Changes In Membrane Structure Research Articles

Microcavity-Supported Lipid Bilayers; Evaluation of Drug-Lipid Membrane Interactions by Electrochemical Impedance and Fluorescence Correlation Spectroscopy.

Many drugs have intracellular or membrane-associated targets, thus understanding their interaction with the cell membrane is of value in drug development. Cell-free tools used to predict membrane interactions should replicate the molecular organization of the membrane. Microcavity array-supported lipid bilayer (MSLB) platforms are versatile biophysical models of the cell membrane that combine liposome-like membrane fluidity with stability and addressability. We used an MSLB herein to interrogate drug-membrane interactions across seven drugs from different classes, including nonsteroidal anti-inflammatories: ibuprofen (Ibu) and diclofenac (Dic); antibiotics: rifampicin (Rif), levofloxacin (Levo), and pefloxacin (Pef); and bisphosphonates: alendronate (Ale) and clodronate (Clo). Fluorescence lifetime correlation spectroscopy (FLCS) and electrochemical impedance spectroscopy (EIS) were used to evaluate the impact of drug on 1,2-dioleyl- sn-glycerophosphocholine and binary bilayers over physiologically relevant drug concentrations. Although FLCS data revealed Ibu, Levo, Pef, Ale, and Clo had no impact on lipid lateral mobility, EIS, which is more sensitive to membrane structural change, indicated modest but significant decreases to membrane resistivity consistent with adsorption but weak penetration of drugs at the membrane. Ale and Clo, evaluated at pH 5.25, did not impact the impedance of the membrane except at concentrations exceeding 4 mM. Conversely, Dic and Rif dramatically altered bilayer fluidity, suggesting their translocation through the bilayer, and EIS data showed that resistivity of the membrane decreased substantially with increasing drug concentration. Capacitance changes to the bilayer in most cases were insignificant. Using a Langmuir-Freundlich model to fit the EIS data, we propose Rsat as an empirical value that reflects permeation. Overall, the data indicate that Ibu, Levo, and Pef adsorb at the interface of the lipid membrane but Dic and Rif interact strongly, permeating the membrane core modifying the water/ion permeability of the bilayer structure. These observations are discussed in the context of previously reported data on drug permeability and log P.

The cationic cell-penetrating KT2 peptide promotes cell membrane defects and apoptosis with autophagy inhibition in human HCT 116 colon cancer cells.

The anticancer activity of cationic antimicrobial peptides (AMPs) has become more interesting because some AMPs have selective recognition against cancer cells. However, their antitumor properties and underlying mechanisms in cancer cells have not been clearly understood. In this study, we evaluated the effects of KT2 (lysine/tryptophan-rich AMP) on the cellular uptake and internalization mechanism, cell viability, surface charge of the cell membrane, membrane integrity, apoptotic cell death, and autophagy in human HCT 116 colon cancer cells. We found that KT2 interacted with the cell membrane of HCT 116 cells and was internalized into HCT 116 cells via clathrin-mediated and caveolae-mediated endocytosis mechanisms. The interaction of KT2 with cells caused cell membrane structure change, elevated membrane permeability, and KT2 also affected the lipid component. The results of atomic force microscopy showed cellular membrane defects of KT2-treated cells. The internalized KT2 induced nuclear condensation and apoptotic cell death. It elevated the apoptotic factor levels including those of cytochrome c and apoptosis-inducing factor. Furthermore, KT2 inhibited autophagy by the suppression of autophagy-related 5, autophagy-related 7, autophagy-related 16 like 1, and Beclin-1 proteins. In conclusion, these results revealed the cytotoxicity of cationic KT2 against HCT 116 cells and may help to clarify the interactions between cationic AMPs and cancer cells.

Biophysical characterization of mycobacterial model membranes and their interaction with rifabutin: Towards lipid-guided drug screening in tuberculosis

Lipid structure critically dictates the molecular interactions of drugs with membranes influencing passive diffusion, drug partitioning and accumulation, thereby underpinning a lipid-composition specific interplay. Spurring selective passive drug diffusion and uptake through membranes is an obvious solution to combat growing antibiotic resistance with minimized toxicities. However, the spectrum of complex mycobacterial lipids and lack thereof of suitable membrane platforms limits the understanding of mechanisms underlying drug-membrane interactions in tuberculosis. Herein, we developed membrane scaffolds specific to mycobacterial outer membrane and demonstrate them as improvised research platforms for investigating anti-tubercular drug interactions. Combined spectroscopy and microscopy results reveal an enhanced partitioning of model drug Rifabutin in trehalose dimycolate-containing mycobacterial membrane systems. These effects are apportioned to specific changes in membrane structure, order and fluidity leading to enhanced drug interaction. These findings on the membrane biophysical consequences of drug interactions will offer valuable insights for guiding the design of more effective antibiotic drugs coupled with tuned toxicity profiles.

Excessive salt intake increases peritoneal solute transport rate via local tonicity-responsive enhancer binding protein in subtotal nephrectomized mice.

High peritoneal transport is associated with high mortality and technical failure in peritoneal dialysis (PD). Baseline peritoneal solute transport rate (PSTR) as measured by the peritoneal equilibration test (PET) within 6 months after PD initiation varies between patients. Sodium is reported to be stored in the skin or muscle of dialysis patients. This study investigated whether excessive salt intake in uremic mice caused peritoneal alterations without exposure to PD fluid. Sham-operated (Sham) and subtotal nephrectomized (Nx) mice were randomly given tap water or 1% sodium chloride (NaCl)-containing water for 8 weeks. PET was then performed to evaluate peritoneal function. Human mesothelial cell line Met-5A was used for in vitro studies. We observed higher PSTR in Nx mice with 1% NaCl-containing drinking water (Nx + salt) compared with those with tap water (Nx + water), along with enhanced angiogenesis and inflammation in the peritoneum. Blockade of interleukin (IL)-6 signaling rescued peritoneal transport function in Nx + salt mice. In cultured Met-5A, additional NaCl in the medium upregulated IL-6 as well as vascular endothelial growth factor-A, associated with increased expression and nuclear translocation of tonicity-responsive enhancer binding protein (TonEBP). Knockdown of TonEBP lowered the induction caused by high tonicity. Peritoneal TonEBP expression was higher in Nx + salt mice, while removal of high-salt diet lowered TonEBP level and improved peritoneal transport function. Excessive dietary salt intake caused peritoneal membrane functional and structural changes under uremic status. TonEBP regulated hypertonicity-related inflammatory changes and might play a crucial role in high baseline peritoneal transport.

Revealing electrical stresses acting on the surface of protoplast cells under electric field

Abstract When cells exposed to an electric field, localized changes in the distribution of the electric field will be induced and these changes in turn lead to electrical stresses on cell surface. The electrical stresses play a key role in the cell membrane structural changes which leads to important phenomena like hydrophilic pores formation on the cell membrane resulting in the cell permeability. In this work, protoplast cell interaction with direct current (DC) electric field is investigated. The electrical stresses acted on the cell membrane in the presence of electric field are investigated numerically by a modified finite difference method, fast Immersed Interface Method (IIM). Exact solution of dielectrophoresis (DEP) force applied on a cell under a non-uniform electric field is obtained to verify numerical solution obtained by the fast IIM. The numerical results reveal that both mismatched permittivity between cell and insulating suspension fluid and the applied voltage are essential for generating and tuning the total stresses exerted on cell surface. This paper can help to analytically study the electroporation phenomenon which its exact mechanism is still unclear.

Effect of pressure profile of shock waves on lipid membrane deformation.

Use of shock waves to temporarily increase the permeability of the cell membrane is a promising approach in drug delivery and gene therapy to allow the translocation of macromolecules and small polar molecules into the cytoplasm. Our understanding of how the characteristics of the pressure profile of shock waves, such as peak pressure and pulse duration, influences membrane properties is limited. Here we study the response of lipid bilayer membranes to shock pulses with different pressure profiles using atomistic molecular dynamics simulations. From our simulation results, we find that the transient deformation/disordering of the membrane depends on both the magnitude and the pulse duration of the pressure profile of the shock pulse. For a low pressure impulse, peak pressure has a dominant effect on membrane structural changes, while for the high pressure impulse, we find that there exists an optimal pulse duration at which membrane deformation/disordering is maximized.

Daphnetin: A Novel Anti-Helicobacter pylori Agent.

Background: Antibiotic-resistant H. pylori was increasingly found in infected individuals, which resulted in treatment failure and required alternative therapeutic strategies. Daphnetin, a coumarin-derivative compound, has multiple pharmacological activities. Methods: The mechanism of daphnetin on H. pylori was investigated focusing on its effect on cell morphologies, transcription of genes related to virulence, adhesion, and cytotoxicity to human gastric epithelial (GES-1) cell line. Results: Daphnetin showed good activities against multidrug resistant (MDR) H. pylori clinical isolates, with minimal inhibitory concentration (MIC) values ranging from 25 to 100 μg/mL. In addition, daphnetin exposure resulted in H. pylori morphological changes. Moreover, daphnetin caused increased translocation of phosphatidylserine (PS), DNA damage, and recA expression, and RecA protein production vs. control group. Of great importance, daphnetin significantly decreased H. pylori adhesion to GES-1 cell line vs. control group, which may be related to the reduced expression of colonization related genes (e.g., babA and ureI). Conclusions: These results suggested that daphnetin has good activity against MDR H. pylori. The mechanism(s) of daphnetin against H. pylori were related to change of membrane structure, increase of DNA damage and PS translocation, and decrease of H. pylori attachment to GES-1 cells.

Reversibility of membrane performance and structure changes caused by extreme cold water temperature and elevated conditioning water temperature

The effects of extreme cold water temperature, elevated cleaning water temperature, cleaning time on membrane performance, and pore size distribution were studied using flat-sheet polyvinylidene fluoride (PVDF) membranes. Extreme cold water temperature led to a decrease in membrane permeability, and membrane pore size shrinkage. The use of periodical warm water (35 °C) membrane conditioning could almost completely recover the membrane permeability and pore size structure loss caused by extreme cold water temperature, while the use of room temperature (23±1 °C) conditioning recovered majority of the membrane permeability and structure loss caused by extreme cold water temperature. Changes in membrane performance correlate well with changes in membrane structure. Membranes periodically conditioned in warmer water temperature showed a higher permeability than that periodically conditioned at room temperature from a long-term operation of three months. The results suggest the change in PVDF membrane performance and structure caused by extreme cold water temperature is almost completely reversible after periodical warm water conditioning, and thus the use of warm water conditioning and/or chemical cleaning will benefit the recovery of membrane performance and structure change caused by extreme cold water temperature in cold regions.

Calcium-Induced Lipid Nanocluster Structures: Sculpturing of the Plasma Membrane.

The plasma membrane of the cell is a complex, tightly regulated, heterogeneous environment shaped by proteins, lipids, and small molecules. Ca2+ ions are important cellular messengers, spatially separated from anionic lipids. After cell injury, disease, or apoptotic events, anionic lipids are externalized to the outer leaflet of the plasma membrane and encounter Ca2+, resulting in dramatic changes in the plasma membrane structure and initiation of signaling cascades. Despite the high chemical and biological significance, the structures of lipid-Ca2+ nanoclusters are still not known. Previously, we demonstrated by solid-state nuclear magnetic resonance (NMR) spectroscopy that upon binding to Ca2+, individual phosphatidylserine lipids populate two distinct yet-to-be-characterized structural environments. Here, we concurrently employ extensive all-atom molecular dynamics (MD) simulations with our accelerated membrane mimetic and detailed NMR measurements to identify lipid-Ca2+ nanocluster conformations. We find that major structural characteristics of these nanoclusters, including interlipid pair distances and chemical shifts, agree with observable NMR parameters. Simulations reveal that lipid-ion nanoclusters are shaped by two characteristic, long-lived lipid structures induced by divalent Ca2+. Using ab initio quantum mechanical calculations of chemical shifts on MD-captured lipid-ion complexes, we show that computationally observed conformations are validated by experimental NMR data. Both NMR measurements of diluted specifically labeled lipids and MD simulations reveal that the basic structural unit that reshapes the membrane is a Ca2+-coordinated phosphatidylserine tetramer. Our combined computational and experimental approach presented here can be applied to other complex systems in which charged membrane-active molecular agents leave structural signatures on lipids.

Novel therapeutic compound acridine\u2013retrotuftsin action on biological forms of melanoma and neuroblastoma

PurposeAs a continuation of our search for anticancer agents, we have synthesized a new acridine-retrotuftsin analog HClx9-[Arg(NO2)-Pro-Lys-Thr-OCH3]-1-nitroacridine (named ART) and have evaluated its activity against melanoma and neuroblastoma lines. Both tumors develop from cells (melanocytes, neurons) of neuroectodermal origin, and both are tumors with high heterogeneity and unsatisfactory susceptibility to chemotherapies. Thus, we analyzed the action of ART on pairs of biological forms of melanoma (amelanotic and melanotic) and neuroblastoma (dopaminergic and cholinergic) with regard to proliferation, mechanism of cell death, and effect on the activity of tricarboxylic acid cycle (TAC) enzymes.MethodsThe cytotoxicity of ART was evaluated by XTT and trypan blue tests. Cell death was estimated by plasma membrane structure changes (phosphatidylserine and calreticulin externalization), caspase activation, presence of ROS (reactive oxygen species), activity of tricarboxylic acid cycle enzymes (pyruvate dehydrogenase complex, aconitase, and isocitrate dehydrogenase), NAD level, and ATP level.ResultsART influences the biological forms of melanoma and neuroblastoma in different ways. Amelanotic (Ab) melanoma (with the inhibited melanogenesis, higher malignancy) and SHSY5Y neuroblastoma (with cholinergic DC cells) were especially sensitive to ART action. The Ab melanoma cells died through apoptosis, while, with SH-SY5Y-DC neuroblastoma, the number of cells decreased but not as a result of apoptosis. With Ab melanoma and SH-SY5Y-DC cells, a diminished activity of TAC enzymes was noticed, along with ATP/NAD depletion.ConclusionOur data show that the biological forms of certain tumors responded in different ways to the action of ART. As a combination of retrotuftsin and acridine, the compound can be an inducer of apoptotic cell death of melanoma, especially the amelanotic form. Although the mechanism of the interrelationships between energy metabolism and cell death is not fully understood, interference of ART with TAC enzymes could encourage the further investigation of its anticancer action.

Chemotherapy induced PRL3 expression promotes cancer growth via plasma membrane remodeling and specific alterations of caveolae-associated signaling

BackgroundThe outcome of cancer therapy is greatly defined by the ability of a tumor cell to evade treatment and re-establish its bulk mass after medical interventions. Consequently, there is an urgent need for the characterization of molecules affecting tumor reoccurrence. The phosphatase of regenerating liver 3 (PRL3) protein was recently emerged among the targets that could affect such a phenomenon.MethodsThe expression induction of PRL3 in melanoma cells treated with chemotherapeutic agents was assessed by western blotting. The effect of PRL3 expression on cancer growth was investigated both in vitro and in vivo. The association of PRL3 with the caveolae structures of the plasma membrane was analyzed by detergent free raft purification. The effect of PRL3 expression on the membrane organization was assayed by electron microscopy and by membrane biophysical measurements. Purification of the plasma membrane fraction and co-immunoprecipitation were used to evaluate the altered protein composition of the plasma membrane upon PRL3 expression.ResultsHere, we identified PRL3 as a genotoxic stress-induced oncogene whose expression is significantly increased by the presence of classical antitumor therapeutics. Furthermore, we successfully connected the presence of this oncogene with increased tumor growth, which implies that tumor cells can utilize PRL3 effects as a survival strategy. We further demonstrated the molecular mechanism that is connected with the pro-growth action of PRL3, which is closely associated with its localization to the caveolae-type lipid raft compartment of the plasma membrane. In our study, PRL3 was associated with distinct changes in the plasma membrane structure and in the caveolar proteome, such as the dephosphorylation of integrin β1 at Thr788/Thr789 and the increased partitioning of Rac1 to the plasma membrane. These alterations at the plasma membrane were further associated with the elevation of cyclin D1 in the nucleus.ConclusionsThis study identifies PRL3 as an oncogene upregulated in cancer cells upon exposure to anticancer therapeutics. Furthermore, this work contributes to the existing knowledge on PRL3 function by characterizing its association with the caveolae-like domains of the plasma membrane and their resident proteins.

Metabolomics Study on the Differences of Endogenous Small Molecule between A549/DDP and A549 Cells Based on High Solution UPLC-TOF-MS

背景与目的顺铂获得性耐药是非小细胞肺癌（non-small cell lung cancer, NSCLC）化疗中至关重要并且有待进一步解决的问题。近年来通过细胞培养获得肿瘤耐药细胞，并将其作为代谢组学研究对象，寻找差异代谢物，获得潜在生物标志物，可以有效地为临床研究和治疗提供参考。本研究旨在通过代谢组学分析获取与顺铂耐药性相关的代谢物信息。方法培养NSCLC细胞A549与其顺铂获得性耐药细胞A549/DDP后进行代谢物提取，通过超高效液相色谱-飞行时间质谱法对两种细胞的内源性小分子进行代谢组学分析，获取代谢差异物。结果通过数据分析处理，获得40种差异代谢物，主要涉及磷脂、脂肪酸、氨基酸和能量代谢相关代谢物。结论A549/DDP细胞的耐药性可能由于细胞膜结构的改变以及相关代谢途径的变化而导致。

DNMT and HDAC inhibitors together abrogate endotoxemia mediated macrophage death by STAT3-JMJD3 signaling.

Acute lung injury (ALI) is a common complication of sepsis that often leads to fatal lung disease without effective therapies. It is known that bone marrow derived macrophages are important in resolving the inflammation and maintaining tissue homeostasis. Here, we hypothesize that treatment in combination of DNA methyl transferase inhibitor (DNMTi) 5-Aza 2-deoxycytidine (Aza) and histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) mitigates the inflammation induced pyroptosis and apoptosis during endotoxemia induced ALI. To test this hypothesis, the mice challenged with a sublethal dose of LPS followed by one-hour post-treatment with a single dose of Aza and TSA intraperitoneally showed a substantial attenuation of apoptosis and inflammation. Importantly, we observed significant changes in the mitochondrial membrane structure, and lower levels of DNA fragmentation, reduced expression of apoptotic and pyroptotic genes both transcriptionally and translationally in LPS induced BMDMs treated by a combination of Aza and TSA than in LPS-induced BMDMs treated with either drug alone. The protection was mediated by an inhibition of JNK-ERK and STAT3-JMJD3 activated pathways. Thus, targeting these important signaling pathways with the combination of Aza and TSA would be a good treatment modality for ALI.

Expression and activity of cathepsin L2 and its effect on the degradation of melanosomes in skin lesions of seborrheic keratosis

Objective To determine the expression of cathepsin L2 (CTSL2) and evaluate its activity in skin lesions of seborrheic keratosis (SK) , to observe the ultrastructural changes of melanosomes in the skin lesions of SK, and to estimate the effect of CTSL2 on the degradation of melanosomes. Methods Twenty patients with SK were enrolled from the Department of Dermatology, Renmin Hospital of Wuhan University. The lesional tissue and the perilesional normal skin were biopsied from each patient. Among 15 of the 20 patients, hematoxylin and eosin (HE) staining and Fontana-Masson silver staining were performed to observe the distribution of melanin granules, transmission electron microscopy (TEM) was conducted to observe the ultrastructural changes of melanosomes, and immunohistochemical staining was performed to estimate the cellular proliferative activity. RT-PCR and fluorogenic substrate cleavage assay were performed in the other 5 patients to determine the mRNA expression of CTSL2 and evaluate its activity, respectively. Sucrose density gradient ultracentrifugation was performed to isolate and purify melanosomes from the retinal pigment epithelium (RPE) harvested from a discarded eyeball of a 35-year old male patient with informed consent. The purified melanosomes were incubated with epidermal lysates of SK lesions, and TEM was used to observe the changes in the membrane structure of melanosomes. Statistical analysis was carried out by paired t test, and a P value < 0.05 was considered statistically significant. Results A large number of melanin granules were deposited in SK lesions, while the linear deposition of melanin granules was only seen in the basal layer of the normal skin. TEM showed that the percentage of damaged melanosomes was much higher in the normal skin (49.00% ± 4.00%) than in the SK lesions (24.33% ± 3.06%) (t= 8.49, P < 0.05) . RT-PCR revealed that the mRNA expression and activity of CTSL2 were both significantly lower in the SK lesions than in the normal skin (mRNA: 0.35 ± 0.09 vs. 0.43 ± 0.08, t= 3.17, P < 0.05; activity: 17.46 ± 0.45 vs. 28.78 ± 0.58, t= 34.29, P < 0.05) . Moreover, TEM also showed that the percentage of damaged melanosome was lower in the SK lesion lysate-treated group (32.33% ± 4.93%) than in the normal skin lysate-treated group (43.00% ± 2.65%, t= 3.30, P < 0.05) . Conclusion Decreased expression of CTSL2 in the SK lesions can affect the degradation of melanosomes by keratinocytes. However, whether CTSL2 directly takes part in the pathogenesis of SK or not is still needed to be further confirmed. Key words: Keratosis, seborrheic; Melanosomes; Cathepsin L

Enhanced oral absorption of pemetrexed by ion-pairing complex formation with deoxycholic acid derivative and multiple nanoemulsion formulations: preparation, characterization, and in vivo oral bioavailability and anticancer effect.

ObjectiveThe current study sought to design an oral delivery system of pemetrexed (PMX), a multitargeted antifolate antimetabolite, by enhancing its intestinal membrane permeability.Materials and methodsPMX was ionically complexed with a permeation enhancer such as Nα-deoxycholyl-l-lysyl-methylester (DCK) and prepared as an amorphous solid dispersion by mixing with dispersants such as 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and poloxamer 188 (P188), forming an HP-beta-CD/PMX/DCK/P188; the complex was incorporated into multiple water-in-oil-in-water nanoemulsions in a supersaturated state (HP-beta-CD/PMX/DCK/P188-NE).ResultsAfter complex formation, the partition coefficient and in vitro membrane permeability of PMX were markedly increased, but it showed similar cytotoxic and inhibitory effects on cancer cell proliferation/migration. Furthermore, the intestinal membrane permeability and epithelial cell uptake of PMX were synergistically improved after HP-beta-CD/PMX/DCK/P188 was incorporated into a nanoemulsion with a size of 14.5±0.45 nm. The in vitro permeability of HP-beta-CD/PMX/DCK/P188-NE across a Caco-2 cell monolayer was 9.82-fold greater than that of free PMX, which might be attributable to the partitioning of PMX to the epithelial cells being facilitated via specific interaction of DCK with bile acid transporters, as well as the enhanced lipophilicity accompanied by surfactant-induced changes in the intestinal membrane structure and fluidity. Therefore, the oral bioavailability of HP-beta-CD/PMX/DCK/P188-NE in rats was evaluated as 26.8%±2.98% which was 223% higher than that of oral PMX. Moreover, oral HP-beta-CD/PMX/DCK/P188-NE significantly suppressed tumor growth in Lewis lung carcinoma cell-bearing mice, and the tumor volume was maximally inhibited by 61% compared with that in the control group.ConclusionThese results imply that HP-beta-CD/PMX/DCK/P188-NE is an effective and promising delivery system for enhancing the oral absorption of PMX. Thus, there is the potential for new medical applications, including applications in metronomic cancer treatment.

Improved fermentation efficiency of S. cerevisiae by changing glycolytic metabolic pathways with plasma agitation

Production of ethanol by the yeast Saccharomyces cerevisiae is a process of global importance. In these processes, productivities and yields are pushed to their maximum possible values leading to cellular stress. Transient and lasting enhancements in tolerance and performance have been obtained by genetic engineering, forced evolution, and exposure to moderate levels of chemical and/or physical stimuli, yet the drawbacks of these methods include cost, and multi-step, complex and lengthy treatment protocols. Here, plasma agitation is shown to rapidly induce desirable phenotypic changes in S. cerevisiae after a single treatment, resulting in improved conversion of glucose to ethanol. With a complex environment rich in energetic electrons, highly-reactive chemical species, photons, and gas flow effects, plasma treatment simultaneously mimics exposure to multiple environmental stressors. A single treatment of up to 10 minutes performed using an atmospheric pressure plasma jet was sufficient to induce changes in cell membrane structure, and increased hexokinase 2 activity and secondary metabolite production. These results suggest that plasma treatment is a promising strategy that can contribute to improving metabolic activity in industrial microbial strains, and thus the practicality and economics of industrial fermentations.

Exploring Molecular-Biomembrane Interactions with Surface Plasmon Resonance and Dual Polarization Interferometry Technology: Expanding the Spotlight onto Biomembrane Structure.

The molecular analysis of biomolecular-membrane interactions is central to understanding most cellular systems but has emerged as a complex technical challenge given the complexities of membrane structure and composition across all living cells. We present a review of the application of surface plasmon resonance and dual polarization interferometry-based biosensors to the study of biomembrane-based systems using both planar mono- or bilayers or liposomes. We first describe the optical principals and instrumentation of surface plasmon resonance, including both linear and extraordinary transmission modes and dual polarization interferometry. We then describe the wide range of model membrane systems that have been developed for deposition on the chips surfaces that include planar, polymer cushioned, tethered bilayers, and liposomes. This is followed by a description of the different chemical immobilization or physisorption techniques. The application of this broad range of engineered membrane surfaces to biomolecular-membrane interactions is then overviewed and how the information obtained using these techniques enhance our molecular understanding of membrane-mediated peptide and protein function. We first discuss experiments where SPR alone has been used to characterize membrane binding and describe how these studies yielded novel insight into the molecular events associated with membrane interactions and how they provided a significant impetus to more recent studies that focus on coincident membrane structure changes during binding of peptides and proteins. We then discuss the emerging limitations of not monitoring the effects on membrane structure and how SPR data can be combined with DPI to provide significant new information on how a membrane responds to the binding of peptides and proteins.

\u03b3-Tocotrienol induces apoptosis in pancreatic cancer cells by upregulation of ceramide synthesis and modulation of sphingolipid transport

BackgroundCeramide synthesis and metabolism is a promising target in cancer drug development. γ-tocotrienol (GT3), a member of the vitamin E family, orchestrates multiple effects that ensure the induction of apoptosis in both, wild-type and RAS-mutated pancreatic cancer cells. Here, we investigated whether these effects involve changes in ceramide synthesis and transport.MethodsThe effects of GT3 on the synthesis of ceramide via the de novo pathway, and the hydrolysis of sphingomyelin were analyzed by the expression levels of the enzymes serine palmitoyl transferase, ceramide synthase-6, and dihydroceramide desaturase, and acid sphingomyelinase in wild-type RAS BxPC3, and RAS-mutated MIA PaCa-2 and Panc 1 pancreatic cancer cells. Quantitative changes in ceramides, dihydroceramides, and sphingomyelin at the cell membrane were detected by LCMS. Modulation of ceramide transport by GT3 was studied by immunochemistry of CERT and ARV-1, and the subsequent effects at the cell membrane was analyzed via immunofluorescence of ceramide, caveolin, and DR5.ResultsGT3 favors the upregulation of ceramide by stimulating synthesis at the ER and the plasma membrane. Additionally, the conversion of newly synthesized ceramide to sphingomyelin and glucosylceramide at the Golgi is prevented by the inhibition of CERT. Modulation ARV1 and previously observed inhibition of the HMG-CoA pathway, contribute to changes in membrane structure and signaling functions, allows the clustering of DR5, effectively initiating apoptosis.ConclusionsOur results suggest that GT3 targets ceramide synthesis and transport, and that the upregulation of ceramide and modulation of transporters CERT and ARV1 are important contributors to the apoptotic properties demonstrated by GT3 in pancreatic cancer cells.

Computational study of solution behavior of magainin 2 monomers

Antimicrobial peptides (AMPs) play crucial role as mediators of the primary host defense against microbial invasion. They are considered a promising alternative to antibiotics for multidrug resistant bacterial strains. For complete understanding of the antimicrobial defense mechanism, a detailed knowledge of the dynamics of peptide-membrane interactions, including atomistic studies on AMPs geometry and both peptide and membrane structural changes during the whole process is a prerequisite. We aim at clarifying the conformation dynamics of small linear AMPs in solution as a first step of in silico protocol for establishing a correspondence between certain amino-acid sequence motifs, secondary-structure elements, conformational dynamics in solution and the intensity and mode of interaction with the bacterial membrane. To this end, we use molecular dynamics simulations augmented by well-tempered metadynamics to study the free-energy landscape of two AMPs with close primary structure and different antibacterial activity – the native magainin 2 (MG2) and an analog (MG2m, with substitutions F5Y and F16W) in aqueous solution. We observe that upon solvation, the initial α-helical structures change differently. The native form remains structured, with three shorter α-helical motifs, connected by random coils, while the synthetic analog tends predominantly to a disordered conformation. Our results indicate the importance of the side-chains at positions 5 and 16 for maintaining the solvated peptide conformation. They also provide a modeling background for recent experimental observations, relating the higher α-helical content in solution (peptide pre-folding) in the case of small linear AMPs to a lower antibacterial activity.

Poly(vinylidene Fluoride-Hexafluoropropylene) Porous Membrane with Controllable Structure and Applications in Efficient Oil/Water Separation.

Poly(vinylidene fluoride-hexafluoropropylene) (PVDF-HFP) porous membranes are fabricated via thermally induced phase separation (TIPS) with mixed diluent (dibutyl phthalate (DBP)/dioctyl phthalate (DOP)). The effects of mixed diluent are discussed in detail in term of morphology, mean pore size, selective wettability, etc. The results show that the membrane structure changes from spherulitic to bicontinuous with the change of DBP/DOP ratio. It is also found that the degree of crystallization decreases with the decrease of DBP/DOP ratio in mixed diluent. When liquid–liquid (L-L) phase separation precedes solid–liquid (S-L) phase separation, the obtained membranes have outstanding hydrophobicity and lipophilicity, excellent mechanical property. Additionally, the PVDF-HFP hybrid membranes are prepared with silica (SiO2) particles and the effect of SiO2 content on structure and properties is discussed. It is found that the PVDF-HFP hybrid membrane with 2 wt % SiO2 (M3-S2) has better properties and higher filtration rate and separation efficiency for surfactant-stabilized water-in-oil emulsion separation. Moreover, the membrane M3-S2 also exhibits excellent antifouling performance for long-running.