Peptide Flexibility and the Hydrophobic Moment are Determinants to Evaluate the Clinical Potential of Magainins.

Using a flexibility prediction algorithm and in silico structural modeling, we have calculated the intrinsic flexibility of several magainin derivatives. In the case of magainin-2 (Mag-2) and magainin H2 (MAG-H2) we have found that MAG-2 is more flexible than its hydrophobic analog, Mag-H2. This affects the degree of bending of both peptides, with a kink around two central residues (R10, R11), whereas, in Mag-H2, W10 stiffens the peptide. Moreover, this increases the hydrophobic moment of Mag-H2, which could explain its propensity to form pores in POPC model membranes, which exhibit near-to-zero spontaneous curvatures. Likewise, the protective effect described in DOPC membranes for this peptide regarding its facilitation in pore formation would be related to the propensity of this lipid to form membranes with negative spontaneous curvature. The flexibility of another magainin analog (MSI-78) is even greater than that of Mag-2. This facilitates the peptide to present a kind of hinge around the central F12 as well as a C-terminal end prone to be disordered. Such characteristics are key to understanding the broad-spectrum antimicrobial actions exhibited by this peptide. These data reinforce the hypothesis on the determinant role of spontaneous membrane curvature, intrinsic peptide flexibility, and specific hydrophobic moment in assessing the bioactivity of membrane-active antimicrobial peptides.

Water as a Link between Membrane and Colloidal Theories for Cells.

This review is an attempt to incorporate water as a structural and thermodynamic component of biomembranes. With this purpose, the consideration of the membrane interphase as a bidimensional hydrated polar head group solution, coupled to the hydrocarbon region allows for the reconciliation of two theories on cells in dispute today: one considering the membrane as an essential part in terms of compartmentalization, and another in which lipid membranes are not necessary and cells can be treated as a colloidal system. The criterium followed is to describe the membrane state as an open, non-autonomous and responsive system using the approach of Thermodynamic of Irreversible Processes. The concept of an open/non-autonomous membrane system allows for the visualization of the interrelationship between metabolic events and membrane polymorphic changes. Therefore, the Association Induction Hypothesis (AIH) and lipid properties interplay should consider hydration in terms of free energy modulated by water activity and surface (lateral) pressure. Water in restricted regions at the lipid interphase has thermodynamic properties that explain the role of H-bonding networks in the propagation of events between membrane and cytoplasm that appears to be relevant in the context of crowded systems.

Development of curcumin-loaded chitosan/pluronic membranes for wound healing applications.

The emergence of new materials with improved antibacterial, anti-inflammatory and healing properties compared to conventional wound dressings has both social and economic appeal. In this study, novel chitosan-based (CTS) membranes containing curcumin (CUR) incorporated in Pluronic (PLU) copolymers were developed and characterized to obtain suitable properties for applications as a wound healing dressing. The mechanical, thermal, swelling, wettability, release and permeation properties were evaluated by DSC, TGA, water contact angle measurements, FTIR, fluorescence and microscopic techniques. Membranes containing PLU and CUR presented wettability close to the ideal range for interaction with cellular components (contact angle ~40-70°), improved mechanical properties, higher thermal stability, high swelling degree (>800%) and CUR release (~60%) compared to samples without PLU addition. A higher retention of CUR in the epidermis than in the dermis layer was observed, which also was confirmed by confocal microscopy. Furthermore, the CTS-PLU membranes loaded with CUR showed to be active against Staphylococcus aureus and Pseudomonas aeruginosa (MIC = 25 and 100 mg mL-1, respectively), the microbial species most present in chronic wounds. Overall, the CTS-PLU-CUR membranes presented suitable properties to act as a new wound healing dressing formulation and in vivo studies should be performed to confirm these benefits.

Silk fibroin/chitosan/alginate multilayer membranes as a system for controlled drug release in wound healing.

In this study, we proposed the use of the biopolymers silk fibroin, chitosan and alginate, which are recognized for their biocompatibility and biodegradability, for the preparation of multilayer membranes aiming at high performance wound dressings with controlled drug delivery. The rationale was to combine in one material the mechanical properties of fibroin, the antimicrobial action of chitosan and the ideal exudate absorption of alginate, reaching a synergic effect of each biopolymer, without losing their individual intrinsic properties. The membranes were prepared by casting and diclofenac sodium was incorporated as model drug into the chitosan solution before the solvent evaporation, being retained in the middle layer of the membrane. Morphological, thermal, mechanical, solubility and barrier properties of the membranes were evaluated, as well as cytotoxicity and microbiological permeation. Results show that the incorporation of the drug did not affect mechanical and barrier properties, as well as microbiological permeation. Drug release was evaluated in vitro using simulated solution of wound exudate at 37 °C and diclofenac sodium was released from the multilayer membrane in 7 h, in which Fickian diffusion was the main mechanism associated. The results show the potential application of the biopolymer multilayer membranes as high-performance wound dressings.

Innovative development of membrane sparger for carbon dioxide supply in microalgae cultures.

The present study was aimed to develop a membrane sparger (MS) integrated into a tubular photobioreactor to promote the increase of the carbon dioxide (CO2 ) fixation by Spirulina sp. LEB 18 cultures. The use of MS for the CO2 supply in Spirulina cultures resulted not only in the increase of DIC concentrations but also in the highest accumulated DIC concentration in the liquid medium (127.4 mg L-1 d-1 ). The highest values of biomass concentration (1.98 g L-1 ), biomass productivity (131.8 mg L-1 d-1 ), carbon in biomass (47.9% w w-1 ), CO2 fixation rate (231.6 mg L-1 d-1 ), and CO2 use efficiency (80.5% w w-1 ) by Spirulina were verified with MS, compared to the culture with conventional sparger for CO2 supply. Spirulina biomass in both culture conditions had high protein contents varying from 64.9 to 69% (w w-1 ). MS can be considered an innovative system for the supply of carbon for the microalgae cultivation and biomass production. Moreover, the use of membrane system might contribute to increased process efficiency with a reduced cost of biomass production.

Interactions of amphipathic α-helical MEG proteins from Schistosomamansoni with membranes.

Micro Exon Gene (MEG) proteins are thought to play major roles in the infection and survival of parasitic Schistosoma mansoni worms in host organisms. Here, the physical chemical properties of two small MEG proteins found in the genome of S. mansoni, named MEG-24 and MEG-27, were examined by a combination of biophysical techniques such as differential scanning calorimetry, tensiometry, circular dichroism, fluorescence, and electron spin resonance spectroscopies. The proteins are surface active and structurally arranged as cationic amphipathic α-helices that can associate with lipid membranes and cause their disruption. Upon adsorption to lipid membranes, MEG-27 strongly affects the fluidity of erythrocyte ghost membranes, whereas MEG-24 forms pores in erythrocytes without modifying the ghost membrane fluidity. Whole-mount in situ hybridization experiments indicates that MEG-27 and MEG-24 transcripts are located in the parasite esophagus and subtegumental cells, respectively, suggesting a relevant role of these proteins in the host-parasite interface. Taken together, these characteristics lead us to propose that these MEG proteins may interact with host cell membranes and potentially modulate the immune process using a similar mechanism as that described for α-helical membrane-active peptides.

Hopanoids, like sterols, modulate dynamics, compaction, phase segregation and permeability of membranes.

In recent years, hopanoids, a group of pentacyclic compounds found in bacterial membranes, are in the spotlight since it was proposed that they induce order in lipid membranes in a similar way cholesterol do in eukaryotes, despite their structural differences. We studied here whether diplopterol (an abundant hopanoid) promoted similar effects on model membranes as sterols do. We analyzed the compaction, dynamics, phase segregation, permeability and compressibility of model membranes containing diplopterol, and compared with those containing sterols from animals, plants and fungi. We also tested the effect that the incubation with diplopterol had on hopanoid-lacking bacteria. Our results show that diplopterol induces phase segregation, increases lipid compaction, and decreases permeability on phospholipid membranes, while retaining membrane fluidity and compressibility. Furthermore, the exposition to this hopanoid decreases the permeability of the opportunistic pathogen Pseudomonas aeruginosa and increases the resistance to antibiotics. All effects promoted by diplopterol were similar to those generated by the sterols. Our observations add information on the functional significance of hopanoids as molecules that play an important role in membrane organization and dynamics in model membranes and in a bacterial system.

A new model for lipid monolayer and bilayers based on thermodynamics of irreversible processes.

Lipid monolayers are used as experimental model systems to study the physical chemical properties of biomembranes. With this purpose, surface pressure/area per molecule isotherms provide a way to obtain information on packing and compressibility properties of the lipids. These isotherms have been interpreted considering the monolayer as a two dimensional ideal or van der Waals gas without contact with the water phase. These modelistic approaches do not fit the experimental results. Based on Thermodynamics of Irreversible Processes (TIP), the expansion/compression process is interpreted in terms of coupled phenomena between area changes and water fluxes between a bidimensional solution of hydrated head groups in the monolayer and the bulk solution. The formalism obtained can reproduce satisfactorily the surface pressure/area per lipid isotherms of monolayer in different states and also can explain the area expansion and compression produced in particles enclosed by bilayers during osmotic fluxes. This novel approach gives relevance to the lipid-water interaction in restricted media near the membrane and provides a formalism to understand the thermodynamic and kinetic response of biointerphases to biological effectors.

Anomalous interaction of tri-acyl ester derivatives of uridine nucleoside with a l-α-dimyristoylphosphatidylcholine biomembrane model: a differential scanning calorimetry study.

OBJECTIVES: Uridine was conjugated with fatty acids to improve the drug lipophilicity and the interaction with phospholipid bilayers. METHODS: The esterification reaction using carbodiimides compounds as coupling agents and a nucleophilic catalyst allowed us to synthesize tri-acyl ester derivatives of uridine with fatty acids. Analysis of molecular interactions between these tri-acyl ester derivatives and l-α-dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV) - as a mammalian cell membrane model - have been performed by differential scanning calorimetry (DSC). KEY FINDINGS: The DSC thermograms suggest that nucleoside and uridine triacetate softly interact with phospholipidic multilamellar vesicles which are predominantly located between the polar phase, whereas the tri-acyl ester derivatives with fatty acids (myristic and stearic acids) present a strongly interaction with the DMPC bilayer due to the nucleoside and aliphatic chains parts which are oriented towards the polar and lipophilic phases of the phospholipidic bilayer, respectively. However, the effects caused by the tri-myristoyl uridine and tri-stearoyl uridine are different. CONCLUSIONS: We show how the structural changes of uridine modulate the calorimetric behaviour of DMPC shedding light on their affinity with the phospholipidic biomembrane model.

Development and evaluation of calcium hydroxide-coated, pericardium-based biomembranes for direct pulp capping.

AIM: The aim of the present study was to develop a bovine pericardium biomembrane (BPB) and to evaluate pulp response in vivo. METHODS: A double-layer bovine BPB/chitosan was manufactured, and the porous chitosan side was coated with calcium hydroxide. The microstructure of the matrices was evaluated with electron microscopy. To test pulp response, cavities were prepared on the occlusal surface of Wistar rats' mandibular left first molars and capped with matrices, followed by appropriate adhesives/composite restorations. The animals were divided into three groups: group 1, calcium hydroxide alone; group 2, BPB without calcium hydroxide; and group 3, BPB coated with calcium hydroxide. Specimens were processed and histologically evaluated at 7, 14, and 30 days, postoperatively. RESULTS: Electron microscopy showed porous chitosan surface and a cohesive calcium hydroxide layer. Histological analysis showed that groups 1 and 3 had mild odontoblast layer disorganization, but normal pulp tissue appearance at 7, 14, and 30 days. At the same time points, group 2 showed a loss of general pulp tissue, pulp necrosis, and periapical abscess in some teeth. CONCLUSION: Coated bovine pericardium-based biomembranes resulted in favorable outcomes in cases of pulp exposure after a 30-day observation period, and might protect against injuries caused by adhesive systems and composites.

Theoretical rationalisation of the photophysics of a TICT excited state of cinnamoyl-coumarin derivatives in homogeneous and biological membrane models.

A new hybrid cinnamoyl-coumarin probe was synthesised to study the formation and dynamics of a twisted internal charge transfer (TICT) excited state in homogeneous and biological membrane models. This probe showed a large bathochromic shift of the fluorescence band with the solvent polarity, which is associated with the decrease in the fluorescence intensity due to fast non-radiative deactivation pathways, ascribed to TICT excited state formation in polar solvents. The calculated potential energy surfaces using density functional theory (DFT) and time dependent-DFT (TD-DFT) along with the energetic barriers calculated using the ABF methodology established the energy requirements for a rotational twisting of the cinnamoyl-coumarin bond for TICT excited state formation. This strategy has allowed estimating the role of the ground state conformation and excited state distribution that, concomitant with fluorescence lifetime measurements, describes in detail dual fluorescence emission from TICT and ICT excited states. Moreover, the high sensitivity of fluorescence lifetimes of the TICT excited state in liposomes allows us to propose the use of this type of probes as a powerful tool for the study of gel and crystalline liquid phases in lipid membrane models. The development of this new approach will allow rationalizing and understanding the photochemical behavior of fluorescent TICT-based probes in constrained biological environments.

Regulation of phase boundaries and phase-segregated patterns in model membranes.

Demixing of components has long been described in model membranes. It is a consequence of non-ideal lateral interactions between membrane components, and it causes the presence of segregated phases, forming patches (domains) of different properties, thus introducing heterogeneity into the membrane. In the present review we first describe the processes through which domains are generated, how they grow, and why they are rounded, striped or fractal-like, as well as why they get distributed forming defined patterns. Next, we focus on the effect of an additive on a lipid mixture, which usually induces shifts in demixing points, thus stabilizing or destabilizing the phase-segregated state. Results found for different model membranes are summarized, detailing the ways in which phase segregation and the generated patterns may be modulated. We focus on which are, from our viewpoint, the most relevant regulating factors affecting the surface texture observed in model membranes. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.

Life and Work of Stress Granules and Processing Bodies: New Insights into Their Formation and Function.

The dynamic formation of stress granules (SGs), processing bodies (PBs), and related RNA organelles regulates diverse cellular processes, including the coordination of functionally connected messengers, the translational regulation at the synapse, and the control of viruses and retrotransposons. Recent studies have shown that pyruvate kinase and other enzymes localize in SGs and PBs, where they become protected from stress insults. These observations may have implications for enzyme regulation and metabolic control exerted by RNA-based organelles. The formation of these cellular bodies is governed by liquid-liquid phase separation (LLPS) processes, and it needs to be strictly controlled to prevent pathogenic aggregation. The intracellular concentration of key metabolites, such as ATP and sterol derivatives, may influence protein solubility, thus affecting the dynamics of liquid organelles. LLPS in vitro depends on the thermal diffusion of macromolecules, which is limited inside cells, where the condensation and dissolution of membrane-less organelles are helped by energy-driven processes. The active transport by the retrograde motor dynein helps SG assembly, whereas the anterograde motor kinesin mediates SG dissolution; a tug of war between these two molecular motors allows transient SG formation. There is evidence that the efficiency of dynein-mediated transport increases with the number of motor molecules associated with the cargo. The dynein-dependent transport may be influenced by cargo size as larger cargos can load a larger number of motors. We propose a model based on this emergent property of dynein motors, which would be collectively stronger during SG condensation and weaker during SG breakdown, thus allowing kinesin-mediated dispersion.

Peritrophic membrane origin in adult bees (Hymenoptera): immunolocalization.

The midgut is a region of the digestive tract of bees with the lumen lined by a peritrophic membrane that is composed of chitin and proteins (peritrophins). The origin of the peritrophins in the midgut of adult bees is unknown. This study used an anti-peritrophin 55-kDa antibody to immunolocalize the sites of the peritrophic membrane synthesis in nine species of adult bees' representatives of different families and sociability levels. In all studied species the peritrophin-55 is produced by digestive cells in the entire midgut in the rough endoplasmic reticulum following transference to Golgi apparatus and released by secretory vesicles, which fuses with the plasma membrane and microvilli. Thus, in the representatives of different groups of bees, the PM is of type I.

Development and glycoprotein composition of the perimicrovillar membrane in Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae).

Hemipterans and thysanopterans (Paneoptera: Condylognatha) differ from other insects by having an intestinal perimicrovillar membrane (PMM) which extends from the base of the microvilli to the intestinal lumen. The development and composition of the PMM in hematophagous Reduviidae depend on factors related to diet. The PMM may also allow the human parasite Trypanosoma cruzi, the etiological agent of human Chagas Disease, to establish and develop in this insect vector. We studied the PMM development in the Mexican vector of Chagas Disease, Triatoma (Meccus) pallidipennis. We describe changes in the midgut epithelial cells of insects in response to starvation, and at different times (10, 15 and 20 days) after bloodfeeding. In starved insects, the midguts showed epithelial cells closely connected to each other but apparently free of PMM with some regions being periodic acid-Schiff (PAS-Schiff) positive. In contrast, the PMM was evident and fully developed in the midgut region of insects 15 days after feeding. After this time, the PMM completely covered the microvilli and reached the midgut lumen. At 15 days following feeding the labeled PAS-Schiff increased in the epithelial apex, suggesting an increase in carbohydrates. Lectins as histochemical reagents show the presence of a variety of glycoconjugates including mannose, glucose, galactosamine, N-acetyl-galactosamine. Also present were N-acetyl-glucosamine and sialic acid which contribute to the successful establishment and replication or T. cruzi in its insect vectors. By means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the formation and structure of the PMM is confirmed at 15 days post feeding. Our results confirmed the importance of the feeding processes in the formation of the PMM and showed the nature of the biochemical composition of the vectors' intestine in this important Mexican vector of Chagas disease.

Development of a membrane adsorber based capture step for the purification of yellow fever virus.

Yellow fever (YF) is an endemic disease in some tropical areas of South America and Africa that presents lethality rate between 20 and 50%. There is no specific treatment and to control this disease a highly effective live-attenuated egg based vaccine is widely used for travelers and residents of areas where YF is endemic. However, recent reports of rare, sometimes fatal, adverse events post-vaccination have raised concerns. In order to increase safety records, alternative strategies should be considered, such as developing a new inactivated vaccine using a cell culture based technology, capable of meeting the demands in cases of epidemic. With this goal, the production of YF virus in Vero cells grown on microcarriers and its subsequent purification by chromatographic techniques was studied. In this work we investigate the capture step of the purification process of the YF virus. At first, virus stability was studied over a wide pH range, showing best results for the alkaline region. Considering this result and the pI of the envelope protein previously determined in silico, a strong anion exchanger was considered most suitable. Due to the easy scalability, simplicity to handle, absence of diffusional limitations and suitability for virus handling of membrane adsorbers, a Q membrane was evaluated. The amount of antigen adsorbed onto the membrane was investigated within the pH range for virus stability, and the best pH for virus adsorption was considered to be 8.5. Finally, studies on gradient and step elution allowed to determine the most adequate salt concentration for washing (0.15M) and virus elution (0.30 M). Under these operating conditions, it was shown that this capture step is quite efficient, showing high product recovery (93.2±30.3%) and efficient DNA clearance (0.9±0.3 ng/dose).

Evaluation of the Defay-Prigogine model for the membrane interphase in relation to biological response in membrane-protein interactions.

Surface water activity appears as a common factor when the interaction of several aqueous soluble and surface active proteins with lipid membranes of different compositions is measured by the changes in surface pressure of a lipid monolayer. The perturbation of the lipid surface caused by aqueous soluble proteins depends on the composition of the hydrocarbon phases, either modified by unsaturated bonds in the acyl chains or by inclusion of cholesterol. The cut-off (critical) surface pressure in monolayers, at which no effect of the proteins is found, is related to the composition of the head group region. The perturbation of surface pressure is produced by proteins when the area per lipid is above just 4% larger than that corresponding to the hydration shell of the phospholipid head groups found in the cut-off. This area excess gives place to regions in which the chemical potential of water changes with respect to bulk water. According to the Defay-Prigogine relation this interfacial water activity is the reason of the surface pressure increase induced by aqueous soluble proteins injected in the subphase. As predicted by solution chemistry, the increase of surface pressure is independent of the protein nature but depends on the water surface state determined by the lipid composition.

Effects of propofol and other GABAergic phenols on membrane molecular organization.

GABA(A) receptor is the main inhibitory receptor of the central nervous system. The phenols propofol and thymol have been shown to act on this receptor. GABA(A) is an intrinsic protein, the activity of which may be affected by physical changes in the membrane. Taking into account the lipophilicity of phenols, their interaction with the membrane and a consequent non-specific receptor modulation cannot be discarded. By using Langmuir films, we analyze the comparative effects on the molecular properties of the membrane exerted by propofol, thymol and other related compounds, the activities of which on the GABA(A) are under investigation in our laboratory. All the compounds were able to expand phospholipid films, by their incorporation into the monolayer being favored by less-packed structures. Nonetheless, they were able to be incorporated at lateral pressures above the equilibrium pressure estimated for a natural membrane. Epifluorescence images revealed their presence between phospholipid molecules, probably at the head-group region. Hence, all results indicated that the phenols studied were clearly able to interact with membranes, suggesting that their anesthetic activity could be the combined result of their interaction with specific receptor proteins and with their surrounding lipid molecules modulating the supramolecular organization of the receptor environment.

Defect dynamics in crystalline buckled membranes.

We study the dynamics of defect annihilation in flexible crystalline membranes suffering a symmetry-breaking phase transition. The kinetic process leading the system toward equilibrium is described through a Brazovskii-Helfrich-Canham Hamiltonian. In membranes, a negative disclination has a larger energy than a positive disclination. Here we show that this energetic asymmetry does not only affect equilibrium properties, like the Kosterlitz-Thouless transition temperature, but also plays a fundamental role in the dynamic of defects. Both unbinding of dislocations and Carraro-Nelson "antiferromagnetic" interactions between disclinations slow down the dynamics below the Lifshitz-Safran regime observed in flat hexagonal systems.

The amino- and carboxyl-terminal fragments of the Bacillus thuringensis Cyt1Aa toxin have differential roles in toxin oligomerization and pore formation.

The Cyt toxins produced by the bacteria Bacillus thuringiensis show insecticidal activity against some insects, mainly dipteran larvae, being able to kill mosquitoes and black flies. However, they also possess a general cytolytic activity in vitro, showing hemolytic activity in red blood cells. These proteins are composed of two outer layers of α-helix hairpins wrapped around a ß-sheet. With regard to their mode of action, one model proposed that the two outer layers of α-helix hairpins swing away from the ß-sheet, allowing insertion of ß-strands into the membrane forming a pore after toxin oligomerization. The other model suggested a detergent-like mechanism of action of the toxin on the surface of the lipid bilayer. In this work, we cloned the N- and C-terminal domains form Cyt1Aa and analyzed their effects on Cyt1Aa toxin action. The N-terminal domain shows a dominant negative phenotype inhibiting the in vitro hemolytic activity of Cyt1Aa in red blood cells and the in vivo insecticidal activity of Cyt1Aa against Aedes aegypti larvae. In addition, the N-terminal region is able to induce aggregation of the Cyt1Aa toxin in solution. Finally, the C-terminal domain composed mainly of ß-strands is able to bind to the SUV liposomes, suggesting that this region of the toxin is involved in membrane interaction. Overall, our data indicate that the two isolated domains of Cyt1Aa have different roles in toxin action. The N-terminal region is involved in toxin aggregation, while the C-terminal domain is involved in the interaction of the toxin with the lipid membrane.