Cells immersed in collagen matrices show a decrease in plasma membrane fluidity as the matrix stiffness increases.

Cells are constantly adapting to maintain their identity in response to the surrounding media's temporal and spatial heterogeneity. The plasma membrane, which participates in the transduction of external signals, plays a crucial role in this adaptation. Studies suggest that nano and micrometer areas with different fluidities at the plasma membrane change their distribution in response to external mechanical signals. However, investigations linking fluidity domains with mechanical stimuli, specifically matrix stiffness, are still in progress. This report tests the hypothesis that the stiffness of the extracellular matrix can modify the equilibrium of areas with different order in the plasma membrane, resulting in changes in overall membrane fluidity distribution. We studied the effect of matrix stiffness on the distribution of membrane lipid domains in NIH-3 T3 cells immersed in matrices of varying concentrations of collagen type I, for 24 or 72 h. The stiffness and viscoelastic properties of the collagen matrices were characterized by rheometry, fiber sizes were measured by Scanning Electron Microscopy (SEM) and the volume occupied by the fibers by second harmonic generation imaging (SHG). Membrane fluidity was measured using the fluorescent dye LAURDAN and spectral phasor analysis. The results demonstrate that an increase in collagen stiffness alters the distribution of membrane fluidity, leading to an increasing amount of the LAURDAN fraction with a high degree of packing. These findings suggest that changes in the equilibrium of fluidity domains could represent a versatile and refined component of the signal transduction mechanism for cells to respond to the highly heterogeneous matrix structural composition. Overall, this study sheds light on the importance of the plasma membrane's role in adapting to the extracellular matrix's mechanical cues.

Use of blend based on an emulsifier, monolaurin, and glycerides of butyric acid in the diet of broilers: impacts on intestinal health, performance, and meat.

The objective of this study was to evaluate whether the addition of a blend based on α-monolaurin mono-, di- and triglycerides of butyric acid, and lysolecithin on the performance even on diets containing reduced inclusion of oil in the diet and without the use of growth-promoting antibiotics of broilers considering the effect on health, performance, and meat. Three treatments were defined: positive control (TP: with enramycin), negative control (TN: no enramycin), and blend (T-FRA: with monolaurin and glycerides of acid butyric minus 0.8% soybean oil). At 21 days, broilers treated with TP and T-FRA obtained the lower feed conversion ratio (FC); at 35 days, T-FRA broilers obtained lower FC than TN broilers. Cholesterol levels were higher in the blood of T-FRA broilers. On day 42, levels of ROS and TBARS were lower in the intestine, muscles, and liver of T-FRA broilers. Moreover, glutathione S-transferase and total non-enzymatic antioxidants were greater at the intestinal and muscular levels. The T-FRA broilers had a lower percentage of lipids in the meat. The MIC indicated that 111mg of the blend/mL inhibited the growth of E. coli; however, the counts of total coliforms and E. coli in the feces and the broilers' litter did not differ between treatments. In conclusion, the addition of the blend T-FRA in broiler diets was able to improve the feed conversion and maintain the other performance parameters even considering a reduction of 0.8% in the inclusion of oil.

Impact of macromolecular crowding on the mesomorphic behavior of lipid self-assemblies.

Using LAURDAN fluorescence we observed that water dynamics measured at the interface of DOPC bilayers can be differentially regulated by the presence of crowded suspensions of different proteins (HSA, IgG, Gelatin) and PEG, under conditions where the polymers are not in direct molecular contact with the lipid interface. Specifically, we found that the decrease in water dipolar relaxation at the membrane interface correlates with an increased fraction of randomly oriented (or random coil) configurations in the polymers, as Gelatin > PEG > IgG > HSA. By using the same experimental strategy, we also demonstrated that structural transitions from globular to extended conformations in proteins can induce transitions between lamellar and non-lamellar phases in mixtures of DOPC and monoolein. Independent experiments using Raman spectroscopy showed that aqueous suspensions of polymers exhibiting high proportions of randomly oriented conformations display increased fractions of tetracoordinated water, a configuration that is dominant in ice. This indicates a greater capacity of this type of structure for polarizing water and consequently reducing its chemical activity. This effect is in line with one of the tenets of the Association Induction Hypothesis, which predicts a long-range dynamic structuring of water molecules via their interactions with proteins (or other polymers) showing extended conformations. Overall, our results suggest a crucial role of water in promoting couplings between structural changes in macromolecules and supramolecular arrangements of lipids. This mechanism may be of relevance to cell structure/function when the crowded nature of the intracellular milieu is considered.

LAURDAN since Weber: The Quest for Visualizing Membrane Heterogeneity.

Any chemist studying the interaction of molecules with lipid assemblies will eventually be confronted by the topic of membrane bilayer heterogeneity and may ultimately encounter the heterogeneity of natural membranes. In artificial bilayers, heterogeneity is defined by phase segregation that can be in the nano- and micrometer range. In biological bilayers, heterogeneity is considered in the context of small (10-200 nm) sterol and sphingolipid-enriched heterogeneous and highly dynamic domains. Several techniques can be used to assess membrane heterogeneity in living systems. Our approach is to use a fluorescent reporter molecule immersed in the bilayer, which, by changes in its spectroscopic properties, senses physical-chemistry aspects of the membrane. This dye in combination with microscopy and fluctuation techniques can give information about membrane heterogeneity at different temporal and spatial levels: going from average fluidity to number and diffusion coefficient of nanodomains. LAURDAN (6-dodecanoyl-2-(dimethylamino) naphthalene), is a fluorescent probe designed and synthesized in 1979 by Gregorio Weber with the purpose to study the phenomenon of dipolar relaxation. The spectral displacement observed when LAURDAN is either in fluid or gel phase permitted the use of the technique in the field of membrane dynamics. The quantitation of the spectral displacement was first addressed by the generalized polarization (GP) function in the cuvette, a ratio of the difference in intensity at two wavelengths divided by their sum. In 1997, GP measurements were done for the first time in the microscope, adding to the technique the spatial resolution and allowing the visualization of lipid segregation both in liposomes and cells. A new prospective to the membrane heterogeneity was obtained when LAURDAN fluorescent lifetime measurements were done in the microscope. Two channel lifetime imaging provides information on membrane polarity and dipole relaxation (the two parameters responsible for the spectral shift of LAURDAN), and the application of phasor analysis allows pixel by pixel understanding of these two parameters in the membrane. To increase temporal resolution, LAURDAN GP was combined with fluctuation correlation spectroscopy (FCS) and the motility of nanometric highly packed structures in biological membranes was registered. Lately the application of phasor analysis to spectral images from membranes labeled with LAURDAN allows us to study the full spectra pixel by pixel in an image. All these methodologies, using LAURDAN, offer the possibility to address different properties of membranes depending on the question being asked. In this Account, we will focus on the principles, advantages, and limitations of different approaches to orient the reader to select the most appropriate technique for their research.

Correlated flickering of erythrocytes membrane observed with dual time resolved membrane fluctuation spectroscopy under different D-glucose concentrations.

A correlated human red blood cell membrane fluctuation dependent on D-glucose concentration was found with dual time resolved membrane fluctuation spectroscopy (D-TRMFS). This new technique is a modified version of the dual optical tweezers method that has been adapted to measure the mechanical properties of red blood cells (RBCs) at distant membrane points simultaneously, enabling correlation analysis. Mechanical parameters under different D-glucose concentrations were obtained from direct membrane flickering measurements, complemented with membrane fluidity measurements using Laurdan Generalized Polarization (GP) Microscopy. Our results show an increase in the fluctuation amplitude of the lipid bilayer, and a decline in tension value, bending modulus and fluidity as D-glucose concentration increases. Metabolic mechanisms are proposed as explanations for the results.

Chemical composition, lipid peroxidation, and fatty acid profile in meat of broilers fed with glycerol monolaurate additive.

The objective of this study was to evaluate the chemical composition and fatty acid profile of broilers fed diets containing glycerol monolaurate (GML) in place of antimicrobials. Groups: T0 group used as control; T100, T200, and T300 groups received diets supplemented with 100, 200, and 300 mg/kg of GML, respectively. The feed mixture used in the poultry feed during the four phases of the production cycle (days 1 to 7; 8 to 21; 22 to 35; and 36 to 42 of birds age) showed similar levels of protein, lipid and ash, as well as fatty acid profiles. Samples of frozen breasts from chickens slaughtered at 42 days of age were used for chemical gross composition and fatty acid analysis. We observed lower lipid levels in the meat of broilers in the T200 and T300 groups than in the T0 group. Lower lipid peroxidation occurred in the meat of animals that consumed GML in respect to control. Total saturated fatty acid percentage was lower, while total polyunsaturated fatty acid percentage was higher in the meat of broilers fed GML than in the control group. We conclude that the increase in GML concentrations alters the lipid profile of broiler meat.

Effects of glycerol monolaurate on growth and physiology of chicks consuming diet containing fumonisin.

Glycerol monolaurate (GML) is composed of lauric acid and glycerol. Research has shown that such organic acids can minimize negative effects caused by mycotoxins. Therefore, the objective of this study was to determine whether adding GML (free or encapsulated) to chick feed minimizes the effects of natural contamination by fumonisin (Fusarium verticillioides), evaluating parameters such as biochemistry, antioxidant properties, histological analysis and chick growth. Were weighed 84 chicks of the Cobb 500 strain and randomly distributed them into six groups of two replicates each (n = 14). The F group consumed feed containing fumonisin (levels 400 ppb), with no performance enhancer; F + ZB- feed with fumonisin (levels 400 ppb) + zinc bacitracin; F + GLM100 - feed with fumonisin (levels 400 ppb) + 100 mg of GML/kg of feed; F + NGLM4 - feed with fumonisin (levels 400 ppb) + 4 mg GML/kg in nanocapsules added to the feed; F + NGLM8 - fumonisin feed (levels 400 ppb) + 8 mg GML/kg in nanocapsules in the feed; and F0 - fumonisin-free feed (negative control) + zinc bacitracin. The body weights of birds fed with feed fumonisin-contaminated feed (F, F + ZB, F + GLM100, F + NGLM4 and F + NGLM8) were significantly lower (P < 0.05) than those of the negative control (F0), despite the use of GML (free and nanoencapsulated). Serum levels of triglycerides, globulins and cholesterol were significantly lower in the F0 group than in the other groups (P < 0.05), except for the F + NGLM8 group. Significantly greater levels of lipid peroxidation were observed in livers in the groups that consumed fumonisin than in the control group (F0) (P < 0.05). Serum levels of reactive oxygen species were significantly lower in groups F + NGLM8 and F0 than in the other treatments (P < 0.05). Superoxide dismutase activity was significantly greater in groups F + NGLM8 and F0 than in groups F, F + ZB and F + NGLM4. Hepatic catalase activity was significantly lower in birds that consumed contaminated feed (F, F + ZB, F + GLM100, F + NGLM4 and F + NGLM8) than in the control group (F0). Greater hepatic glutathione S-transferase activity was observed in the F + NGLM8 group than in the F0 group. Despite changes in cellular lesions in the liver, no histological changes were observed in the liver or intestines, even though visually there was yellowing of the liver. Taken together, the data suggest that free or nano-encapsulated GML did not minimize oxidative stress caused by fumonisin, and consequently, these birds had less weight gain.

Mono laurina na prevenção e tratamento de pacientes com COVID-19 / Mono laurine in the prevention and treatment of patients with Covid-19

OBJETIVO: Esta nota técnica tem por objetivo apresentar informações sobre o uso da monolaurina na prevenção e no tratamento de pacientes com COVID-19. DOS FATOS: Trata-se de despacho proveniente do Centro de Operações de Emergências em Saúde Pública (COE) e encaminhado ao Departamento de Gestão e Incorporação de Tecnologias e Inovação em Saúde (DGITIS/SCTIE/MS). O despacho em questão apresenta um e-mail encaminhado pelo profissional Dr. Ronaldo Amaral de Paiva (vinculado à Universidade Federal de Viçosa), intitulado "Ações técnicas efetivas para o combate à Covid-19", que traz informações acerca do uso da monolaurina no tratamento da COVID-19 para análise no âmbito de suas competências e medidas julgadas pertinentes. BUSCA NA LITERATURA E SELEÇÃO DOS ESTUDOS: Com base na pergunta PICO estruturada, foram realizadas buscas nas bases de dados Medline (via PubMed) e Embase com acesso em 05 de maio de 2020. As estratégias de busca estão descritas conforme o Quadro 2 abaixo e mostram que não foram identificados estudos científicos sobre a questão de pesquisa. As plataformas de registros de ensaios clínicos ClinicalTrial.gov e International Clinical Trials Registry Platform (ICTRP), da Organização Mundial de Saúde (OMS), também foram consultadas. Foram utilizados os termos de busca: SARS-COV-2, COVID-19, 2019 novel coronavirus, 2019-nCoV, severe acute respiratory syndrome coronavirus 2, Wuhan coronavirus, COVID, monolaurin e glycerol monolaurate. Da mesma forma, nenhum estudo foi identificado em ambas as plataformas de registro. CONCLUSÕES: Embora haja evidências substanciais de ação antiviral de monoglicerídeos em vírus de RNA envelopados, não foram identificadas evidências científicas que corroborem o uso da monolaurina na prevenção ou tratamento de pacientes da COVID-19. Portanto, não foi possível aferir informações sobre um potencial efeito da substância sobre as membranas protetoras do SARS-CoV-2. Sendo assim, conclui-se que não há eficácia comprovada do uso na prevenção e tratamento da COVID-19. É prudente alertar sobre o crescente número de informações on-line, pretensamente fundadas em evidências científicas, que disseminam suposições de que produtos naturais, como o óleo de coco (23,24), teriam um efeito "protetor" contra a infecção pelo SARS-CoV-2. Não há qualquer regulamentação por nenhuma agência de vigilância sanitária nacional ou internacional para esses produtos que inclua essa finalidade de uso. Haja vista a precariedade de evidências acerca do tema, o presente documento será atualizado à medida que elas forem identificadas.

Diversity of Lipase-Producing Microorganisms from Tropical Oilseeds Elaeis guineensis, Ricinus communis, and Jatropha curcas L. from Costa Rica.

Tropical oleaginous seeds are an unexplored source for the discovery of novel lipolytic microorganisms, which could be applied to the bioremediation of agro-industrial oily wastes and solve numerous environmental issues. Such wastes hold potential to be revalorized towards a variety of products through microbial bioremediation. In this study, we investigate the microbial diversity and lipase activity from bacterial and fungal isolates obtained from the oil seeds of Elaeis guineensis, Ricinus communis, and Jatropha curcas L. from Costa Rica. A total of 27 strains were confirmed as lipase-producing strains via fluorogenic and colorimetric agar plate assays. The diversity of the isolates comprises 12 fungal ascomycetes from the genera Aspergillus and Fusarium and 15 bacterial isolates classified into four genera: Serratia, Proteus, Pseudomonas, and Bacillus. Microbial isolates from E. guineensis showed the highest diversity of lipolytic microorganisms (6 genera) followed by J. curcas (4 genera) and R. communis (2 genera). Isolates showing the highest activity in agar plates were tested further by submerged fermentation and the specific lipase activity was measured with 4-nitrophenyl laurate as substrate. Accordingly, the highest specific lipase activity was demonstrated by Bacillus pumilus B5 (24.98 U mg-1), Serratia marcescens B10 (17.65 U mg-1), Pseudomonas mendocina B16 (8.62 U mg-1), and Bacillus pumilus B1 (5.72 U mg-1) in submerged fermentation. These findings indicate the presence of a specialized microbial diversity in tropical oil seeds and highlight their potential to be applied in the bioremediation of agro-industrial oily wastes.

Glycerol monolaurate nanocapsules for biomedical applications: in vitro toxicological studies.

The glycerol monolaurate (GML) is a surfactant used in the food industry and has potent antimicrobial activity against many microorganisms; however, the use of GML is not expanded due its high melting point and poor solubility in water. The aim of the study was to produce, characterize, and evaluate in vitro the cytotoxicity of GML and GML nanocapsules. The GML nanocapsules were produced and characterized by a mean diameter, zeta potential, and polydispersity index. The cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) release, thiobarbituric acid reactive substances (TBARS), and hemolytic activity. The genotoxicity was verified by comet assay. The physicochemical parameters showed a mean diameter of 192.5 ± 2.8 nm, a polydispersity index of 0.061 ± 0.018, and a zeta potential about - 21.9 ± 1 mV. The viability test demonstrated the protector effect of GML nanocapsule compared with the GML on peripheral blood mononuclear cells (PBMC) and VERO cells (isolated from kidney epithelial cells extracted from an African green monkey). A reduction in lipid peroxidation and lactate dehydrogenase release in GML nanocapsule-exposed cells compared with GML treated cells was observed. The damage on erythrocytes was addressed in treatment with GML, while the treatment with GML nanocapsules did not cause an effect. Moreover, the comet assay showed that the GML-caused genotoxicity and GML nanocapsules do not demonstrate damage. The study showed the reduction of toxicity of GML nanocapsules by many methods used in antimicrobial therapy.

Effects of artepillin C on model membranes displaying liquid immiscibility.

It has been hypothesized that the therapeutic effects of artepillin C, a natural compound derived from Brazilian green propolis, are likely related to its partition in the lipid bilayer component of biological membranes. To test this hypothesis, we investigated the effects of the major compound of green propolis, artepillin C, on model membranes (small and giant unilamelar vesicles) composed of ternary lipid mixtures containing cholesterol, which display liquid-ordered (lo) and liquid-disordered (ld) phase coexistence. Specifically, we explored potential changes in relevant membrane parameters upon addition of artepillin C presenting both neutral and deprotonated states by means of small angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and confocal and multiphoton excitation fluorescence microscopy. Thermotropic analysis obtained from DSC experiments indicated a loss in the lipid cooperativity of lo phase at equilibrium conditions, while at similar conditions spontaneous formation of unilamellar vesicles from SAXS experiments showed that deprotonated artepillin C preferentially located at the surface of the membrane. Time-resolved experiments using fluorescence microscopy showed that at doses above 100 µM, artepillin C in its neutral state interacted with both liquid-ordered and liquid-disordered phases, inducing curvature stress and promoting dehydration at the membrane interface.

Effect of cholesterol on the surface polarity and hydration of lipid interphases as measured by Laurdan fluorescence: New insights.

Comparison of the behavior of Laurdan in gel and in the liquid crystalline DPPC bilayers with that observed in chloroform and OctOH allow concluding that changes in the membrane lipid order cannot be ascribed to changes in viscosity of the local environment. Cholesterol acts as a spacer below the transition temperature of DPPC, promoting a disorder state in the acyl chain region. No evidence of water entrance has been detected with Laurdan up to 30% Cholesterol in DPPC in this condition. In contrast, Chol displaces to longer values the wavelength of Laurdan in membranes in the liquid crystalline state. This decrease in polarity occurs above 5% Chol and is directly related to the water extrusion produced by Chol. This effect is similar to that occurring in liquid crystalline membranes subjected to hypertonic stress. The behavior is comparable to that of Laurdan in OctOH at different water ratios below 5% Chol/DPPC. At higher ratios, other changes are evident.

Glycerol monolaurate in the diet of broiler chickens replacing conventional antimicrobials: Impact on health, performance and meat quality.

Glycerol monolaurate (GML), known as lauric acid, is a chemical compound formed from lauric acid and glycerol that presents strong antimicrobial activity. Therefore, our hypothesis is that MGL can replace conventional antimicrobials, being a new alternative to poultry farming. The aim of this study was to evaluate whether the addition of GML as a replacement for antibiotics could have positive effects on health and performance of broiler chickens. For this, 240, one-day-old, Cobb 500 broiler chicks were weighed and randomly distributed into four groups with four repetitions each (n = 15). The control group, T0, received a basal diet containing antibiotic (60 ppm of bacitracin), while the T100, T200, and T300 groups received a basal diet supplemented with 100, 200, and 300 mg/kg of GML, respectively. The birds were weighed at intervals of seven days, as well as at the end of the experiment (day 42). Blood samples were collected for evaluating animal health, stool for counting bacteria and coccidian, as well as muscle (chest) to measure meat quality, respectively. At the end of the experiment (day 42), body weight, weight gain, and daily weight gain of broiler chickens in the T300 group were higher than the T0 group (P < 0.05). Indeed, feed conversion was lower compared to T0. Animals that received diets containing GML showed lower amounts of Eimeria spp. oocysts on day 42 in comparison to the control group. Low total bacterial counts on day 21 of the experiment were also observed in the treated groups. Conversely, plasma levels of total protein, globulins, uric acid, and glucose were higher in animals that received GML when compared to the control group. It was also observed higher carcass yields in the breast muscle of the T100 group when compared to other groups. Lower water holding capacity was observed in breast meat of animals of the groups T100, T200, and T300 when compared to T0. Histopathological findings were compatible with coccidiosis, and the degree of these lesions did not differ among groups. Based on these results, GML in the diets of broiler chickens, showing potent antimicrobial effect, growth promoter capacity, and lack of toxicity. Therefore, GML is a promising alternative to replace conventional antimicrobials used in the diets of broiler chickens.

Effects of artepillin C on model membranes displaying liquid immiscibility

It has been hypothesized that the therapeutic effects of artepillin C, a natural compound derived from Brazilian green propolis, are likely related to its partition in the lipid bilayer component of biological membranes. To test this hypothesis, we investigated the effects of the major compound of green propolis, artepillin C, on model membranes (small and giant unilamelar vesicles) composed of ternary lipid mixtures containing cholesterol, which display liquid-ordered (lo) and liquid-disordered (ld) phase coexistence. Specifically, we explored potential changes in relevant membrane parameters upon addition of artepillin C presenting both neutral and deprotonated states by means of small angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and confocal and multiphoton excitation fluorescence microscopy. Thermotropic analysis obtained from DSC experiments indicated a loss in the lipid cooperativity of lo phase at equilibrium conditions, while at similar conditions spontaneous formation of unilamellar vesicles from SAXS experiments showed that deprotonated artepillin C preferentially located at the surface of the membrane. Time-resolved experiments using fluorescence microscopy showed that at doses above 100 µM, artepillin C in its neutral state interacted with both liquid-ordered and liquid-disordered phases, inducing curvature stress and promoting dehydration at the membrane interface.

Binding properties of sterol carrier protein 2 (SCP2) characterized using Laurdan.

Sterol carrier protein 2 (SCP2) binds lipids with high affinity and broad specificity. The overall hydrophobicity, fluidity, and dipolar dynamics of the binding site of SCP2 from Yarrowia lipolytica were characterized using the environmentally-sensitive fluorescent probe Laurdan. The study revealed a binding site with an overall polarity similar to that of dichloromethane and an internal phase comparable to that of phospholipid membranes with coexisting solid-ordered and liquid-crystalline states. The fluorescence properties of bound Laurdan also revealed that the binding site of SCP2 can accommodate competitively more than one ligand, with micro and nanomolar dissociation constants. The much higher affinity for the second than for the first ligand implies that the most prominent SCP2 species in the cellular context are those occupied by two ligands. Thus SCP2 may carry a highly populated lipid in the background and a second one, specific for the functional purpose of SCP2. Our findings are important for the characterization of SCP2 biological functions and the design of specific inhibitors.

Stationary phase based on cellulose dodecanoate physically immobilized on silica particles for high-performance liquid chromatography.

The chemical agent free preparation of a stationary phase using a natural macromolecule was the focus of this paper. Thermal immobilization of cellulose dodecanoate on silica particles was used for the preparation of a stationary phase without the use of chemical reagents. Cellulose modification was performed to produce a hydrophobic macromolecule with solubility in common organic solvents. The new stationary phase was characterized morphologically and physico-chemically, presenting as spherical particles immobilized with a thin cellulose dodecanoate layer. The degree of substitution of cellulose dodecanoate was 1.7, which resulted in a separation mechanism in reversed phase mode, but with lower hydrophobicity and higher steric selectivity, which are properties from cellulose. These characteristics resulted in a stationary phase with intrinsic selectivity that was able to separate mixtures of polar drugs, homologs of an anionic surfactant and omeprazole isomers, which are not well resolved in typical C18 phases. Considering that cellulose is a natural polymer and the preparation method of stationary phase involves only physical processes of silica modification, the final material presents as a stationary phase with specific retention properties coming from both dodecanoate and cellulose.

Direct visualization of the lateral structure of giant vesicles composed of pseudo-binary mixtures of sulfatide, asialo-GM1 and GM1 with POPC.

We compared the lateral structure of giant unilamellar vesicles (GUVs) composed of three pseudo binary mixtures of different glycosphingolipid (GSL), i.e. sulfatide, asialo-GM1 or GM1, with POPC. These sphingolipids possess similar hydrophobic residues but differ in the size and charge of their polar head group. Fluorescence microscopy experiments using LAURDAN and DiIC18 show coexistence of micron sized domains in a molar fraction range that depends on the nature of the GSLs. In all cases, experiments with LAURDAN show that the membrane lateral structure resembles the coexistence of solid ordered and liquid disordered phases. Notably, the overall extent of hydration measured by LAURDAN between the solid ordered and liquid disordered membrane regions show marked similarities and are independent of the size of the GSL polar head group. In addition, the maximum amount of GSL incorporated in the POPC bilayer exhibits a strong dependence on the size of the GSL polar head group following the order sulfatide>asialo-GM1>GM1. This observation is in full harmony with previous experiments and theoretical predictions for mixtures of these GSL with glycerophospholipids. Finally, compared with previous results reported in GUVs composed of mixtures of POPC with the sphingolipids cerebroside and ceramide, we observed distinctive curvature effects at particular molar fraction regimes in the different mixtures. This suggests a pronounced effect of these GSL on the spontaneous curvature of the bilayer. This observation may be relevant in a biological context, particularly in connection with the highly curved structures found in neural cells.

Study of rabbit erythrocytes membrane solubilization by sucrose monomyristate using laurdan and phasor analysis.

The study of surfactant and bio membranes interaction is particularly complex due to the diversity in lipid composition and the presence of proteins in natural membranes. Even more difficult is the study of this interaction in vivo since cellular damage may complicate the interpretation of the results, therefore for most of the studies in this field either artificial or model systems are used. One of the model system most used to study biomembranes are erythrocytes due to their relatively simple structure (they lack nuclei and organelles having only the plasma membrane), their convenient experimental manipulation and availability. In this context, we used rabbit erythrocytes as a model membrane and Laurdan (6-lauroyl-2-dimethylaminonaphthalene) as the fluorescent probe to study changes promoted in the membrane by the interaction with the sucrose monoester of myristic acid, ß-d-fructofuranosyl-6-O-myristoyl-α-d-glucopyranoside (MMS). Surfactant and erythrocytes interaction was studied by measuring hemoglobin release and the changes in water content in the membrane sensed by Laurdan. Using two-photon excitation, three types of measurements were performed: Generalized Polarization (analyzed as average GP values), Fluorescence Lifetime Imaging, FLIM (analyzed using phasor plots) and Spectral imaging (analyzed using spectral phasor). Our data indicate that at sublytical concentration of surfactant (20µM MMS), there is a decrease of about 35% in erythrocytes size, without changes in Laurdan lifetime or emission spectra. We also demonstrate that as hemolysis progress, Laurdan lifetime increased due to the decrease in hemoglobin (strong quencher of Laurdan emission) content inside the erythrocytes. Under these conditions, Laurdan spectral phasor analyses can extract the information on the water content in the membrane in the presence of hemoglobin. Our results indicate an increase in membrane fluidity in presence of MMS.

Production, purification and partial characterization of four lipases from a thermophile isolated from Deception Island.

Four lipases were purified from ID17, a thermophilic bacterium belonging to Geobacillus genus isolated from Deception Island, Antarctica. Lipase activity was detected by opacity test and p-nitrophenyl laurate methods. Lipase production was better in a medium containing tryptone as the carbon and nitrogen source, without non-ionic detergents and pH 7.5. Proteins were ultrafiltered from supernatant and separated using anion exchange and size exclusion chromatography resulting in four distinct fractions with lipase activity (called Lip1-4). Purified lipases showed an optimal pH at 9.0, 9.5, 10.0 and 8.0 and temperature at 65, 70, 75 and 80 °C for Lip1-4, respectively. Lip1 and Lip2 showed higher activity using p-nitrophenol decanoate as substrate, whereas Lip3 and Lip4 prefer p-nitrophenol laurate. Based on their molecular weight Lip1 and Lip2 are trimeric and pentameric proteins, respectively, whereas Lip3 and Lip4 are monomeric proteins. Lip1 was exceptionally thermostable maintaining 70 % of its activity after incubating it at 70 °C for 8 h. Based on their characteristics, the four lipases obtained from ID17 are good candidates to understand the mechanisms of lipase stability and to be used in different types of industrial applications.

The new fluorescent membrane probe Ahba: a comparative study with the largely used Laurdan.

Lipid bilayers have been largely used as model systems for biological membranes. Hence, their structures, and alterations caused on them by biological active molecules, have been the subject of many studies. Accordingly, fluorescent probes incorporated into lipid bilayers have been extensively used for characterizing lipid bilayer fluidity and/or polarity. However, for the proper analysis of the alterations undergone by a membrane, a comprehensive knowledge of the fluorescent properties of the probe is fundamental. Therefore, the present work compares fluorescent properties of a relative new fluorescent membrane probe, 2-amino-N-hexadecyl-benzamide (Ahba), with the largely used probe 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan), using both static and time resolved fluorescence. Both Ahba and Laurdan have the fluorescent moiety close to the bilayer surface; Ahba has a rather small fluorescent moiety, which was shown to be very sensitive to the bilayer surface pH. The main goal was to point out the fluorescent properties of each probe that are most sensitive to structural alterations on a lipid bilayer. The two probes were incorporated into bilayers of the well-studied zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC), which exhibits a gel-fluid transition around 23 °C. The system was monitored between 5 and 50 °C, hence allowing the study of the two different lipid structures, the gel and fluid bilayer phases, and the transition between them. As it is known, the fluorescent emission spectrum of Laurdan is highly sensitive to the bilayer gel-fluid transition, whereas the Ahba fluorescence spectrum was found to be insensitive to changes in bilayer structure and polarity, which are known to happen at the gel-fluid transition. However, both probes monitor the bilayer gel-fluid transition through fluorescence anisotropy measurements. With time-resolved fluorescence, it was possible to show that bilayer structural variations can be monitored by Laurdan excited state lifetimes changes, whereas Ahba lifetimes were found to be insensitive to bilayer structural modifications. Through anisotropy time decay measurements, both probes could monitor structural bilayer changes, but the limiting anisotropy was found to be a better parameter than the rotational correlation time. It is interesting to have in mind that the relatively small fluorophore of Ahba (o-Abz) could possibly be bound to a phospholipid hydrocarbon chain, not disturbing much the bilayer packing and being a sensitive probe for the bilayer core.