Evaluation of 1,9-Dimethyl-Methylene Blue nanoencapsulation using rhamnolipid nanoparticles to potentiate the Photodynamic Therapy technique in Candida albicans: In vitro study.

With the rapid development of nanotechnology, various functional nanomaterials have shown exciting potential in biomedical areas such as drug delivery, antitumor, and antibacterial therapy. These nanomaterials improve the stability and selectivity of loaded drugs, reduce drug-induced side effects, realize controlled and targeted drug release, and increase therapeutic efficacy. The increased resistance to antifungal microbicides in medical practice and their side effects stimulate interest in new therapies, such as Photodynamic Therapy (PDT), which do not generate resistance in microorganisms and effectively control the pathology. The present study aimed to evaluate, in vitro, the efficacy of photodynamic therapy on Candida albicans using 1,9-Dimethyl-Methylene Blue (DMMB) as photosensitizer, red LED (λ630), and nanoencapsulation of DMMB (RL-NPs/DMMB) using rhamnolipids produced by Pseudomonas aeruginosa to evaluate if there is better performance of DMMB + RL particles compared to DMMB alone via the characterization of DMMB + RL and colony forming count. The tests were carried out across six experimental groups (Control, DMMB, RL-NPs, RL-NPs/DMMB, PDT and PDT + RL-NPs/DMMB) using in the groups with nanoparticles, DMMB (750 ng/mL) encapsulated with rhamnolipids in a 1:1 ratio, the light source consisted of a prototype built with a set of red LEDs with an energy density of 20 J/cm2. The results showed that applying PDT combined with encapsulation (RL-NPs/DMMB) was a more practical approach to inhibit Candida albicans (2 log reduction) than conventional applications, with a possible clinical application protocol.

In Search of a Molecular View of Peptide-Lipid Interactions in Membranes.

Lipid bilayer membranes are often represented as a continuous nonpolar slab with a certain thickness bounded by two more polar interfaces. Phenomena such as peptide binding to the membrane surface, folding, insertion, translocation, and diffusion are typically interpreted on the basis of this view. In this Perspective, I argue that this membrane representation as a hydrophobic continuum solvent is not adequate to understand peptide-lipid interactions. Lipids are not small compared to membrane-active peptides: their sizes are similar. Therefore, peptide diffusion needs to be understood in terms of free volume, not classical continuum mechanics; peptide solubility or partitioning in membranes cannot be interpreted in terms of hydrophobic mismatch between membrane thickness and peptide length; peptide folding and translocation, often involving cationic peptides, can only be understood if realizing that lipids adapt to the presence of peptides and the membrane may undergo considerable lipid redistribution in the process. In all of those instances, the detailed molecular interactions between the peptide residues and the lipid components are essential to understand the mechanisms involved.

Photodynamic antimicrobial therapy (A-PDT) using 1,9-Dimethyl-Methylene Blue zinc chloride double salt - DMMB and λ640 ± 5ηm LED light in patients undertaking orthodontic treatment.

Orthodontic treatment involves the use of apparatuses that impairs oral hygiene making patients susceptible to periodontal diseases and caries. To prevent increased antimicrobial resistance A-PDT has shown itself a feasible option. The aim of this investigation was to assess the efficiency of A-PDT employing 1,9-Dimethyl-Methylene Blue zinc chloride double salt - DMMB as a photosensitizing agent combined with red LED irradiation (λ640 ± 5 ηm) against oral biofilm of patients undertaking orthodontic treatment. Twenty-one patients agreed to participate. Four biofilm collections were carried out on brackets and gingiva around inferior central incisors; first was carried out before any treatment (Control); second followed five minutes of pre-irradiation, the third was immediately after the first AmPDT, and the last after a second AmPDT. Then, a microbiological routine for microorganism growth was carried out and, after 24-h, CFU counting was performed. There was significant difference between all groups. No significant difference was seen between Control and Photosensitizer and AmpDT1 and AmPDT2 groups. Significant differences were observed between Control and AmPDT1 and AmPDT2 groups, Photosensitizer and AmPDT1 and AmPDT2 groups. It was concluded that double AmPDT using DMBB in nano concentration and red LED was capable to meaningfully decrease the number of CFUs in orthodontic patients.

Production and viscosity of Xanthan Gum are increased by LED irradiation of X. campestris cultivated in medium containing produced water of the oil industry.

Oil recovery is a challenge and microbial enhanced oil recovery is an option. We theorized that the use of produced water (PW) with photo-stimulation could influence both production and viscosity of Xanthan gum. This study aimed at the evaluation of the effect of photo-stimulation by λ630 ± 1 ηm LED light on the biosynthesis of Xanthan gum produced by Xanthomonas campestris IBSBF 2103 strain reusing PW of the oil industry. We assessed the effect of photo-stimulation by LED light (λ630 nm) on the biosynthesis of Xanthan gum produced by X. campestris in medium containing produced water. Different energy densities applied during the microbial growth phase were tested. The highest production was achieved when using 12 J/cm2 LED light (p < 0.01). Three protocols were assessed: Non-irradiated (Control), Irradiation with LED light during the growth phase (LEDgrowth) and Irradiation with LED light during both growth and production phases (LED growth+production). Both the amount and viscosity of the xanthan gum was significantly higher (p < 0.01) in the group LEDgrowth+production. The study showed that LED irradiation (λ630 ± 1 ηm) during both the growth and production phases of the biopolymer increased both the production and viscosity of Xanthan gum.

The Antibiotic Peptide Daptomycin Functions by Reorganizing the Membrane.

The mechanism of the antimicrobial peptide daptomycin is reviewed and discussed. Daptomycin is a last-resort antibiotic in current use against drug-resistant bacterial infections. Many models have been proposed for its function, most based on the observation that it increases membrane permeability and causes leakage of contents, such as ions and small molecules from bacterial cells and lipid vesicles. However, daptomycin is actually not efficient at permeabilizing or translocating across membranes, contrary to many well-known antimicrobial peptides. There is strong evidence that daptomycin binds preferentially to membranes in active division regions of bacterial cells and that it causes large membrane reorganization in terms of the distribution of lipids and proteins, both in cells and in model membranes. Those observations support the alternative hypothesis for the mechanism of daptomycin that its primary effect is in inducing membrane reorganization and that other events, such as increased membrane leakage and depolarization, are secondary consequences, not essential to its function.

Effects of photo-stimulation with laser or LED on the composition of Xanthan gum produced in media containing distilled water or dialyzed or not produced water by means of Raman spectroscopy.

Oil is expected to continue to be one of the most important sources of energy in the world and world's energy matrix for the foreseeable future. However, high demand for energy and the decline of the production of oil fields makes oil recovery a challenge. Most techniques used for the recovery process are expensive, non-sustainable and technically difficult to implement. In this context, microbial enhanced oil recovery (MEOR) represents an attractive alternative. It employs products derived from the metabolism of microorganisms that produce biopolymers. Certain bacteria species (e.g., Xanthomonas campestris) produce polysaccharides (exopolysaccharides - EPS) such as the well-known Xanthan gum (XG). We hypothesized that the use of produced water (PW) water in combination photo-stimulation with laser/LED could influence the production and composition of XG. Raman spectroscopy has been used for qualitative and quantitative evaluation of the biochemical composition of XG biopolymer under light stimulation. X. campestris cultures in either distilled water or dialysis-produced water were studied under the absence or presence of laser irradiation (λ = 660 nm, CW, spot size 0.040 cm2, 40 mW, 444 s, 8.0 J/cm2) or LED (λ = 630 nm ± 2 nm, CW, spot size 0.50 cm2, 140 mW, 500 s, 12 J/cm2). XG produced by these cultures was analyzed by Raman spectroscopy at 1064 nm excitation and subjected to principal component analysis (PCA). Results of the exploratory analysis and ANOVA general linear model (GLM) suggested that the extent of XG and pyruvate (pyruvyl mannose) production was affected differentially in X. campestris when cultured in distilled water plus LED photo-stimulation versus dialysis-produced water plus LED photo-stimulation. XG production increased in the distilled water culture. In contrast, both pyruvate acetyl mannose content went up in the dialysis-water culture. These results open a wide field of opportunities in the use of metal-enriched cultures in combination with photo-biomodulation to direct and optimize bacterial production of compounds (i.e., XG) that may be of great benefit in the implementation of sustainable practices for oil extraction.

Composition of Xanthan gum produced by Xanthomonas campestris using produced water from a carbonated oil field through Raman spectroscopy.

Produced water (PW) is a by-product generated throughout oil exploration. Geological formation and geographical location of the reservoir influence its physical, chemical and biological characteristics. Xanthan gum (XG), an exopolysaccharide (EPS) produced by Xanthomonas campestris, has been widely used in enhanced oil recovery (EOR) technology because of its high viscosity, pseudoplastic behavior, stability in function of salinity, temperature and alkaline conditions. The production of XG may be affected by the composition of the PW, where the acetyl and pyruvyl radicals may be present in the mannoses. The aim of this study was to evaluate the composition of XG produced by X. campestris, particularly the amount of Xanthan, acetyl and pyruvyl groups, in culture mediums containing distilled (DW) or produced (PW) water in different concentrations, by means of dispersive Raman spectroscopy (1064 nm). The spectra of XG showed peaks referred to the main constituents of the Xanthan (glucose, mannose and glucuronic acid). Spectral features assigned to pyruvyl were seen in all samples mainly at ~1010 cm-1, with higher intensity when using DW and 25% PW. PCA loadings showed that the peaks assigned to pyruvyl are consistent to presence of sodium pyruvate (~1040/~1050 and ~ 1432 cm-1) and were higher in the samples obtained in 25% PW. ANOVA GLM applied to Raman peaks of interest (~1010 and ~ 1090 cm-1) and to PCA scores (Score 1 to Score 3) showed that both were influenced by the type of water used in the culture medium, where the XG were strongly reduced in the groups PW compared to DW while the pyruvyl content increased proportionally with the concentration of PW. The results suggest that the composition of the water used in the bacteria's culture medium influenced the composition of XG, including the amount of Xanthan and particularly the pyruvyl content, and therefore needs to be considered when using this approach of injecting XG in oil fields as pyruvyl content affects viscosity.

Enhancement of photodynamic inactivation of planktonic cultures of Staphylococcus aureus by DMMB-AuNPs.

Photodynamic inactivation is a promising method for the treatment of infectious diseases. Nanotechnology through gold nanoparticles, as a tool to improve the delivery of photosensitizer is an attractive approach to enhance photodynamic inactivation of bacteria. Moreover, gold nanoparticles enchance the absorption of light due to their plasmon resonance. The aim of this study was to evaluate in vitro photodynamic inactivation effects of 1.9-Dimethyl-Methylene Blue (DMMB)-AuNPs associated with the red LED (λ630 ηm ± 20 ηm, 125 mW, 12 J / cm², 192 s) on S. aureus strain. Eight experimental groups were studied: Control, LED, AuNPs, AuNPs + LED, DMMB, DMMB + LED, DMMB + AuNPs, DMMB + AuNPs + LED. After incubation, the number of bacteria surviving each treatment was determined and then enumerated by viable counting (CFU / mL). The logarithm of CFU / mL (CFU/mL log10) was calculated. All experiments realized in triplicate. The statistical analyses included one-way ANOVA tests, Tukey's multiple comparisons and nonlinear regression, p values <0.05 were considered statistically significant. According to results, the photodynamic inactivation of S. aureus on groups DMMB + LED and DMMB-AuNPs + LED, showed a significant reduction of the microbial load (p < 0.0001) when compared to the Control group. The decimal reduction (RD) of these groups were 99.96 % (RD = 3) and 99.994 % (RD = 4) respectively. In conclusion, these findings demonstrated that photodynamic inactivation is enhanced by using DMMB-AuNPs on S. aureus.

Oral microbiological control by photodynamic action in orthodontic patients.

BACKGROUND: Orthodontics involves diagnosis and treatment of dental and skeletal malocclusions. Orthodontic apparatus may repair these malocclusions but may also impair oral hygiene making patients prone to develop both periodontal diseases and caries. Antimicrobial agents may be used to prevent this.To avoid increased antimicrobial resistance to available drugs, A-PDT (Antimicrobial Photodynamic Therapy) appears as a viable alternative. OBJECTIVE: This work aimed to evaluate the efficacy of A-PDT on reducing the number of colony forming units (CFU) through the use of phenothiazine compound (methylene blue+ toluidine blue) as a photosensitizer, associated with red LED (λ640±5ηm) irradiation in orthodontic patients. METHODOLOGY: Twenty-one patients consented to participate in the study. Three biofilm collections were performed around the brackets and gums of the inferior central incisors; first before any intervention (Control); second after 5min of pre-irradiation and the last one immediately after AmPDT. Subsequently, a microbiological routine for microorganism growth period were performed and CFU counting after a 24h done. RESULTS: The data showed that the AmPDT was able to reduce CFU count around 90% when compared to Control group (p=0.007) and also between the A-PDT and Photosensitizer groups (p=0.010). However, there were no differences between the Control and Photosensitizer groups. CONCLUSION: A-PDT associated with the use of phenothiazine compounds and red LED was able to significantly reduce the number of CFUs in orthodontic patients.

Glucose and Nitrogen Amendments Can Mitigate Wastewater-Borne Bacteria Competition Effect Against Algal Growth in Wastewater-Based Systems.

The reuse of wastewater is important for reducing costs involved with algal lipid production. However, nutrient limitations, wastewater-borne microbes, and mixotrophic growth can significantly affect biomass yields and lipid/biomass ratios. This research compared the growth performances of both Chlorella vulgaris and Pseudokirchneriella subcapitata on domestic wastewater effluent. The experiments were conducted in the presence and absence of wastewater-borne bacteria, while additionally assessing the impact of distinct nitrate and glucose supplementations. When compared to the sterilized controls, the presence of wastewater-borne bacteria in the effluent reduced C. vulgaris and P. subcapitata total biomass production by 37% and 46%, respectively. In the corresponding treatments supplemented with glucose and nitrate, total biomass production increased by 12% and 61%, respectively. The highest biomass production of 1.11 and 0.72 g · L-1 was, however, observed in the sterilized treatments with both glucose and nitrate supplementations for C. vulgaris and P. subcapitata, respectively. Lipid to biomass ratios were, on average, threefold higher when only nitrate was introduced in the sterilized treatments for both species (0.4 and 0.5, respectively). Therefore, the combination of nitrate and glucose supplementation is shown to be an important strategy for enhancing algal lipid and biomass production when those algae are grown in the presence of wastewater-borne bacteria. On the other hand, in the absence of wastewater-borne bacteria, only nitrate supplementation can significantly improve lipid/biomass ratios.

How to Determine Lipid Interactions in Membranes from Experiment Through the Ising Model.

The determination and the meaning of interactions in lipid bilayers are discussed and interpreted through the Ising model. Originally developed to understand phase transitions in ferromagnetic systems, the Ising model applies equally well to lipid bilayers. In the case of a membrane, the essence of the Ising model is that each lipid is represented by a site on a lattice and that the interaction of each site with its nearest neighbors is represented by an energy parameter ω. To calculate the thermodynamic properties of the system, such as the enthalpy, the Gibbs energy, and the heat capacity, the partition function is derived. The calculation of the configurational entropy factor in the partition function, however, requires approximations or the use of Monte Carlo (MC) simulations. Those approximations are described. Ultimately, MC simulations are used in combination with experiment to determine the interaction parameters ω in lipid bilayers. Several experimental approaches are described, which can be used to obtain interaction parameters. They include nearest-neighbor recognition, differential scanning calorimetry, and Förster resonance energy transfer. Those approaches are most powerful when used in combination of MC simulations of Ising models. Lipid membranes of different compositions are discussed, which have been studied with these approaches. They include mixtures of cholesterol, saturated (ordered) phospholipids, and unsaturated (disordered) phospholipids. The interactions between those lipid species are examined as a function of molecular properties such as the degree of unsaturation and the acyl chain length. The general rule that emerges is that interactions between different lipids are usually unfavorable. The exception is that cholesterol interacts favorably with saturated (ordered) phospholipids. However, the interaction of cholesterol with unsaturated phospholipids becomes extremely unfavorable as the degree of unsaturation increases.

Net Interactions That Push Cholesterol Away from Unsaturated Phospholipids Are Driven by Enthalpy.

The exchangeable unsaturated phospholipids c1-Phos and c3-Phos, which bear one and three permanent kinks, respectively, in their acyl chains, are mimics of the biologically important, low-melting phosphatidylcholines (PCs) having one and three cis double bonds in their sn-2 chains (i.e., 16:0,18:1 PC and 16:0,18:3 PC, respectively). The net interaction of an exchangeable form of cholesterol (Chol) with c1-Phos and with c3-Phos has been investigated using the nearest-neighbor recognition method. These interactions were found to be unfavorable in both cases having a positive free energy, ω, for replacing like by unlike nearest-neighbor contacts. The values for this free energy (or interaction parameter) were 165 cal/mol between Chol and c1-Phos and 395 cal/mol between Chol and c3-Phos. We now report the temperature dependence of these interactions in liquid-disordered bilayers. Their experimentally determined temperature dependencies, in combination with Monte Carlo simulations, revealed that the interaction parameter ω is dominated in both cases by enthalpy. These findings have important implications for the distribution of lipids in natural membranes and for the formation of lipid rafts.

Heat Capacity of DPPC/Cholesterol Mixtures: Comparison of Single Bilayers with Multibilayers and Simulations.

The excess heat capacity (Δ C p) of mixtures of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (Chol) is examined in detail in large unilamellar vesicles (LUVs), both experimentally, using differential scanning calorimetry (DSC), and theoretically, using a three-state Ising model. The model postulates that DPPC can access three conformational states: gel, liquid-disordered (Ld), and liquid-ordered (Lo). The Lo state, however, is only available if coupled with interaction with an adjacent Chol. Δ C p was calculated using Monte Carlo simulations on a lattice and compared to experiment. The DSC results in LUVs are compared with literature data on multilamellar vesicles (MLVs). The enthalpy change of the complete phase transition from gel to Ld is identical in LUVs and MLVs, and the melting temperatures ( Tm) are similar. However, the DSC curves in LUVs are significantly broader, and the maxima of Δ C p are accordingly smaller. The parameters in the Ising model were chosen to match the DSC curves in LUVs and the nearest-neighbor recognition (NNR) data. The model reproduces the NNR data very well. It also reproduces the phase transition in DPPC, the freezing point depression induced by Chol, and the broad component of Δ C p in DPPC/Chol LUVs. However, there is a sharp component, between 5 and 15 mol % Chol, that the model does not reproduce. The broad component of Δ C p becomes dominant as Chol concentration increases, indicating that it involves melting of the Lo phase. Because the simulations reproduce this component, the conclusions regarding the nature of the phase transition at high Chol concentrations and the structure of the Lo phase are important: there is no true phase separation in DPPC/Chol LUVs. There are large domains of gel and Lo phase coexisting below Tm of DPPC, but above Tm the three states of DPPC are mixed with Chol, although clusters persist.

Bacterial glycerol oxidation coupled to sulfate reduction at neutral and acidic pH.

Glycerol is a main co-product of biodiesel production. Crude glycerol may serve as a cheap and attractive substrate in biotechnological applications, e.g. for the production of valuable chemicals or as an electron donor for reduction processes. In this work, sulfate reduction with glycerol was studied at neutral and acidic pH using bioreactor sludge samples and Tinto River sediments as a source of inoculum, respectively. Communities of sulfate-reducing bacteria (SRB) and fermentative bacteria were co-enriched at both pH values. Molecular analyses revealed that sequences belonging to Desulfomicrobium genus were dominant in the cultures enriched at pH 7, while Desulfosporosinus sequences dominated in the culture enriched at pH 4. Glycerol conversion was coupled to sulfate reduction, but the substrate was incompletely oxidized to acetate in the neutrophilic enrichments, and acetate, lactate, and 1,3-propanediol under low pH conditions. Two strains belonging to Desulfomicrobium and Proteiniphilum genera were isolated from the neutrophilic enrichments, but the first isolate was not able to use glycerol, which suggests a syntrophic relationship between glycerol-degrading fermentative bacteria and SRB. A Clostridium strain able to grow with glycerol was isolated from the low pH enrichment. Our data indicate that glycerol promotes the growth of sulfate-reducing communities to form sulfide, which can be used to precipitate and recover heavy metals.

Daptomycin-Phosphatidylglycerol Domains in Lipid Membranes.

Daptomycin is an acidic, 13-amino acid, cyclic polypeptide that contains a number of nonproteinogenic residues and is modified at its N-terminus with a decanoyl chain. It has been in clinical use since 2003 against selected drug-resistant Staphylococcus aureus and Enterococcus spp infections. In vitro, daptomycin is active against Gram-positive pathogens at low concentrations but its antibiotic activity depends critically on the presence of calcium ions. This dependence has been thought to arise from binding of one or two Ca2+ ions to daptomycin as a required step in its interaction with the bacterial membrane. Here, we investigated the interaction of daptomycin with giant unilamellar vesicles (GUVs) composed 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG). We used fluorescence confocal microscopy to monitor binding of the peptide to GUVs and follow its effect on the membrane of the vesicle. We found that in the absence of POPG or Ca2+ daptomycin does not bind measurably to the lipid membrane. In the presence of 20-30% PG in the membrane and 2 mM Ca2+, daptomycin induces the formation of membrane domains rich in acidic lipids. This effect is not induced by Ca2+ alone. In addition, daptomycin causes GUV collapse, but it does not translocate across the membrane to the inside of intact POPC/POPG vesicles. We conclude that pore formation is probably not the mechanism by which the peptide functions. On the other hand, we found that daptomycin coclusters with the anionic phospholipid POPG and the fluorescent probes used, leading to extensive formation of daptomycin-POPG domains in the membrane.

Coarse-grained simulations of hemolytic peptide δ-lysin interacting with a POPC bilayer.

δ-lysin, secreted by a Gram-positive bacterium Staphylococcus aureus, is a 26-residue membrane active peptide that shares many common features with antimicrobial peptides (AMPs). However, it possesses a few unique features that differentiate itself from typical AMPs. In particular, δ-lysin has zero net charge, even though it has many charged residues, and it preferentially lyses eukaryotic cells over bacterial cells. Here, we present the results of coarse-grained molecular dynamics simulations of δ-lysin interacting with a zwitterionic membrane over a wide range of peptide concentrations. When the peptides concentration is low, spontaneous dimerization of peptides is observed on the membrane surface, but deep insertion of peptides or pore formation was not observed. However, the calculated free energy of peptide insertion suggests that a small fraction of peptides is likely to be present inside the membrane at the peptide concentrations typically seen in dye efflux experiments. When the simulations with multiple peptides are carried out with a single pre-inserted transmembrane peptide, spontaneous pore formation occurs with a peptide-to-lipid ratio (P/L) as low as P/L=1:42. Inter-peptide salt bridges among the transmembrane peptides seem to play a role in creating compact pores with very low level of hydration. More importantly, the transmembrane peptides making up the pore are constantly pushed to the opposite side of the membrane when the mass imbalance between the two sides of membrane is significant. Thus, the pore is very dynamic, allowing multiple peptides to translocate across the membrane simultaneously.

Charge Distribution Fine-Tunes the Translocation of α-Helical Amphipathic Peptides across Membranes.

Hundreds of cationic antimicrobial and cell-penetrating peptides (CPPs) form amphipathic α-helices when bound to lipid membranes. Here, we test two hypotheses for the differences in the ability of these peptides to translocate across membranes. The first, which we now call the hydrophobicity hypothesis, is that peptide translocation is determined by the Gibbs energy of insertion into the bilayer from the membrane interface. The second, which we call the charge-distribution hypothesis, is that translocation is determined by whether the distribution of cationic residues in the peptide can transiently stabilize a high-energy inserted intermediate by forming salt bridges to the phosphates of lipid headgroups. To test these hypotheses, we measured translocation of two series of peptide variants. The first series was based on TP10W, a peptide derived from the amphipathic CPP transportan 10; the second was based on DL1a, a synthetic peptide derived from staphylococcal δ-lysin. The peptides in those two series had small sequence changes relative to TP10W and DL1a: either single-residue substitutions or two-residue switches, which were designed to increase or decrease translocation differently according to the two hypotheses. We found that with regard to the changes introduced in the sequences, five out of six peptide variants translocated in agreement with the charge-distribution hypothesis, whereas none showed agreement with the hydrophobicity hypothesis. We conclude that large effects on translocation are probably determined by hydrophobicity, but the fine tuning appears to arise from the distribution of cationic residues along the peptide sequence.

GUVs melt like LUVs: the large heat capacity of MLVs is not due to large size or small curvature.

The excess heat capacity functions (ΔCp) associated with the main phase transition of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs) are very different. Two explanations are possible. First, the difference in vesicle size (curvature) results in different gel-fluid interactions in the membrane; those interactions have a large effect on the cooperativity of the phase transition. Second, there is communication between the bilayers in an MLV when they undergo the gel-fluid transition; this communication results in thermodynamic coupling of the phase transitions of the bilayers in the MLV and, consequently, in an apparent increase in the cooperativity of the transition. To test these hypotheses, differential scanning calorimetry was performed on giant unilamellar vesicles (GUVs) of pure dipalmitoylphosphatidylcholine. The ΔCp curve of GUVs was found to resemble that of the much smaller LUVs. The transition in GUVs and LUVs is much broader (half-width ∼1.5°C) than in MLVs (∼0.1°C). This similarity in GUVs and LUVs indicates that their size has little effect on gel-fluid interactions in the phase transition. The result suggests that coupling between the transitions in the bilayers of an MLV is responsible for their apparent higher cooperativity in melting.

Eliminating the roughness in cholesterol's ß-face: does it matter?

One of the long-standing issues surrounding cholesterol (Chol) relates to its two-faced character. In particular, the consequences of its having a rough ß-face and a smooth α-face on its structural influence in cell membranes has remained elusive. In this study, direct comparisons have been made between cholesterol and a "smoothened" analog, DChol (i.e., 18,19-dinorcholesterol) using model membranes and a combination of nearest-neighbor recognition, differential scanning calorimetry, fluorescence, and monolayer measurements. Taken together, these results indicate that subtle differences exist between the interaction of these two sterols with the different states of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Chol has a greater condensing power than DChol, but only slightly so, i.e., on the order of a few tens of calories per mole.