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Background: Medium-chain fatty acids (MCFAs) are commonly used to enhance the caloric content of infant formulas. We previously reported that pigs fed MCFA developed hepatic steatosis when compared to those fed isocaloric long-chain fatty acid (LCFA) rich formula., Objectives: The objectives of this study were to investigate: 1) whether MCFA and LCFA feeding affect hepatic fatty acid oxidation, and 2) how fat type alters the expression of hepatic fatty acid metabolic genes., Methods: Twenty-six, 7-d-old pigs were fed a low-energy control (CONT) formula, or 2 isocaloric high-energy formulas rich in LCFA or MCFA for 22 days. Livers were collected for examining ex vivo fatty acid oxidation, fatty acid content, and mRNA expression of fatty acid metabolic genes., Results: Liver fat was 20% for pigs in the MCFA compared with 2.9% and 4.6% for those in the CONT and LCFA groups (P < 0.05). MCFA-fed pigs had greater amounts of hepatic laurate, myristate, palmitate, and palmitoleate (14, 34, 49, and 9.3 mg · g -1 ) than those fed LCFA and CONT (1.8, 1.9, 19, 1.5 mg · g -1 ) formulas (P ≤ 0.05). Hepatic laurate and palmitate oxidation was reduced for pigs fed MCFA (29 mmol · mg -1 · h -1 ) compared with those fed CONT (54 mmol · mg -1 · h -1 ) and LCFA (51 mmol · mg -1 · h -1 ) formulas (P < 0.05). Expression of fatty acid synthase 3 (FASN-3), fatty acid binding protein 1 (FABP-1), and acetyl-CoA carboxylase 1 (ACACA-1) were 8-, 6-, and 2-fold greater for pigs in the MCFA than those in the LCFA and CONT groups (P < 0.05)., Conclusions: Feeding MCFA resulted in hepatic steatosis compared with an isocaloric formula rich in LCFA. Steatosis occurred concomitantly with reduced fatty acid oxidation but greater mRNA expression of fatty acid synthetic and catabolic genes., (Copyright © 2024 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Pseudomonas aeruginosa can survive in a myriad of environments, partially due to modifications of its lipid A, the membrane anchor of lipopolysaccharide. We previously demonstrated that divergent late acyltransferase paralogs, HtrB1 and HtrB2, add acyloxyacyl laurate to lipid A 2- and 2'-acyl chains, respectively. The genome of P. aeruginosa also has genes which encode two dioxygenase enzymes, LpxO1 and LpxO2, that individually hydroxylate a specific secondary laurate. LpxO1 acts on the 2'-acyloxyacyl laurate (added by HtrB2), whereas LpxO2 acts on the 2-acyloxyacyl laurate (added by HtrB1) in a site-specific manner. Furthermore, while both enzyme pairs are evolutionarily linked, phylogenomic analysis suggests the LpxO1/HtrB2 enzyme pair as being of ancestral origin, present throughout the Pseudomonas lineage, whereas the LpxO2/HtrB1 enzyme pair likely arose via horizontal gene transfer and has been retained in P. aeruginosa over time. Using a murine pulmonary infection model, we showed that both LpxO1 and LpxO2 enzymes are functional in vivo , as direct analysis of in vivo lipid A structure from bronchoalveolar lavage fluid revealed 2-hydroxylated lipid A. Gene expression analysis reveals increased lpxO2 but unchanged lpxO1 expression in vivo , suggesting differential regulation of these enzymes during infection. We also demonstrate that loss-of-function mutations arise in lpxO1 and lpxO2 during chronic lung infection in people with cystic fibrosis (CF), indicating a potential role for pathogenesis and airway adaptation. Collectively, our study characterizes lipid A 2-hydroxylation during P. aeruginosa airway infection that is regulated by two distinct lipid A dioxygenase enzymes.IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infection in hospitalized and chronically ill individuals. During infection, P. aeruginosa undergoes adaptive changes to evade host defenses and therapeutic interventions, increasing mortality and morbidity. Lipid A structural alteration is one such change that P. aeruginosa isolates undergo during chronic lung infection in CF. Investigating genetic drivers of this lipid A structural variation is crucial in understanding P. aeruginosa adaptation during infection. Here, we describe two lipid A dioxygenases with acyl-chain site specificity, each with different evolutionary origins. Further, we show that loss of function in these enzymes occurs in CF clinical isolates, suggesting a potential pathoadaptive phenotype. Studying these bacterial adaptations provides insight into selection pressures of the CF airway on P. aeruginosa phenotypes that persist during chronic infection. Understanding these adaptive changes may ultimately provide clinicians better control over bacterial populations during chronic infection., Competing Interests: The authors declare no conflict of interest.

Lipid droplets (LD) are important regulators of lipid metabolism and are implicated in several diseases. However, the mechanisms underlying the roles of LD in cell pathophysiology remain elusive. Hence, new approaches that enable better characterization of LD are essential. This study establishes that Laurdan, a widely used fluorescent probe, can be used to label, quantify, and characterize changes in cell LD properties. Using lipid mixtures containing artificial LD we show that Laurdan GP depends on LD composition. Accordingly, enrichment in cholesterol esters (CE) shifts Laurdan GP from ∼0.60 to ∼0.70. Moreover, live-cell confocal microscopy shows that cells present multiple LD populations with distinctive biophysical features. The hydrophobicity and fraction of each LD population are cell type dependent and change differently in response to nutrient imbalance, cell density, and upon inhibition of LD biogenesis. The results show that cellular stress caused by increased cell density and nutrient overload increased the number of LD and their hydrophobicity and contributed to the formation of LD with very high GP values, likely enriched in CE. In contrast, nutrient deprivation was accompanied by decreased LD hydrophobicity and alterations in cell plasma membrane properties. In addition, we show that cancer cells present highly hydrophobic LD, compatible with a CE enrichment of these organelles. The distinct biophysical properties of LD contribute to the diversity of these organelles, suggesting that the specific alterations in their properties might be one of the mechanisms triggering LD pathophysiological actions and/or be related to the different mechanisms underlying LD metabolism., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The effects of GML (Glycerol monolaurate) supplementation with two level (0.5 and 1.0 g kg -1 ) on the productive performance and flesh quality of large yellow croaker (360 per group) were investigated during feeding (23,50-days) and fasting stage (23,70-days). The GML supplementation significantly increased body weight after 23-days and crude protein, inosinic acid, and yellowness after 50-days. Moreover, it increased hardness, springiness, and chewiness by increasing the collagen content, myofiber density, and decreasing myofiber diameter. The high GML supplementation increased the total free amino acids, delicate amino acids, monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (n-3 PUFA), and EPA + DHA, whereas it decreased the content of saturated fatty acids/unsaturated fatty acids (SFA/UFA). During fasting, better body shape and color were shown were shown at high GML supplementation. Conclusively, high dose GML supplementation exerted promising effects on the productive performance and flesh quality of large yellow croaker., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Rhamnolipids are becoming an important class of glycolipid biosurfactants. Herein, we describe for the first time the enzymatic synthesis of rhamnose fatty acid esters by the transesterification of rhamnose with fatty acid vinyl esters, using lipase from Pseudomonas stutzeri as a biocatalyst. The use of this lipase allows excellent catalytic activity in the synthesis of 4- O -acylrhamnose (99% conversion and full regioselectivity) after 3 h of reaction using tetrahydrofuran (THF) as the reaction media and an excess of vinyl laurate as the acyl donor. The role of reaction conditions, such as temperature, the substrates molar ratio, organic reaction medium and acyl donor chain-length, was studied. Optimum conditions were found using 35 °C, a molar ratio of 1:3 (rhamnose:acyldonor), solvents with a low logP value, and fatty acids with chain lengths from C4 to C18 as acyl donors. In hydrophilic solvents such as THF and acetone, conversions of up to 99-92% were achieved after 3 h of reaction. In a more sustainable solvent such as 2-methyl-THF (2-MeTHF), high conversions were also obtained (86%). Short and medium chain acyl donors (C4-C10) allowed maximum conversions after 3 h, and long chain acyl donors (C12-C18) required longer reactions (5 h) to get 99% conversions. Furthermore, scaled up reactions are feasible without losing catalytic action and regioselectivity. In order to explain enzyme regioselectivity and its ability to accommodate ester chains of different lengths, homology modelling, docking studies and molecular dynamic simulations were performed to explain the behaviour observed.

Imaging of biological membranes by environmentally sensitive solvatochromic probes, such as Laurdan, provides information about the organization of lipids, their ordering, and their uneven distribution. To address a key drawback of Laurdan linked to its rapid internalization and subsequent labeling of internal membranes, we redesigned it by introducing a membrane anchor group based on negatively charged sulfonate and dodecyl chain. The obtained probe, Pro12A, stains exclusively the outer leaflet of lipid bilayers of liposomes, as evidenced by leaflet-specific fluorescence quenching with a viologen derivative, and shows higher fluorescence brightness than Laurdan. Pro12A also exhibits stronger spectral change between liquid-ordered and liquid-disordered phases in model membranes and distinguishes better lipid domains in giant plasma membrane vesicles (GPMVs) than Laurdan. In live cells, it stains exclusively the cell plasma membranes, in contrast to Laurdan and its carboxylate analogue C-Laurdan. Owing to its outer leaflet binding, Pro12A is much more sensitive to cholesterol extraction than Laurdan, which is redistributed within both plasma membrane leaflets and intracellular membranes. Finally, its operating range in the blue spectral region ensures the absence of crosstalk with a number of orange/red fluorescent proteins and dyes. Thus, Pro12A will enable accurate multicolor imaging of lipid organization of cell plasma membranes in the presence of fluorescently tagged proteins of interest, which will open new opportunities in biomembrane research.

Lipase-catalyzed synthesis of xylo-oligosaccharides esters from pure xylobiose, xylotriose and xylotetraose in the presence of vinyl laurate was investigated. The influence of different experimental parameters such as the loading of lipase, the reaction duration or the use of a co-solvent was studied and the reaction conditions were optimized with xylobiose. Under the best conditions, a regioselective esterification occurred to yield a monoester with the acyl chain at the OH-4 of the xylose unit at the non-reducing end. Surface-active properties of these pure xylo-oligosaccharides fatty esters have been evaluated. They display interesting surfactant activities that differ according to the degree of polymerization (DP) of the glycone moiety., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The probiotic activity of skin Staphylococcus epidermidis ( S. epidermidis ) bacteria can elicit diverse biological functions via the fermentation of various carbon sources. Here, we found that polyethylene glycol (PEG)-8 Laurate, a carbon-rich molecule, can selectively induce the fermentation of S. epidermidis , not Cutibacterium acnes ( C. acnes ), a bacterium associated with acne vulgaris. The PEG-8 Laurate fermentation of S. epidermidis remarkably diminished the growth of C. acnes and the C. acnes -induced production of pro-inflammatory macrophage-inflammatory protein 2 (MIP-2) cytokines in mice. Fermentation media enhanced the anti- C. acnes activity of a low dose (0.1%) clindamycin, a prescription antibiotic commonly used to treat acne vulgaris, in terms of the suppression of C. acnes colonization and MIP-2 production. Furthermore, PEG-8 Laurate fermentation of S. epidermidis boosted the activity of 0.1% clindamycin to reduce the sizes of C. acnes colonies. Our results demonstrated, for the first time, that the PEG-8 Laurate fermentation of S. epidermidis displayed the adjuvant effect on promoting the efficacy of low-dose clindamycin against C. acnes . Targeting C. acnes by lowering the required doses of antibiotics may avoid the risk of creating drug-resistant C. acnes and maintain the bacterial homeostasis in the skin microbiome, leading to a novel modality for the antibiotic treatment of acne vulgaris.

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.

The vaginal microbiota influences sexual transmission of human immunodeficiency virus type 1 (HIV-1). Colonization of the vaginal tract is normally dominated by Lactobacillus species. Both Lactobacillus and Enterococcus faecalis may secrete reutericyclin, which inhibits the growth of a variety of pathogenic bacteria. Increasing evidence suggests a potential therapeutic role for an analogue of reutericyclin, glycerol monolaurate (GML), against microbial pathogens. Previous studies using a macaque vaginal simian immunodeficiency virus (SIV) transmission model demonstrated that GML reduces transmission and alters immune responses to infection in vitro Previous studies showed that structural analogues of GML negatively impact other enveloped viruses. We sought to expand understanding of how GML inhibits HIV-1 and other enveloped viruses and show that GML restricts HIV-1 entry post-CD4 engagement at the step of coreceptor binding. Further, HIV-1 and yellow fever virus (YFV) particles were more sensitive to GML interference than particles "matured" by proteolytic processing. We show that high-pressure-liquid-chromatography (HPLC)-purified reutericyclin and reutericyclin secreted by Lactobacillus inhibit HIV-1. These data emphasize the importance and protective nature of the normal vaginal flora during viral infections and provide insights into the antiviral mechanism of GML during HIV-1 infection and, more broadly, to other enveloped viruses. IMPORTANCE A total of 340 million sexually transmitted infections (STIs) are acquired each year. Antimicrobial agents that target multiple infectious pathogens are ideal candidates to reduce the number of newly acquired STIs. The antimicrobial and immunoregulatory properties of GML make it an excellent candidate to fit this critical need. Previous studies established the safety profile and antibacterial activity of GML against both Gram-positive and Gram-negative bacteria. GML protected against high-dose SIV infection and reduced inflammation, which can exacerbate disease, during infection. We found that GML inhibits HIV-1 and other human-pathogenic viruses (yellow fever virus, mumps virus, and Zika virus), broadening its antimicrobial range. Because GML targets diverse infectious pathogens, GML may be an effective agent against the broad range of sexually transmitted pathogens. Further, our data show that reutericyclin, a GML analog expressed by some lactobacillus species, also inhibits HIV-1 replication and thus may contribute to the protective effect of Lactobacillus in HIV-1 transmission., (Copyright © 2020 Welch et al.)

Bactrocera frauenfeldi (Schiner) (Diptera: Tephritidae) is a polyphagous fruit fly pest species that is endemic to Papua New Guinea and has become established in several Pacific Islands and Australia. Despite its economic importance for many crops and the key role of chemical-mediated sexual communication in the reproductive biology of tephritid fruit flies, as well as the potential application of pheromones as attractants, there have been no studies investigating the identity or activity of rectal gland secretions or emission profiles of this species. The present study (1) identifies the chemical profile of volatile compounds produced in rectal glands and released by B. frauenfeldi , (2) investigates which of the volatile compounds elicit an electroantennographic or electropalpographic response, and (3) investigates the potential function of glandular emissions as mate-attracting sex pheromones. Rectal gland extracts and headspace collections from sexually mature males and females of B. frauenfeldi were analysed by gas chromatography-mass spectrometry. Male rectal glands contained ( E , E )-2-ethyl-8-methyl-1,7-dioxaspiro [5.5]undecane as a major component and ( E , E )-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane as a moderate component. Minor components included palmitoleic acid, palmitic acid, and ethyl oleate. In contrast, female rectal glands contained ( E , E )-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and ethyl laurate as major components, ethyl myristate and ethyl palmitoleate as moderate components, and 18 minor compounds including amides, esters, and spiroacetals. Although fewer compounds were detected from the headspace collections of both males and females than from the gland extractions, most of the abundant chemicals in the rectal gland extracts were also detected in the headspace collections. Gas chromatography coupled electroantennographic detection found responses to ( E , E )-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane from the antennae of both male and female B. frauenfeldi . Responses to ( E , E )-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane were elicited from the antennae of females but not males. The two spiroacetals also elicited electropalpographic responses from both male and female B. frauenfeldi . Ethyl caprate and methyl laurate, found in female rectal glands, elicited responses in female antennae and palps, respectively. Y-maze bioassays showed that females were attracted to the volatiles from male rectal glands but males were not. Neither males nor females were attracted to the volatiles from female rectal glands. Our findings suggest ( E , E )-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and ( E , E )-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane as components of a sex-attracting pheromone in B. frauenfeldi .

Regulatory assessment of the bioaccumulation from water is commonly based on bioconcentration factors (BCFs) derived from fish flow-through tests. Such experiments require many laboratory animals and are time-consuming and costly. An alternative test setup for organic, neutral compounds using the amphipod Hyalella azteca was recently suggested, resulting in BCF values which show a strong correlation with fish BCF data. In the present study, the bioconcentration potential of the ionic compound laurate was elucidated in H. azteca. The sodium salt of 1- 14 C laurate was applied to H. azteca in a flow-through and a semistatic approach. Because of rapid biodegradation, a semistatic approach with frequent medium replacements was required to ensure a stable medium concentration. Laurate was also rapidly metabolized by H. azteca. A large proportion of the total radioactivity measured in the amphipod tissue was not extractable, suggesting that mineralized laurate was accumulated in the calcified exoskeleton of H. azteca. This was confirmed in a further study using carbonate [ 14 C]. A lipid-normalized (5.0%) Hyalella BCF of 8.9 was calculated for laurate, measured as free fatty acids. The results of the bioconcentration studies with H. azteca confirm the low bioaccumulation potential of the test item previously observed in fish. However, more organic ionic compounds with various properties need to be tested to assess whether a general correlation between fish and Hyalella BCF data exists. Environ Toxicol Chem 2020;39:310-322. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC., (© 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.)

The electroporation of cells is nowadays used for a large variety of purposes, from basic research to cancer therapy and food processing. Understanding molecular mechanisms of the main processes involved in electroporation is thus of significant interest. In the present work, we propose an experimental system to record in real time the evolution of any cell parameter which can be evaluated by fluorescence (before, during and after application of the electroporation pulses to cells in suspension). The system is based on the design of adequate electroporation electrodes, compatible with a standard spectrofluorometer cuvette housing. The electric field intensity generated when pulses are applied was carefully characterized for different geometries of the electrodes, to choose a construction ensuring the greatest homogeneity of the field in combination with the best possible illumination of the sample. As an example of the method's application, we present here generalized polarization kinetics for a varying number of electroporation pulses applied to a cell suspension; the general polarization parameter is strongly correlated to water presence in the hydrophobic membrane core. The system may be used for many other fluorescence measurements useful for the characterization of the electroporation process.

This study aimed to investigate whether exogenous application of carnitine stimulates transportation of fatty acids into mitochondria, which is an important part of fatty acid trafficking in cells, and mitochondrial respiration in the leaves of maize seedlings grown under normal and cold conditions. Cold stress led to significant increases in lipase activity, which is responsible for the breakdown of triacylglycerols, and carnitine acyltransferase (carnitine acyltransferase I and II) activities, which are responsible for the transport of activated long-chain fatty acids into mitochondria. While exogenous application of carnitine has a similar promoting effect with cold stress on lipase activity, it resulted in further increases in the activity of carnitine acyltransferases compared to cold stress. The highest activity levels for these enzymes were recorded in the seedlings treated with cold plus carnitine. In addition, these increases were correlated with positive increases in the contents of free- and long-chain acylcarnitines (decanoyl-l-carnitine, lauroyl-l-carnitine, myristoyl-l-carnitine, and stearoyl-l-carnitine), and with decreases in the total lipid content. The highest values for free- and long-chain acylcarnitines and the lowest value for total lipid content were recorded in the seedlings treated with cold plus carnitine. On the other hand, carnitine with and without cold stress significantly upregulated the expression level of citrate synthase, which is responsible for catalysing the first reaction of the citric acid cycle, and cytochrome oxidase, which is the membrane-bound terminal enzyme in the electron transfer chain, as well as lipase. All these results revealed that on the one hand, carnitine enhanced transport of fatty acids into mitochondria by increasing the activities of lipase and carnitine acyltransferases, and, on the other hand, stimulated mitochondrial respiration in the leaves of maize seedlings grown under normal and cold conditions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Understanding the interactions between feed additives and the functional properties of egg white protein (EWP) may offer novel insights into the effects of feed additives on laying hens and may provide an alternative for modification of the functional properties of EWP by using laying hens as bioreactors. Glycerol monolaurate (GML) is widely used in the food industry as an effective antibacterial emulsifier. In this work, the effects of three doses of dietary GML supplementation (150, 300, and 450 mg kg -1 hen) on the functional properties of EWP were investigated., Results: The hardness of EWP gels was significantly improved by 300 and 450 mg kg -1 GML supplementation. Foaming capacity (FC) and foaming stability (FS) were increased after GML treatment; 450 mg kg -1 GML supplementation showed the most significant improvements, with 44.82% in FC and 23.39% in FS. Stabilization of EWP-oil emulsions was also improved, supported by a slowed creaming process and the formation of smaller oil droplets. The heat denaturation temperature and rheological properties were also modified by dietary GML supplementation, implying improved thermal stability., Conclusion: Our study demonstrated that GML supplementation has the potential to modify the functional properties of EWP, broadening the application of GML and providing a new perspective for evaluation of the efficacy of feed additives. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Erythorbyl laurate is a potential food additive as a multi-functional emulsifier having antioxidant and antimicrobial activities. In this study, a gas-solid-liquid multiphase system (GSL-MPS) was established to enhance the production yield of erythorbyl laurate in a lipase-catalyzed solvent-free synthesis. The significant reaction variables were optimized as follows: substrate molar ratio of 2:1 (lauric acid:erythorbic acid) and enzyme concentration of 120 mg/mL (840 PLU/mL). Under these conditions, the maximum production yield in GSL-MPS was 13.974 mg/mL, which is 8.60- and 4.26-fold higher than the yields obtained in an organic solvent monophase system (OS-MPS) and a solid-liquid biphase system (SL-BPS), respectively. Moreover, the operational stability of the immobilized lipase was significantly improved in GSL-MPS compared with OS-MPS. These results indicate that GSL-MPS can be an enzymatic reaction system facilitating efficient production of ester compounds as a means of increasing production yields and the reusability of the immobilized lipase., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Efficient enzymatic synthesis of d-xylose and l-arabinose lauryl mono- and diesters has been achieved by transesterification reactions catalysed by immobilized Candida antarctica lipase B as biocatalyst, in organic medium in the presence of d-xylose or l-arabinose and vinyllaurate at 50 °C. In case of l-arabinose, one monoester and one diester were obtained in a 57% overall yield. A more complex mixture was produced for d-xylose as two monoesters and two diesters were synthesized in a 74.9% global yield. The structures of all these pentose laurate esters was solved. Results demonstrated that the esterification first occurred regioselectively onto the primary hydroxyl groups. Pentose laurate esters exhibited interesting features such as low critical aggregation concentrations values all inferior to 25 μM. Our study demonstrates that the enzymatic production of l-arabinose and d-xylose-based esters represents an interesting approach for the production of green surfactants from lignocellulosic biomass-derived pentoses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The properties and the antioxidant activity of a series of hydroxytyrosyl esters having different carbon chain lengths (C4, C8, C12 and C18) have been measured in phosphatidylcholine model membrane (liposomes) using specific probes for the bilayer and liposome lumen microenvironment, i.e., 1,6-diphenyl-1,3,5-hexatriene (DPH) and 2',7'-dichlorodihydrofluorescein (H 2 DCF), respectively. Antioxidants self-assembly and their interaction with liposomes has been evaluated by light scattering, fluorescence, turbidimetry, gel filtration chromatography and microfiltration measurements, allowing the determination of critical aggregation concentration, bound fraction, capacity of crossing the lipid bilayer. The distribution of hydroxytyrosyl long chain esters has been proved to depend quite specifically on their lipophilic chain length, and this turns to have deep effects on their antioxidant behaviour. Shedding new light on the cut off effect and antioxidant behaviour of phenolipids, this study also put forward the relevance of cell-free liposome-based cellular models, like giant liposomes, for further characterization of analogous systems., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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, β-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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study, the coding sequence of the lipase from Proteus sp. SW1 was optimized via codon optimization and subjected to expression in Pichia pastoris GS115. The maximum enzyme yield was 387 mg/L in the supernatants of the shake-flask culture. The purified recombinant lipase exhibited a specific activity of 130 U/mg toward p-nitrophenyl Laurate. Its optimum pH and temperature were 8.0 and 40°C, respectively. It was highly stable and even activated in water-miscible solvents, showing over 102% residual activity after 24 h incubation in ethanol, acetone, isopropanol and acetonitrile. In addition, the enzyme showed promoted activity with the increasing concentrations of methanol/ethanol and exhibited the maximum activity at 80%. In a solvent-free system for biodiesel synthesis with a one-step addition of methanol, the recombinant lipase displayed a 87% conversion rate toward palm oil at the high water content of 80%. The highly improved expression level and activity of the recombinant lipase may contribute to enable its commercial-scale production, and the unique properties would make it a particularly promising biocatalyst for biodiesel production in the future., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The HSL-like lipase encoding gene (Blip) from the polyextremophile Bacillus halodurans C-125 has been heterologously expressed in E.coli BL21(DE3). The enzyme is a monomer of ~42 kDa. It has extremely high thermal stability with a t 1/2 of 35 min at 100 °C. Thermal denaturation/renaturation studies by CD and fluorescence analysis revealed complete refolding of the protein back to its native conformation even after 30 min at 90 °C. Blip prefers substrates with mid to long chain fatty acids. It has a higher catalytic efficiency on para-nitrophenyl fatty acyl esters as opposed to triacylglycerides (k cat /K m with pNP-palmitate as a substrate was 2.52 × 10 5 mM -1  min -1 while that with glyceryl tripalmitin was 4.06 × 10 2 mM -1  min -1 , respectively). The enzyme also has a unique selectivity for hydrolysis of unsaturated fatty acyl esters. The enzyme catalyses the synthesis of pNP-laurate with an optimized conversion of 95.94 ± 0.24%. A simple procedure for purification of the product has been developed that led to 89.91 ± 0.33% product recovery.

Immobilization of lipase was successfully achieved on the surface of magnetically separable Fe 3 O 4 /graphene oxide (GO) via a post-modification. This post modification was achieved in alternation to glutaraldehyde post-modification. The activity of immobilized lipase had not a significant loss in the activity while on the other hand, it is simply extractable (by keeping its major activity) from reaction crude by a magnet. Each step of immobilization was carefully monitored by characterization and all were successfully proved. SEM, TEM, XRD, EDX, and FTIR were used to characterize the support and immobilization process., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The diffusible signaling factor (DSF)-based quorum sensing (QS) system has emerged as a widely conserved cell-cell communication mechanism in Gram-negative bacteria. Typically, signals from the DSF family are cis-2-unsaturated fatty acids which regulate diverse biological functions. Recently, substantial progress has been made on the characterization of new members of this family of signals. There have also been new developments in the understanding of the biosynthesis of these molecules where dual enzymatic activities of the DSF synthase and the use of various substrates have been described. The recent discovery of a naturally occurring DSF turnover mechanism and its regulation provides a new dimension in our understanding of how DSF-dependent microorganisms modulate virulence gene expression in response to changes in the surrounding environment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The current research work represents solvent free enzymatic synthesis of fatty acid ethyl ester; ethyl laurate. Immobilized lipase Fermase CALB™10,000 was used for the synthesis of ethyl laurate from ethanol and lauric acid. The influence of process parameters such as catalyst loading, speed of agitation, mole ratio, molecular sieves and temperature were studied. Fermase CALB™10,000 offered a conversion of 92.46 % at 60 °C in 4 h at optimized condition. The experimental data was best fitted by the Random Bi-Bi model with inhibition of both the substrates i.e. lauric acid and ethanol. The following kinetic parameters were retrieved from the model: V max  = 1.243 × 10 3  mmol/min/g enzyme; K A  = 0.1283 mmol; K B  = 8.51 mmol; K iA  = 5.098 mmol; and SSE = 0.0142. The activation energy for the enzymatic esterification was also determined and calculated to be 28.49 kJ/mol. A maximum conversion of 71 % was obtained after 5 successive reuse of Fermase CALB™10,000 lipase.

Sugar esters are valuable compounds composed of various sugars and fatty acids that can be used as antibacterial agents and emulsifiers in toothpaste and canned foods. For example, fructose fatty acid esters suppress growth of Streptococcus mutans, a typical pathogenic bacterium causing dental caries. In this study, fructose laurate ester was chosen as a target material and was synthesized by a transesterification reaction using Candida antarctica lipase B. We performed a solvent screening experiment and found that a t-butanol/dimethyl sulfoxide mixture was the best solvent to dissolve fructose and methyl laurate. Fructose laurate was synthesized by transesterification of fructose (100 mM) with methyl laurate (30 mM) in t-butanol containing 20% dimethyl sulfoxide. The conversion yield was about 90%, which was calculated based on the quantity of methyl laurate using high-performance liquid chromatography. Fructose monolaurate (Mr 361) was detected in the reaction mixture by high-resolution mass spectrometry. The inhibitory effect of fructose laurate on growth of oral or food spoilage microorganisms, including S. mutans, Bacillus coagulans, and Geobacillus stearothermophilus, was evaluated.

Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

The thermostable NAD(+)-dependent alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH) was exploited with regard to the biocatalytic synthesis of ω-oxo lauric acid methyl ester (OLAMe), a key intermediate for biobased polyamide 12 production, from the corresponding long-chain alcohol. Recombinant BsADH was produced in Escherichia coli as a homogeneous tetrameric enzyme and showed high activity towards the industrially relevant substrate ω-hydroxy lauric acid methyl ester (HLAMe) with K M = 86 μM and 44 U mg(-1). The equilibrium constant for HLAMe oxidation to the aldehyde (OLAMe) with NAD(+) was determined as 2.16 × 10(-3) from the kinetic parameters of the BsADH-catalyzed forward and reverse reactions. Since BsADH displayed limited stability under oxidizing conditions, the predominant oxidation-prone residue Cys257 was mutated to Leu based on sequence homology with related enzymes and computational simulation. This substitution resulted in an improved BsADH variant exhibiting prolonged stability and an elevated inactivation temperature. Semi-preparative biocatalysis at 60 °C using the stabilized enzyme, employing butyraldehyde for in situ cofactor regeneration with only catalytic amounts of NAD(+), yielded up to 23 % conversion of HLAMe to OLAMe after 30 min. In contrast to other oxidoreductases, no overoxidation to the dodecanoic diacid monomethyl ester was detected. Thus, the mutated BsADH offers a promising biocatalyst for the selective oxidation of fatty alcohols to yield intermediates for industrial polymer production.

Pseudomonas aeruginosa (PA) is an opportunistic Gram-negative pathogen associated with nosocomial infections, acute infections and chronic lung infections in patients with cystic fibrosis. The ability of PA to cause infection can be attributed to its ability to adapt to a multitude of environments. Modification of the lipid A portion of lipopolysaccharide (LPS) is a vital mechanism Gram-negative pathogens use to remodel the outer membrane in response to environmental stimuli. Lipid A, the endotoxic moiety of LPS, is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria making it a critical factor for bacterial adaptation. One way PA modifies its lipid A is through the addition of laurate and 2-hydroxylaurate. This secondary or late acylation is carried out by the acyltransferase, HtrB (LpxL). Analysis of the PA genome revealed the presence of two htrB homologs, PA0011 (htrB1) and PA3242 (htrB2). In this study, we were able to show that each gene identified is responsible for site-specific modification of lipid A. Additionally, deletions of either gene altered resistance to specific classes of antibiotics, cationic antimicrobial peptides and increased membrane permeability suggesting a role for these enzymes in maintaining optimal membrane organization and integrity., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Intraspecific olfactory signals known as pheromones play important roles in insect mating systems. In the model Drosophila melanogaster, a key part of the pheromone-detecting system has remained enigmatic through many years of research in terms of both its behavioral significance and its activating ligands. Here we show that Or47b-and Or88a-expressing olfactory sensory neurons (OSNs) detect the fly-produced odorants methyl laurate (ML), methyl myristate, and methyl palmitate. Fruitless (fru(M))-positive Or47b-expressing OSNs detect ML exclusively, and Or47b- and Or47b-expressing OSNs are required for optimal male copulation behavior. In addition, activation of Or47b-expressing OSNs in the male is sufficient to provide a competitive mating advantage. We further find that the vigorous male courtship displayed toward oenocyte-less flies is attributed to an oenocyte-independent sustained production of the Or47b ligand, ML. In addition, we reveal that Or88a-expressing OSNs respond to all three compounds, and that these neurons are necessary and sufficient for attraction behavior in both males and females. Beyond the OSN level, information regarding the three fly odorants is transferred from the antennal lobe to higher brain centers in two dedicated neural lines. Finally, we find that both Or47b- and Or88a-based systems and their ligands are remarkably conserved over a number of drosophilid species. Taken together, our results close a significant gap in the understanding of the olfactory background to Drosophila mating and attraction behavior; while reproductive isolation barriers between species are created mainly by species-specific signals, the mating enhancing signal in several Drosophila species is conserved.

Sugar esters of fatty acids have many applications as biocompatible and biodegradable emulsifiers, which are determined by their degrees of esterification (DE). Direct esterification of fructose with lauric acid in organic media used commercial immobilized Candida antarctica lipase B (CALB) was investigated for DE. Significant difference of DE was observed between 2-methyl-2-butanol (2M2B) and methyl ethyl ketone (MEK), as di-ester/mono-ester molar ratio of 1.05:1 in 2M2B and 2.79:1 in MEK. Fourier transform infrared (FTIR) spectra showed that the secondary structure of the enzyme binding mono-ester presented distinct difference in 2M2B and MEK. Contents of β-turn and antiparallel β-sheet of CALB in 2M2B were 26.9% and 16.2%, respectively, but 19.1% and 13.2% in MEK. To understand the relationship between the conformational changes and differences of DE, mono-ester and fatty acid were directly employed for synthesis of di-ester. The maximum initial velocity of di-ester synthesis in MEK was 0.59 mmolg(enzyme)(-1)h(-1), which was 2.19-fold as greater as that in 2M2B, indicating that CALB conformation in MEK was preferred for the synthesis of di-ester. These results demonstrated that the conformation of CALB binding mono-ester affected by organic solvents essentially determined DE., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Oxidized polyvinyl alcohol hydrolase (OPH) catalyzes the cleavage of C-C bond in β-diketone. It belongs to the α/β-hydrolase family and contains a unique lid region that covers the active site. The lid is the most variable region when pOPH from Pseudomonas sp. VM15C and sOPH from Sphingopyxis sp. 113P3 are compared. The wild-type enzymes and the pOPH mutants W255A, W255Y and W255F were analyzed for lipase activity by using p-nitrophenyl (pNP) esters as the substrates. The wild-type enzymes showed increased Km and decreased kcat/Km with the acyl chain length, and the mutants showed reduced kcat/Km for pNP acetate, indicating the importance of Trp255 in sequestering the active site from solvent. The significantly lower activity for pNP butyrate can be a result of product inhibition, as suggested by the complex crystal structures, in which butyric acid, DMSO or PEG occupied the same substrate-binding cleft. The mutant activity was retained with pNP caprylate and pNP laurate as the substrates, reflecting the amphipathic nature of the cleft. Moreover, the disulfide bond formation of Cys257/267 is important for the activity of pOPH, but it is not essential for sOPH, which has a shorter lid structure., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A whole-cell based method was developed for the regioselective synthesis of arabinocytosine laurate. Among the seven kinds of bacteria strains tested in the acylation reaction, Pseudomonas fluorescens gave the highest productivity and a higher 5'-regioselectivity than 99%. Compared with pure organic solvents, the use of organic solvent mixtures greatly promoted the yield of the whole-cell catalyzed reaction, but showed little influence on the 5'-regioselectivity. Of all the tested solvent mixtures, the best reaction result was found in isopropyl ether/pyridine followed by isopentanol/pyridine. However, the whole-cells showed much lower thermostability in isopropyl ether/pyridine than in THF-pyridine. To better understand the toxic effects of the organic solvents on P. fluorescens whole-cells and growing cells were further examined. Significant influences of organic solvents on the biomass of the cells were found, which differed depending on the type of solvents used. SEM analysis visually revealed the changes in the surface morphology of whole-cells and growing cells cultured in media containing various organic solvents, in terms of surface smoothness, bulges and changed cell sizes. Results demonstrated that organic toxicity to cell structure played an important role in whole-cell mediated catalysis.

This paper presents the molecular identification of a newly isolated bacterial strain producing a novel and organic solvent stable lipase, statistical optimization of fermentation medium, and its application in the synthesis of ethyl laurate. On the basis of nucleotide homology and phylogenetic analysis of 16S rDNA sequence, the strain was identified as Bacillus safensis DVL-43 (Gen-bank accession number KC156603). Optimization of fermentation medium using Plackett-Burman design and response surface methodology led to 11.4-fold increase in lipase production. The lipase from B. safensis DVL-43 exhibited excellent stability in various organic solvents. The enzyme retained 100% activity after 24h incubation in xylene, DMSO and toluene, each solvent being used at a concentration of 25% (v/v). The use of partially purified DVL-43 lipase as catalyst in the synthesis of ethyl laurate, an esterification product of lauric acid and ethanol, resulted in 80% esterification in 12h under optimized conditions. The formation of ethyl laurate was confirmed using TLC and (1)H NMR. Organic solvent stable lipases exhibiting potential application in enzymatic esterification are in great demand in flavor, fine chemicals and pharma industries. We could not find any report on lipase production from B. safensis strain and its application in esterification., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Purpose: In vitro skin permeation studies have been used extensively in the development and optimisation of delivery of actives in vivo. However, there are few reported correlations of such in vitro studies with in vivo data. The aim of this study was to investigate the skin permeation of a model active, niacinamide, both in vitro and in vivo., Methods: Conventional diffusion cell studies were conducted in human skin to determine niacinamide permeation from a range of vehicles which included dimethyl isosorbide (DMI), propylene glycol (PG), propylene glycol monolaurate (PGML), N-methyl 2-pyrrolidone (NMP), Miglyol 812N® (MG), and mineral oil (MO). Single, binary or ternary systems were examined. The same vehicles were subsequently examined to investigate niacinamide delivery in vivo. For this proof-of-concept study one donor was used for the in vitro studies and one volunteer for the in vivo investigations to minimise biovariability. Analysis of in vitro samples was conducted using HPLC and in vivo uptake of niacinamide was evaluated using Confocal Raman spectroscopy (CRS)., Results: The amount of niacinamide permeated through skin in vitro was linearly proportional to the intensity of the niacinamide signal determined in the stratum corneum in vivo. A good correlation was observed between the signal intensities of selected vehicles and niacinamide signal intensity., Conclusions: The findings provide further support for the use of CRS to monitor drug delivery into and across the skin. In addition, the results highlight the critical role of the vehicle and its disposition in skin for effective dermal delivery.

This mini-review reports on applications of particular multiphoton excitation microscopy-based methodologies employed in our laboratory to study skin. These approaches allow in-depth optical sectioning of the tissue, providing spatially resolved information on specific fluorescence probes' parameters. Specifically, by applying these methods, spatially resolved maps of water dipolar relaxation (generalized polarization function using the 6-lauroyl-2-(N,N-dimethylamino)naphthale probe), activity of protons (fluorescence lifetime imaging using a proton sensitive fluorescence probe--2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) and diffusion coefficients of distinct fluorescence probes (raster imaging correlation spectroscopy) can be obtained from different regions of the tissue. Comparative studies of different tissue strata, but also between equivalent regions of normal and abnormal excised skin, including applications of fluctuation correlation spectroscopy on transdermal penetration of liposomes are presented and discussed. The data from the different studies reported reveal the intrinsic heterogeneity of skin and also prove these strategies to be powerful noninvasive tools to explore structural and dynamical aspects of the tissue., (Copyright © 2013 Elsevier B.V. All rights reserved.)

1'-O-Lauroylsucrose and 6'-O-lauroylsucrose were formed through hydrolysis of the C-6″ galactose group of 1'-O-lauroylraffinose and 6'-O-lauroylraffinose, respectively, in the presence of α-galactosidase. The enzymatic hydrolysis of 1'-O-lauroylraffinose and 6'-O-lauroylraffinose is discussed in detail. Acetic acid-sodium acetate was chosen as the buffer solution of the enzymatic hydrolysis reaction. The optimum conditions for the enzymatic hydrolysis reaction were as follows: buffer solution, pH 3.8; enzymatic time, 48 h; and enzymatic temperature, 37 °C. Under the optimal process conditions, the efficiency of α-galactosidase was ca. 82.6%. The isomers were fully compared in solubility, hydrophile-lipophile balance (HLB) values, critical micelle concentration (CMC), and thermal stability. The results showed that all lauroylsucrose isomers have similar solubilities in polar solvent, HLB values, CMC values, and thermal stabilities.

Erythorbyl laurate was continuously synthesized by esterification in a packed-bed enzyme reactor with immobilized lipase from Candida antarctica. Response surface methodology based on a five-level three-factor central composite design was adopted to optimize conditions for the enzymatic esterification. The reaction variables, such as reaction temperature (10-70°C), substrate molar ratio ([lauric acid]/[erythorbic acid], 5-15), and residence time (8-40 min) were evaluated and their optimum conditions were found to be 56.2°C, 14.3, and 24.2 min, respectively. Under the optimum conditions, the molar conversion yield was 83.4%, which was not significantly different (P < 0.05) from the value predicted (84.4%). Especially, continuous water removal by adsorption on an ion-exchange resin in a packed-bed enzyme reactor improved operational stability, resulting in prolongation of half-life (2.02 times longer compared to the control without water-removal system). Furthermore, in the case of batch-type reactor, it exhibited significant increase in initial velocity of molar conversion from 1.58% to 2.04%/min., (© 2013 American Institute of Chemical Engineers.)

Since monolaurin, a monoglyceride formed in the human body in small quantities, has proven effective both in vitro and in vivo against certain strains of Staphylococcus aureus, an important question arises whether consuming a substance high in lauric acid content, such as coconut oil could increase intrinsic monolaurin production to levels that would be successful in overcoming staphylococcal and other microbial invaders. Both a cup plate method and a microdilution broth culture system were employed to test bacteriostatic and bactericidal effects of the test agents in vitro. To test effectiveness in vivo, female C3H/he mice (10-12 per group) were orally administered sterile saline (regular control), vancomycin (positive control), aqueous monolaurin, or two varieties of coconut oil (refined, bleached, deodorized coconut oil and virgin coconut oil) for 1 week before bacterial challenge and 30 days after. A final group received both monolaurin and vancomycin. In contrast to monolaurin, the coconut oils did not show bactericidal activity in vitro. In vivo, the groups receiving vancomycin, monolaurin, or the combination showed some protection--50-70% survival, whereas the protection from the coconut oils were virtually the same as control--0-16% survival. Although we did not find that the two coconut oils are helpful to overcome S. aureus infections, we corroborated earlier studies showing the ability of monolaurin to do such.

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 experiments conducted on tonoplast of Beta vulgaris L. roots were performed to identify detergent-resistant lipid-protein microdomains (DRMs, interpreted as lipid rafts).The presence of DRMs can be found when dynamic clustering of sphingolipids, sterols, saturated fatty acids is registered, and the insolubility of these microdomains in nonionic detergents at low temperatures is proven. The elucidation of tonoplast microdomains has been based on results obtained with the aid of high-speed centrifuging in the sucrose gradient. The experiments have shown that tonoplast microdomains are rich in sphingolipids, free sterols and saturated fatty acids (such a lipid content is also typical of lipid-protein microdomains of other membranes), while only few phospholipids are present in tonoplast microdomains. The presence of microdomains has been confirmed by fluorescence and confocal microscopy using filipin and Laurdan as fluorescent probes. The experiments with Laurdan have shown that tonoplast microdomains are characterized by a high order compared to characteristics of the rest of the tonoplast. Thus, the presence of detergent-resistant lipid-protein microdomains in the tonoplast has been demonstrated.

This study determined the membrane fluidity of clostridial endospores during treatment with heat and pressure with nisin or reutericyclin. Heating (90°C) reduced laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) general polarization, corresponding to membrane fluidization. Pressure (200 MPa) stabilized membrane order. Reutericyclin and nisin exhibit divergent effects on heat- and pressure-induced spore inactivation and membrane fluidity.

Short-chain-length medium-chain-length polyhydroxyalkanoate (SCL-MCL PHA) copolymers are promising as bio-plastics with properties ranging from thermoplastics to elastomers. In this study, the hybrid pathway for the biosynthesis of SCL-MCL PHA copolymers was established in recombinant Escherichia coli by co-expression of β-ketothiolase (PhaARe) and NADPH-dependent acetoacetyl-CoA reductase (PhaBRe) from Ralstonia eutropha together with PHA synthases from R. eutropha (PhaCRe), Aeromonas hydrophila (PhaCAh), and Pseudomonas putida (PhaC2Pp) and with (R)-specific enoyl-CoA hydratases from P. putida (PhaJ1Pp and PhaJ4Pp), and A. hydrophila (PhaJAh). When glycerol supplemented with dodecanoate was used as primary carbon source, E. coli harboring various combinations of PhaABCJ produced SCL-MCL PHA copolymers of various monomer compositions varying from C4 to C10. In addition, polymer property analysis suggested that the copolymers produced from this recombinant source have thermal properties (lower glass transition and melting temperatures) superior to polyhydroxybutyrate homopolymer.

A method for measuring the fluidity of inner membranes of populations of endospores of Clostridium spp. with a fluorescent dye was developed. Cells of Clostridium beijerinckii ATCC 8260 and Clostridium sporogenes ATCC 7955 were allowed to sporulate in the presence of 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) on a soil-based media. Labeling of endospores with LAURDAN did not affect endospore viability. Removal of the outer membranes of endospores was done using a chemical treatment and confirmed using transmission electron microscopy (TEM). Two-photon confocal laser scanning microscopy (CLSM), and generalized polarization (GP) measurements were used to assess fluorescence of endospores. Lipid composition analysis of cells and endospores was done to determine whether differences in GP values are attributable to differences in membrane composition. Removal of the outer membranes of endospores did not significantly impact GP values. Decoated, labeled endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 exhibited GP values of 0.77±0.031 and 0.74±0.027 respectively. Differences in ratios of fatty acids between cells and endospores are unlikely to be responsible for high GP values observed in endospores. These GP values indicate high levels of lipid order and the exclusion of water from within inner membranes of endospores., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This work reports that cellulose laurate could be directly produced by mechanical activation-strengthened solid phase synthesis (MASPS) in a customized stirring mill with using bagasse pulp and lauric acid as materials in an environmentally friendly way. Cellulose laurates with different degree of substitution were obtained under different synthesis conditions without the use of organic co-reagents and solvents. The characterization results showed that cellulose laurates had great changes in surface morphologies and crystal structures compared with bagasse pulp because of the intense milling and introduction of laurate groups, but still retained the cellulose I crystalline form of the native cellulose. MASPS could be considered as a simple, efficient and green method for the production of long chain cellulose esters., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Bovine β-lactoglobulin (βlg) is able to recognize a wide variety of hydrophobic ligands. Although binding promiscuity is characteristic of highly hydrophobic interactions, the structural plasticity of the βlg binding cavity entrance seems to be crucial for the interaction with polar moieties of different ligands. On the other hand, thermodynamic studies have shown that βlg can associate to cognate ligands with distinctly different binding energetics, as in the case of the closely related molecules lauric acid (LA) and dodecyl sulfate (DS). In the recognition of LA, βlg shows a classical hydrophobic signature (entropically driven), whereas the interaction of βlg with DS exhibits a nonclassical hydrophobic signature (enthalpically driven). To gain insights into these opposed binding behaviors, MD simulations were carried out on βlg in apo-form and bound to DS or LA. Overall, the results suggested that the distinct energetic signatures of these ligands come from distinct optimizations of both hydrophilic and hydrophobic contacts with the protein., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.

Cytochrome P450 BM3 is a versatile enzyme, which holds great promise for applications in biocatalysis and biomedicine. We here report on the generation of a hybrid DNA-protein device based on the two subdomains of BM3, the reductase domain BMR and the porphyrin domain BMP. Both subdomains were fused genetically to the HaloTag protein, a self-labeling enzyme, allowing for the bioconjugation with chloroalkane-modified oligonucleotides. The subdomain-DNA-chimeras could be reassembled by complementary oligonucleotides, thus leading to reconstitution of the monooxygenase activity of BM3 holoenzyme, as demonstrated by conversion of the reporter substrate 12-pNCA. Arrangement of the two chimeras on a switchable DNA scaffold allowed one to control the distance between both subdomains, as indicated by the DNA-dependent activity of the holoenzyme. Furthermore, a switchable chimeric device was constructed, in which monooxygenase activity could be turned off by DNA strand displacement. This study demonstrates that P450 BM3 engineering and strategies of DNA nanotechnology can be merged to open up novel ways for the development of novel screening systems or responsive catalysts with potential applications in drug delivery.

The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. Previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apAAP) from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering. After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site, three variants with enhanced activity towards pNP-C12 were identified. Additionally, a combined mutant W474V/F488G/R526V/T560W was generated, which had the highest catalytic efficiency (k (cat)/K (m)) for pNP-C12, about 71-fold higher than the wild type. Its activity was further increased by solvent engineering, resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30% DMSO. The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation. It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center, which may facilitate binding and subsequent hydrolysis of bulky substrates. In conclusion, the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.

Prefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralleled cell morphology changes, indicating progressive cell compaction and death. Upon protein aggregation, we observed increased membrane water penetration as reported by Laurdan generalized polarization imaging., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)