing ratio of 0, AFM image showed the formation of two distinct phases of the membrane, i.e., liquid-disordered phase (ld) and solid-ordered phase (so) membrane. However, with Chol/MPLL mixing ratio of 0.5 and above, only liquid-ordered phase (lo) membrane was formed. Also, two distinct breakthrough forces corresponding to ld and so were observed for Chol/MPLL mixing ratio of 0, whereas only one breakthrough force was observed for membranes with Chol/MPLL mixing ratio of 0.5 and above. No significant difference in the membrane surface roughness was measured with increasing Chol content for these membranes; however, breakthrough force and KA for lo membrane increased when Chol/MPLL mixing ratio was increased from 0.5 to 1. Interestingly above the Chol/MPLL mixing ratio of 1, both breakthrough force and KA decreased, indicating the formation of CBDs. Furthermore, these results showed that membrane elasticity increases at high Chol content, suggesting that high Chol content in lens membrane might be responsible for maintaining lens membrane elasticity.

Zebrafish Melanoma-Derived Interstitial EVs Are Carriers of ncRNAs That Induce Inflammation.

Extracellular vesicles (EVs) are membranous particles released by all cell types. Their role as functional carrier of bioactive molecules is boosted by cells that actively secrete them in biological fluids or in the intercellular space (interstitial EVs, iEVs). Here we have optimised a method for the isolation and characterization of zebrafish iEVs from whole melanoma tissues. Zebrafish melanoma iEVs are around 140 nm in diameter, as determined by nanoparticle tracking and transmission electron microscopy (TEM) analysis. Western blot analysis shows enrichment for CD63 and Alix in the iEV fraction, but not in melanoma cell lysates. Super resolution and confocal microscopy reveal that purified zebrafish iEVs are green fluorescent protein positive (GFP+), indicating that they integrate the oncogene GFP-HRASV12G used to induce melanoma in this model within their vesicular membrane or luminal content. Analysis of RNA-Seq data found 118 non-coding (nc)RNAs differentially distributed between zebrafish melanoma and their iEVs, with only 17 of them being selectively enriched in iEVs. Among these, the RNA components of RNAses P and MRP, which process ribosomal RNA precursors, mitochondrial RNAs, and some mRNAs, were enriched in zebrafish and human melanoma EVs, but not in iEVs extracted from brain tumours. We found that melanoma iEVs induce an inflammatory response when injected in larvae, with increased expression of interferon responsive genes, and this effect is reproduced by MRP- or P-RNAs injected into circulation. This suggests that zebrafish melanoma iEVs are a source of MRP- and P-RNAs that can trigger inflammation in cells of the innate immune system.

Bilayer Forming Phospholipids as Targets for Cancer Therapy.

Phospholipids represent a crucial component for the structure of cell membranes. Phosphatidylcholine and phosphatidylethanolamine are two phospholipids that comprise the majority of cell membranes. De novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine occurs via the Kennedy pathway, and perturbations in the regulation of this pathway are linked to a variety of human diseases, including cancer. Altered phosphatidylcholine and phosphatidylethanolamine membrane content, phospholipid metabolite levels, and fatty acid profiles are frequently identified as hallmarks of cancer development and progression. This review summarizes the research on how phospholipid metabolism changes over oncogenic transformation, and how phospholipid profiling can differentiate between human cancer and healthy tissues, with a focus on colorectal cancer, breast cancer, and non-small cell lung cancer. The potential for phospholipids to serve as biomarkers for diagnostics, or as anticancer therapy targets, is also discussed.

Impact of cholesterol and sphingomyelin on intrinsic membrane permeability

Transwell experiments with Caco-2 or MDCK cells are the gold standard for determining the intestinal permeability of chemicals. The intrinsic membrane permeability (P0), that can be extracted from these experiments, might be comparable to P0 measured in black lipid membrane (BLM) experiments and P0 predicted by the solubility-diffusion model. Unfortunately, the overlap between experimental P0,Caco-2/MDCK and P0,BLM data is very small. So far, differences between both approaches have been attributed to the cholesterol and sphingomyelin content of cell membranes, but the database is too sparse to thoroughly test this theory. To create a diverse dataset, we measured P0,BLM of ten chemicals in BLM experiments using DPhPC and DPhPC/cholesterol/sphingomyelin membranes. The results were compared to predicted BLM data and experimental Caco-2/MDCK data obtained from literature. While P0,BLM of all chemicals was well predicted by the solubility-diffusion model, P0,Caco-2/MDCK was only predictable for rather hydrophilic compounds with logarithmic hexadecane/water partition coefficients below −0.5. The effect of cholesterol and sphingomyelin on P0,BLM was negligibly small.

Mutations of the nACh Receptor M4 Helix Reveal Different Phenotypes in Different Expression Systems: Could Lipids be Responsible?

The role of the outermost helix (M4) in the pentameric ligand-gated ion channel (pLGIC) family is currently not fully understood. It is known that M4 is important for receptor assembly, possibly via interactions with neighboring M1 and M3 helices. M4 can also transmit information on the lipid content of the membrane to the gating mechanism, and it may form a link to the extracellular domain via the Cys-loop. Our previous study examining the α4β2 nACh receptor M4 helix using HEK cells indicated M4 here is more sensitive to change than those of other pLGIC. Many of these other studies, however, were performed in Xenopus oocytes. Here we examine the nine previously identified nonfunctional α4β2 nACh receptor M4 mutant receptors using this system. The data reveal that seven of these mutant receptors do function when expressed in oocytes, with only 2, the conserved Asp at the intracellular end of M4 and a Phe in the center, having a similar phenotype (nonfunctional) in both HEK cells and oocytes. The oocyte data are more consistent with studies in other pLGIC and demonstrate the importance of the expression system used. Of the many differences between these two expression systems, we suggest that the different lipid content of the plasma membrane is a possible candidate for explaining these discrepancies.