Membrane Concentration Research Articles

Multifunctional Heterogeneous Ion-Exchange Membranes for Ion and Microbe Removal in Low-Salinity Water.

Here, multifunctional heterogeneous ion-exchange metal nanocomposite membranes were prepared for surface water desalination and bacterial inactivation under low-pressure (0.05 MPa) filtration conditions. Ultrafiltration (UF) heterogeneous ion exchange membranes (IEMs) were modified with different concentrations of AgNO3 and CuSO4 solutions using the intermatrix synthesis (IMS) technique to produce metal nanocomposite membranes. Scanning electron microscopy (SEM) images revealed that the metal nanoparticles (MNPs) (Ag and Cu) were uniformly distributed on the surface and the interior of the nanocomposite membranes. With increasing metal precursor solution concentration (0.01 to 0.05 mol·L-1), the metal content of Ag and Cu nanocomposite membranes increased from 0.020 to 0.084 mg·cm-2 and from 0.031 to 0.218 m·cm-2 respectively. Results showed that the hydrodynamic diameter diameters of Ag and Cu nanoparticles (NPs) increased from 62.42 to 121.10 nm and from 54.2 to 125.7 nm respectively, as the metal precursor concentration loaded increased. The leaching of metals from metal nanocomposite membranes was measured in a dead-end filtration system, and the highest leaching concentration levels were 8.72 ppb and 5.32 ppb for Ag and Cu, respectively. The salt rejection studies indicated that ionic selectivity was improved with increasing metal content. Bacterial filtration showed higher antibacterial activity for metal nanocomposite membranes, reaching 3.6 log bacterial inactivation.

Improved biomedical bioactivity of polyvinyl alcohol/polyethylene oxide composite system-based nanofiber membranes via incorporating Antarctic krill protein

Polyvinyl alcohol (PVA) and polyethylene oxide (PEO) nanofiber membranes with high porosity and specific surface area supply a suitable structural environment for cell growth, however the difficulty of cells adhesion to and growth has tremendously limited their application in biomedical materials. In this work, by incorporating Antarctic krill protein (AKP), the problem in cells adhesion and growth is successfully addressed for PVA/PEO nanofiber membranes. Meanwhile, it is worth noting that the problem that AKP is difficult to prepare nanofibers by electrospinning process is also effectively avoided. The intermolecular interactions ensure the uniform dispersion of AKP in the nanofiber membranes. And both the crystal structure content of nanofiber membranes and the ordered secondary structure content of AKP reach the maximum values of 37.41 % and 52.17 % at the AKP content of 20 %. The tensile strength is improved by 13.46 % notably. More importantly, the introduction of AKP enhances cell proliferation and viability and adds more recognition sites for cell attachment, solving the problem of difficult cell adhesion and growth. Thus, as-prepared nanofiber membranes containing AKP can supply suitable structural environment and excellent biocompatibility, for cell growth and reproduction. It is expected to be an essential candidate in biomedical materials with potential application prospects.

Structure of a cholinergic cell membrane.

While it has long been established that cell membranes are complex assemblies of proteins and bilayer-forming lipids, the inherent mobility and wide-ranging heterogeneity of the lipids have limited our ability to understand cell-membrane structure at a molecular level. Consequently, little is yet known about the protein-lipid and lipid-lipid interplay that exists in situ. Here I use cryo-EM and exploit the regularity and high protein content of the postsynaptic cell membrane of Torpedo to determine its protein-lipid structure. This membrane has a relatively simple composition, most densely populated by a single protein (nicotinic acetylcholine receptor), embedded in a cholesterol-rich phospholipid bilayer. Moreover in tubular vesicles, which bud from the isolated membranes, the protein arranges on a regular surface lattice, as it does in vivo at the Torpedo synapse and at the neuromuscular junction. Density maps obtained previously from such vesicles revealed that cholesterol attaches to specific sites on the protein in both leaflets of the bilayer, and assembles into protein-bridging aggregates or microdomains.The present study succeeds in resolving longer-range details of the lipids. Thus in the inner leaflet of the bilayer, which has the highest cholesterol concentration, the lipids are now seen to organise into close-packed linear arrays, building within the hydrophobic core a static sterol-hydrocarbon ‘skin’. This hitherto unobserved hydrophobic-core structure has far-reaching implications in terms of how cholesterol-rich membranes are constructed and function.

Effects of cholesterol on membrane binding by Synaptotagmin-7 C2 domains.

Synaptotagmin-7 (Syt-7) is a calcium-dependent membrane binding protein that triggers membrane fusion and content release of large dense core vesicles in neuroendocrine cells, including insulin granules in pancreatic β-cells. Syt-7 is also important in synaptic facilitation for neurotransmitter release. Defining factors involved in Syt-7 membrane binding is important for understanding basic endocrine and neuronal biology as well as disease pathology. Syt-7 contains two C-terminal C2 domains that bind anionic lipid membranes with strong calcium sensitivity. In particular, the C2A domain binds physiological membranes in the presence of low micromolar calcium via a combination of electrostatic and hydrophobic interactions, while C2B interacts with both phosphatidylserine and phosphatidyl-4,5-bisphosphate via conserved lysine and arginine residues in the calcium-binding loops and polybasic regions. However, it remains unknown how other lipids fine-tune Syt-7 membrane binding in cells. Here, we present molecular dynamics simulation data which suggest that cholesterol may preferentially accumulate at the sites of C2 domain membrane interaction. Additionally, we used stopped flow fluorescence to measure kinetics of binding and release from synthetic liposomes, and our preliminary results indicate an increased dissociation rate when cholesterol is removed. Results will be presented for ongoing experiments with Syt-7 C2A, C2B, and C2AB domains. Overall, the study will improve our understanding of the factors that control the membrane affinity and calcium sensitivity of Syt-7.

Isolation, identification, biological characteristics, and antifungal efficacy of sodium bicarbonate combined with natamycin on Aspergillus niger from Shengzhou nane (Prunus salicina var. taoxingli) fruit.

The fungi causing fruit rot were isolated from symptomatic Shengzhou nane (Prunus salicina var. taoxingli) fruit and were identified as Aspergillus niger by biological characteristics and molecular analysis of the internal transcribed spacer region (rDNA-ITS) and translation elongation factor-1α (TEF-1α) sequences. Optimal growth conditions for A. niger were 30°C, pH 5.0-6.0, and fructose and peptone as carbon and nitrogen sources. The effects of sodium bicarbonate (SBC), natamycin (NT), and combined treatments on A. niger inhibition were investigated. Treatment with 4.0 g/L sodium bicarbonate (SBC) + 5.0 mg/L natamycin (NT) inhibited mycelial growth and spore germination as completely as 12.0 mg/L SBC or 25.0 mg/L NT. SBC and NT treatments disrupted the structural integrity of cell and mitochondria membranes and decreased enzyme activities involved in the tricarboxylic acid (TCA) cycle, mitochondrial membrane potential (MMP), ATP production in mitochondria, and ergosterol content in the plasma membrane, thus leading to the inhibition of A. niger growth. Moreover, experimental results in vivo showed that the rot lesion diameter and decay rate of Shengzhou nane fruit treated with SBC and NT were significantly reduced compared with the control. The results suggest that the combination treatment of SBC and NT could be an alternative to synthetic fungicides for controlling postharvest Shengzhou nane decay caused by A. niger.

Involvement of the Membrane Nanodomain Protein, AtFlot1, in Vesicular Transport of Plasma Membrane H+-ATPase in Arabidopsis thaliana under Salt Stress.

The aim of this study was to elucidate whether the membrane nanodomain protein AtFlot1 is involved in vesicular transport pathways and regulation of the P-type H+-ATPase content in plasma membrane of A. thaliana under salt stress. Transmission electron microscopy revealed changes in the endosomal system of A. thaliana root cells due to knockout mutation SALK_205125C (Atflot1ko). Immunoblotting of the plasma membrane-enriched fractions isolated from plant organs with an antibody to the H+-ATPase demonstrated changes in the H+-ATPase content in plasma membrane in response to the Atflot1ko mutation and salt shock. Expression levels of the main H+-ATPase isoforms, PMA1 and PMA2, as well as endocytosis activity of root cells determined by endocytic probe FM4-64 uptake assay, were unchanged in the Atflot1ko mutant. We have shown that AtFlot1 participates in regulation of the H+-ATPase content in the plasma membrane. We hypothesized that AtFlot1 is involved in both exocytosis and endocytosis, and, thus, contributes to the maintenance of cell ion homeostasis under salt stress. The lack of a pronounced Atflot1ko phenotype under salt stress conditions may be due to the assumed ability of Atflot1ko to switch vesicular transport to alternative pathways. Functional redundancy of AtFlot proteins may play a role in the functioning of these alternative pathways.

Severe COVID-19-A Review of Suggested Mechanisms Based on the Role of Extracellular Matrix Stiffness.

The severity of COVID-19 commonly depends on age-related tissue stiffness. The aim was to review publications that explain the effect of microenvironmental extracellular matrix stiffness on cellular processes. Platelets and endothelial cells are mechanosensitive. Increased tissue stiffness can trigger cytokine storm with the upregulated expression of pro-inflammatory cytokines, such as tumor necrosis factor alpha and interleukin IL-6, and tissue integrity disruption, leading to enhanced virus entry and disease severity. Increased tissue stiffness in critically ill COVID-19 patients triggers platelet activation and initiates plague formation and thrombosis development. Cholesterol content in cell membrane increases with aging and further enhances tissue stiffness. Membrane cholesterol depletion decreases virus entry to host cells. Membrane cholesterol lowering drugs, such as statins or novel chitosan derivatives, have to be further developed for application in COVID-19 treatment. Statins are also known to decrease arterial stiffness mitigating cardiovascular diseases. Sulfated chitosan derivatives can be further developed for potential use in future as anticoagulants in prevention of severe COVID-19. Anti-TNF-α therapies as well as destiffening therapies have been suggested to combat severe COVID-19. The inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells pathway must be considered as a therapeutic target in the treatment of severe COVID-19 patients. The activation of mechanosensitive platelets by higher matrix stiffness increases their adhesion and the risk of thrombus formation, thus enhancing the severity of COVID-19.

Nanosecond pulsed electric field suppresses growth and reduces multi-drug resistance effect in pancreatic cancer

Nanosecond pulsed electric fields (nsPEF) have been shown to exert anticancer effects; however, little is known about the mechanisms triggered in cancer cells by nanosecond-length pulses, especially when low, sub-permeabilization voltage is used. In this study, three human pancreatic cancer cell lines were treated with nsPEF and molecular changes at the cellular level were analyzed. Further, we assessed the efficacy of paclitaxel chemotherapy following nsPEF treatment and correlated that with the changes in the expression of multi-drug resistance (MDR) proteins. Finally, we examined the influence of nsPEF on the adhesive properties of cancer cells as well as the formation and growth of pancreatic cancer spheroids. Cell line response differed with the application of a 200 ns, 100 pulses, 8 kV/cm, 10 kHz PEF treatment. PEF treatment led to (1) the release of microvesicles (MV) in EPP85-181RDB cells, (2) electropermeabilization in EPP85-181RNOV cells and (3) cell shrinkage in EPP85-181P cells. The release of MV’s in EPP85-181RDB cells reduced the membrane content of P-gp and LRP, leading to a transient increase in vulnerability of the cells towards paclitaxel. In all cell lines we observed an initial reduction in size of the cancer spheroids after the nsPEF treatment. Cell line EPP85-181RNOV exhibited a permanent reduction in the spheroid size after nsPEF. We propose a mechanism in which the surface tension of the membrane, regulated by the organization of actin fibers, modulates the response of cancer cells towards nsPEF. When a membrane’s surface tension remains low, we observed some cells form protrusions and release MVs containing MDR proteins. In contrast, when cell surface tension remains high, the cell membrane is being electroporated. The latter effect may be responsible for the reduced tumor growth following nsPEF treatment.

Genomic study and lipidomic bioassay of Leeuwenhoekiella parthenopeia: A novel rare biosphere marine bacterium that inhibits tumor cell viability.

The fraction of low-abundance microbiota in the marine environment is a promising target for discovering new bioactive molecules with pharmaceutical applications. Phenomena in the ocean such as diel vertical migration (DVM) and seasonal dynamic events influence the pattern of diversity of marine bacteria, conditioning the probability of isolation of uncultured bacteria. In this study, we report a new marine bacterium belonging to the rare biosphere, Leeuwenhoekiella parthenopeia sp. nov. Mr9T, which was isolated employing seasonal and diel sampling approaches. Its complete characterization, ecology, biosynthetic gene profiling of the whole genus Leeuwenhoekiella, and bioactivity of its extract on human cells are reported. The phylogenomic and microbial diversity studies demonstrated that this bacterium is a new and rare species, barely representing 0.0029% of the bacterial community in Mediterranean Sea metagenomes. The biosynthetic profiling of species of the genus Leeuwenhoekiella showed nine functionally related gene cluster families (GCF), none were associated with pathways responsible to produce known compounds or registered patents, therefore revealing its potential to synthesize novel bioactive compounds. In vitro screenings of L. parthenopeia Mr9T showed that the total lipid content (lipidome) of the cell membrane reduces the prostatic and brain tumor cell viability with a lower effect on normal cells. The lipidome consisted of sulfobacin A, WB 3559A, WB 3559B, docosenamide, topostin B-567, and unknown compounds. Therefore, the bioactivity could be attributed to any of these individual compounds or due to their synergistic effect. Beyond the rarity and biosynthetic potential of this bacterium, the importance and novelty of this study is the employment of sampling strategies based on ecological factors to reach the hidden microbiota, as well as the use of bacterial membrane constituents as potential novel therapeutics. Our findings open new perspectives on cultivation and the relationship between bacterial biological membrane components and their bioactivity in eukaryotic cells, encouraging similar studies in other members of the rare biosphere.

Mimicking effects of cholesterol in lipid bilayer membranes by self-assembled amphiphilic block copolymers.

The effect of cholesterol on biological membranes is important in biochemistry. In this study, a polymer system is used to simulate the consequences of varying cholesterol content in membranes. The system consists of an AB-diblock copolymer, a hydrophilic homopolymer hA, and a hydrophobic rigid homopolymer C, corresponding to phospholipid, water, and cholesterol, respectively. The effect of the C-polymer content on the membrane is studied within the framework of a self-consistent field model. The results show that the liquid-crystal behavior of B and C has a great influence on the chemical potential of cholesterol in bilayer membranes. The effects of the interaction strength between components, characterized by the Flory-Huggins parameters and the Maier-Saupe parameter, were studied. Some consequences of adding a coil headgroup to the C-rod are presented. Results of our model are compared to experimental findings for cholesterol-containing lipid bilayer membranes.

Design of high voltage gain converter for fuel cell based EV application with hybrid optimization MPPT controller

From the previously available articles, the fuel cell dependent power supply systems are increasing every year. Also, these systems are playing a major focus on the microgrid renewable energy networks. However, the fuel cell gives less output power extraction at different operating temperature values. In addition, the working point of the fuel cell vary with respect to the hydrogen supply, and water membrane content. In order to achieve the high output power of the fuel stack, a Maximum Power Point Tracking (MPPT) is used to track the peak power point of the proposed system. In this article, Artificial Neuro Fuzzy Inference System (ANFIS) controller is utilized in order to generate the smooth output voltage waveform of the fuel cell stack. The optimization of ANFIS controller have been done by using the Genetic Algorithm (GA). The advantages of ANFIS based MPPT controller are high output power extraction, and less distortions of converter voltages. Here, the Proton Exchange Membrane Fuel Cell (PEMFC) device is used as a source. The features of PEMFC are fast start-up, and high efficient. The PEMFC generates less voltage which is enhanced by using the interleaved non-isolated boost converter. The proposed fuel cell based interleaved system is analyzed by using the MATLAB/Simulink software.

Elevated Adipocyte Membrane Phospholipid Saturation Does Not Compromise Insulin Signaling.

Increased saturated fatty acid (SFA) levels in membrane phospholipids have been implicated in the development of metabolic disease. Here, we tested the hypothesis that increased SFA content in cell membranes negatively impacts adipocyte insulin signaling. Preadipocyte cell models with elevated SFA levels in phospholipids were generated by disrupting the ADIPOR2 locus, which resulted in a striking twofold increase in SFA-containing phosphatidylcholines and phosphatidylethanolamines, which persisted in differentiated adipocytes. Similar changes in phospholipid composition were observed in white adipose tissues isolated from the ADIPOR2-knockout mice. The SFA levels in phospholipids could be further increased by treating ADIPOR2-deficient cells with palmitic acid and resulted in reduced membrane fluidity and endoplasmic reticulum stress in mouse and human preadipocytes. Strikingly, increased SFA levels in differentiated adipocyte phospholipids had no effect on adipocyte gene expression or insulin signaling invitro. Similarly, increased adipocyte phospholipid saturation did not impair white adipose tissue function invivo, even in mice fed a high-saturated fat diet at thermoneutrality. We conclude that increasing SFA levels in adipocyte phospholipids is well tolerated and does not affect adipocyte insulin signaling invitro and invivo.

Preparation and Characterization of Poly(Lactic Acid)/Poly (ethylene glycol)-Poly(propyl glycol)-Poly(ethylene glycol) Blended Nanofiber Membranes for Fog Collection.

Fog is a resource with great potential to capture fresh water from the atmosphere, regardless of the geographical and hydrological conditions. Micro-sized fog collection requires materials with hydrophilic/phobic patterns. In this study, we prepared hydrophilic poly(lactic acid) (PLA)/poly(ethylene glycol)-poly(propyl glycol)-poly(ethylene glycol) (PEG-PPG-PEG) blended nanofiber membranes with various PEG-PPG-PEG concentrations by electrospinning. Changes in the morphological and chemical properties, surface wettability, and thermal stability of the PLA/PEG-PPG-PEG composite nanofiber membranes were confirmed using field-emission scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, contact angle testing, and thermogravimetric analysis. As the PEG-PPG-PEG content of the nanofiber membranes increased, their hydrophilicity increased. Water stability, membrane porosity, and water transport rate tests were also conducted to observe the behavior of the hydrophilic PLA nanocomposite membranes in aqueous media. Finally, we applied the PLA-based membranes as fog collectors. As the PEG-PPG-PEG content of the nanofiber membranes increased, their ability to collect fog increased by over 40% compared with that collected by a pure PLA membrane. The prepared membranes not only improve the ability of fog collectors to harvest water but also broaden the use of PLA-based membranes in multiple applications, including tissue engineering, drug delivery, scaffolds, and pharmaceuticals.

The potential of Bacillus subtilis and phosphorus in improving the growth of wheat under chromium stress.

Hexavalent chromium (Cr+6 ) is one of the most toxic heavy metals that have deteriorating effects on the growth and quality of the end product of wheat. Consequently, this research was designed to evaluate the role of Bacillus subtilis and phosphorus fertilizer on wheat facing Cr+6 stress. The soil was incubated with Bacillus subtilis and phosphorus fertilizer before sowing. The statistical analysis of the data showed that the co-application of B. subtilis and phosphorus yielded considerably more significant (p < 0.05) results compared with an individual application of the respective treatments. The co-treatment improved the morphological, physiological and biochemical parameters of plants compared with untreated controls. The increase in shoot length, root length, shoot fresh weight and root fresh weight was 38.17%, 29.31%, 47.89% and 45.85%, respectively, compared with untreated stress-facing plants. The application of B. subtilis and phosphorus enhanced osmolytes content (proline 39.98% and sugar 41.30%), relative water content and stability maintenance of proteins (86.65%) and cell membranes (66.66%). Furthermore, augmented production of antioxidants by 67.71% (superoxide dismutase), 95.39% (ascorbate peroxidase) and 60.88% (catalase), respectively, were observed in the Cr+6 - stressed plants after co-application of B. subtilis and phosphorus. It was observed that the accumulation of Cr+6 was reduced by 54.24%, 59.19% and 90.26% in the shoot, root and wheat grains, respectively. Thus, the combined application of B. subtilis and phosphorus has the potential to reduce the heavy metal toxicity in crops. This study explored the usefulness of Bacillus subtilis and phosphorus application on wheat in heavy metal stress. It is a step toward the combinatorial use of plant growth-promoting rhizobacteria with nutrients to improve the ecosystems' health.

Intraspecific variation in ultrastructure and secretion of the resin canals in Anacardium humile (Anacardiaceae).

The study combines a range of light and electron microscopy methods to access variation in secretion and ultrastructure in the secretory canals in the above- and belowground stems of Anacardium humile, which here serves as a model system. The aboveground stem canals show epithelial cells with ultrastructural characteristics typical of cells active in secretion, while in the belowground stems, the subcellular characteristics are typical of cells with low rates of metabolism. The secretory canals of the belowground stems show uniformity in size and shape, a large central vacuole, a cytoplasm reduced to a thin layer at the cell periphery, and a reduced population of organelles. The aboveground stem canals had voluminous nuclei with evident nucleoli, a very dense cytoplasm with free ribosomes, polyribosomes, mitochondria with developed cristae, and ellipsoid plastids with electron-opaque droplets surrounded by a periplastid reticulum. The vacuoles were of different sizes and often had membranous contents and the dictyosomes were very developed with dilated ends to the cisternae, rough endoplasmic reticulum, and numerous vesicles. The results show that particularities in above- and belowground environment have significant implications for ultrastructural morphology and functioning of secretory canals in the stems of A. humile.

CSL112 Increases Plasma Cholesterol Efflux Capacity and Reduces Erythrocyte Membrane Cholesterol Content in Blood Samples from Patients with Sickle Cell Disease

Background: Lipid homeostasis is abnormal in sickle cell disease (SCD) and contributes to elevated erythrocyte cholesterol content which has been implicated in sickling. The highly oxidative environment in blood from patients with SCD alters high-density lipoprotein (HDL) composition and impairs HDL particle remodeling. Specifically, it negatively impacts on the capacity of HDL particles to mediate apolipoprotein A-I (apoA-I) exchange and protect against inflammation. Low apoA-I levels and altered composition of HDL have been well-documented at steady state, associated with high risk features of SCD, including endothelial dysfunction and risk of pulmonary hypertension. The low levels and activity of apoA-I fall even further during vaso-occlusive events in SCD. This diminished pool of apoA-I activity may mediate reduced capacity of HDL to extract cholesterol from erythrocyte membranes and increase risk of vascular dysfunction. Purpose: In this study we aimed to determine whether plasma derived apoA-I (CSL112; apolipoprotein A-I (human)) could elevate the cholesterol efflux capacity (CEC) of plasma from SCD patients and thereby reduce erythrocyte membrane cholesterol content. Methods: All SCD subjects (n=14) had confirmed HbSS genotype except one who was with HbS/β0-thalassemia, healthy control subjects (n=13) were not race-matched. CSL112 was incubated with fresh blood derived from control subjects and SCD patients for 6 hours at 37°C. The cholesterol content of ghost membranes of isolated erythrocytes was measured. HDL particle size distribution was assessed by native gel electrophoresis followed by western blotting. The ex vivo capacity of apoB-depleted plasma to efflux cholesterol was determined using [3H]cholesterol-loaded RAW264.7 macrophages. Results: CEC of SCD plasma was attenuated (75.9 ± 7.4%, n=14) compared to controls (100%, n=13), with a predominant decrease of ATP binding cassette transporter A1 (ABCA1)-dependent CEC (77.7 ± 9.2% vs 100%, respectively). Consistently, SCD plasma showed a trend towards lower levels of pre-β1 HDL (78.5 ± 6.5%) as compared to controls (100 %). Incubation with CSL112 raised plasma levels of small HDL species and pre-β1 HDL, resulting in a more than 3.5 fold increase of CEC in both patients and controls. Treatment of SCD blood with CSL112 reduced erythrocyte cholesterol content to a greater extent compared to control blood (% reduction: 12.8% ± 3.1% in SCD, 6.3% ± 5.4% in controls). Conclusion: Our data suggest that reduced levels of lipid-poor pre-β1 HDL in SCD plasma reflect impaired HDL particle remodeling and account for a decreased CEC. CSL112 may acutely generate HDL species which optimally support ABCA1-dependent cholesterol efflux and thereby improve HDL function, normalize erythrocyte membrane cholesterol content and correct the imbalance in cellular cholesterol homeostasis in SCD patients. ApoA-I as a potential therapeutic could have implications in the pathobiologic understanding and future clinical management of vaso-occlusive crisis and of chronic vascualopathy such as pulmonary hypertension.

The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study

Number and brightness (N&B) analysis is a fluorescence spectroscopy technique to quantify oligomerization of the mobile fraction of proteins. Accurate results, however, rely on a good knowledge of nonfluorescent states of the fluorescent labels, especially of fluorescent proteins, which are widely used in biology. Fluorescent proteins have been characterized for confocal, but not camera-based, N&B, which allows, in principle, faster measurements over larger areas. Here, we calibrate camera-based N&B implemented on a total internal reflection fluorescence microscope for various fluorescent proteins by determining their propensity to be fluorescent. We then apply camera-based N&B in live CHO-K1 cells to determine the oligomerization state of the epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase that is a crucial regulator of cell proliferation and survival with implications in many cancers. EGFR oligomerization in resting cells and its regulation by the plasma membrane microenvironment are still under debate. Therefore, we investigate the effects of extrinsic factors, including membrane organization, cytoskeletal structure, and ligand stimulation, and intrinsic factors, including mutations in various EGFR domains, on the receptor’s oligomerization. Our results demonstrate that EGFR oligomerization increases with removal of cholesterol or sphingolipids or the disruption of GM3-EGFR interactions, indicating raft association. However, oligomerization is not significantly influenced by the cytoskeleton. Mutations in either I706/V948 residues or E685/E687/E690 residues in the kinase and juxtamembrane domains, respectively, lead to a decrease in oligomerization, indicating their necessity for EGFR dimerization. Finally, EGFR phosphorylation is oligomerization dependent, involving the extracellular domain (550–580 residues). Coupled with biochemical investigations, camera-based N&B indicates that EGFR oligomerization and phosphorylation are the outcomes of several molecular interactions involving the lipid content and structure of the cell membrane and multiple residues in the kinase, juxtamembrane, and extracellular domains.

Synergistic antifungal mechanism of effective components from essential oil against Penicillium roqueforti

Essential oil (EO) has significant antifungal activity. However, there is limited information on the mechanism of the synergistic antifungal effect of the effective components of EO against fungi. In the present study, molecular electrostatic potential and molecular docking were used for the first time to investigate the synergistic antifungal mechanism of eugenol and citral small molecule (CEC) against Penicillium roqueforti. The results showed that the CEC treatment made the activity of β-(1,3)-glucan synthase (GS) and chitin synthase (CS) decreas by 20.2% and 11.1%, respectively, and the contents of which decreased by 85.0% and 27.9%, respectively compared with the control group. Molecular docking revealed that CEC small molecules could bind to GS and CS through different amino acid residues, inhibiting their activity and synthesis. The CEC can combine with tryptophan, tyrosine, and phenylalanine in the cell membrane, causing damage to the cell membrane. The binding sites between small molecules and amino acids were mainly around the OH group. In addition, CEC affected the energy metabolism system and inhibited the glycolysis pathway. Simultaneously, CEC treatment reduced the ergosterol content in the cell membrane by 58.2% compared with the control group. Finally, changes in β-galactosidase, metal ion leakage, and relative conductivity confirmed the destruction of the cell membrane, which resulted in the leakage of cell contents. The above results showed that CEC can kill P. roqueforti by inhibiting energy metabolism and destroying the integrity of the cell membrane.

PEO-b-PBD Diblock Copolymers Induce Packing Defects in Lipid/Hybrid Membranes and Improve Insertion Rates of Natively Folded Peptides.

Hybrid membranes assembled from biological lipids and synthetic polymers are a promising scaffold for the reconstitution and utilization of membrane proteins. Recent observations indicate that inclusion of small fractions of polymer in lipid membranes can improve protein folding and function, but the exact structural and physical changes a given polymer sequence imparts on a membrane often remain unclear. Here, we use all-atom molecular dynamics simulations to study the structure of hybrid membranes assembled from DOPC phospholipids and PEO-b-PBD diblock copolymers. We verified our computational model using new and existing experimental data and obtained a detailed picture of the polymer conformations in the lipid membrane that we can relate to changes in membrane elastic properties. We find that inclusion of low polymer fractions induces transient packing defects into the membrane. These packing defects act as insertion sites for two model peptides, and in this way, small amounts of polymer content in lipid membranes can lead to large increases in peptide insertion rates. Additionally, we report the peptide conformational space in both pure lipid and hybrid membranes. Both membranes support similar alpha helical peptide structures, exemplifying the biocompatibility of hybrid membranes.

Influence of a Hydride-Forming Multi-Component Alloy on the Carbonization Behavior of Vulcanized Elastomer Composites

In this work, composites based on a hydride-forming fiber of a multi-principal-component (MPC) Ti20Zr20Nb20V20Hf20 alloy, carbosil, dicumylperoxide and nitrile butadiene rubber (NBR) matrix were obtained. The composites were prepared in a three-stage process including the mixing of elastomeric compounds via a rubber mixing mill and low-temperature vulcanization by heat pressing. Using dynamical mechanical analysis (DMA) and differential scanning calorimetry (DSC) the effect of the metallic filler on the process of carbonization of the composite elastomeric matrix was studied. The microstructure and gas separation properties of the resulting composites were also studied. The results showed that the filler content increase in the elastomeric matrix leads to a noticeable and gradual decrease in the activation energy of the carbonization process, reduces the temperature of this process, and accelerates the growth of the elastic modulus during carbonization. It was shown that the finely dispersed fiber of the MPC acts as an activation center for the process of thermal-oxidative degradation in the elastomeric matrix accompanying the onset of carbonization. The gas permeability values were found to be relatively low and no visible correlation with the MPC alloy content in the composite membrane was observed.