Complete Characterization of the O-Antigen from the LPS of Aeromonas bivalvium

Aeromonas species are found in the aquatic environment, drinking water, bottled mineral water, and different types of foods, such as meat, fish, seafood, or vegetables. Some of these species are primary or opportunistic pathogens for invertebrates and vertebrates, including humans. Among the pathogenic factors associated with these species, there are the lipopolysaccharides (LPSs). LPSs are the major components of the external leaflet of Gram-negative bacterial outer membrane. LPS is a glycoconjugate, generally composed of three portions: lipid A, core oligosaccharide, and O-specific polysaccharide or O-antigen. The latter, which may be present (smooth LPS) or not (rough LPS), is the most exposed part of the LPS and is involved in the pathogenicity by protecting infecting bacteria from serum complement killing and phagocytosis. The O-antigen is a polymer of repeating oligosaccharide units with high structural variability, particularly the terminal sugar, that confers the immunological specificity to the O-antigen. In this study, we established the structure of the O-chain repeating unit of the LPS from Aeromonas bivalvium strain 868 E^T (=CECT 7113^T = LMG 23376^T), a mesophilic bacterium isolated from cockles (Cardium sp.) and obtained from a retail market in Barcelona (Spain), whose biosynthesis core LPS cluster does not contain the waaE gene as most of Aeromonas species. After mild acid hydrolysis, the lipid A was removed by centrifugation and the obtained polysaccharide was fully characterized by chemical analysis and NMR spectroscopy. The polymer consists of a heptasaccharide repeating unit containing D-GalNAc, L-Rha, D-GlcNAc, and D-FucNAc residues.     

Structure of the O-Antigen and the Lipid A from the Lipopolysaccharide of Fusobacterium nucleatum ATCC 51191

Fusobacterium nucleatum is a common member of the oral microbiota. However, this symbiont has been found to play an active role in disease development. As a Gram-negative bacterium, F. nucleatum has a protective outer membrane layer whose external leaflet is mainly composed of lipopolysaccharides (LPSs). LPSs play a crucial role in the interaction between bacteria and the host immune system. Here, we characterised the structure of the O-antigen and lipid A from F. nucleatum ssp. animalis ATCC 51191 by using a combination of GC-MS, MALDI and NMR techniques. The results revealed a novel repeat of the O-antigen structure of the LPS, [→4)-β-d -GlcpNAcA-(1→4)-β-d -GlcpNAc3NAlaA-(1→3)-α-d -FucpNAc4NR-(1→], (R=acetylated 60 %), and a bis-phosphorylated hexa-acylated lipid A moiety. Taken together these data showed that F. nucleatum ATCC 51191 has a distinct LPS which might differentially influence recognition by immune cells.     

1H and 13C NMR studies of melon and head blubber of the striped dolphin (Stenella coeruleoalba)

Intact portions of melon, the echolocation organ of the striped dolphin (Stenella coeruleoalba), and the corresponding raw oils were analyzed by means of one- and two dimensional ¹H and ¹³C NMR techniques. For comparative purposes the tissue and the raw oil of head blubber were also examined. Complete assignments of the spectra were obtained. Furthermore, dynamics of the lipid components was investigated by means of ¹³C NMR spin lattice relaxation time (T ₁). Analysis of the data revealed that lipid molecules in the tissue compartments experience a liquid-like microenvironment and that T ₁ values depend on the lipid composition and/or organization in the intact tissue framework. In particular, a dependence of the T ₁ values on the wax esters content in melon intact tissues was found. A possible correlation between dynamic parameters and sound propagation properties has been hypothesized.     

The Lipid A Structure from the Marine Sponge Symbiont Endozoicomonas sp. HEX 311

Endozoicomonas sp. HEX311 is a Gram-negative bacterium known to establish a commensal interaction with the marine demosponge Suberites domuncula. The molecular bases of the sponge–microbe interaction events are still poorly defined. Nevertheless, it has been proved that S. domuncula possesses an innate immune system with similarities to the mammalian one and is able to recognize the main component of the Gram-negative bacteria cell wall: the lipopolysaccharide. Whether this recognition occurs in a structure-dependent manner, which is typical for mammalian immune system receptors, is still under investigation. Herein, we report the Endozoicomonas sp. HEX311 lipid A structure obtained by a combination of data attained from chemical, MALDI MS, and MS² approaches. The lipid A is a complex family of species decorated by pyrophosphate and phosphate units and carrying (R)-3-hydroxydodecanoic acid, (R)-3-hydroxytetradecanonic acid, iso-2-hydroxyundecanoic acid, iso-(R)-3-hydroxyundecanoic acid, and iso-nonanoic acid as acyl chains.     

Simple dilatometer for polymer density and thermal expansivity measurements

A dilatometer is described to study the temperature dependence of density (ρ) of solid and semiliquid polymers and the following linear relations have been established. Atactic poly(vinylisobutyl ether) (25–90°C): ρ = 0.9166 ? 7.15 × 10^?4 × T. Isotactic poly(vinylisobutyl ether) (25–70°C): ρ = 0.9184 ? 7.13 × 10^?4 × T. Poly(n-butyl methacrylate) (90–150°C): ρ = 1.0622 ? 8.41 × 10^?4 × T. Poly(dimethyl siloxane) (30–51°C, using Lipkins pycnometer): ρ = 0.9846 ? 8.81 × 10^?4 × T; where ρ is in g.cm^?3, temperature T is in Celsius, and the linearity correlation coefficient r is better than 0.9998. Their volume–temperature plots are also linear. As the plots of polyn-butyl methacrylate curved slightly near its glass transition (20°C), the quadratic equation ρ = 1.0402 ? 4.79 × 10^?4 × T ? 1.46 × 10^?6 × T² (standard deviation = 1.57 × 10^?3) has been suggested for the entire range of 30–150°C scrutinized in this study. The data have been utilized to derive thermal expansivity and some equation-of-state parameters of the polymers at the reference temperature (ca. 20°C).     

Thermophiles as Potential Source of Novel Endotoxin Antagonists: the Full Structure and Bioactivity of theLipo‐oligosaccharide from Thermomonas hydrothermalis

Thermomonas hydrothermalis is a Gram‐negative thermophilic bacterium that is able to live at 50 °C. This ability is attributed to chemical modifications, involving those to bacterial cell‐wall components, such as proteins and (glyco)lipids. As the main component of the outer membrane of Gram‐negative bacteria, lipopolysaccharides (LPSs) are exposed to the environment, thus they can undergo structural chemical changes to allow thermophilic bacteria to live at their optimal growth temperature. Furthermore, as one of the major target of the eukaryotic innate immune system, LPS elicits host immune response in a structure‐dependent mode; thus the uncommon chemical features of thermophilic bacterial LPSs might exert a different biological action on the innate immune system—an antagonistic effect, as shown in studies of LPS structure–activity relationship in the ongoing research into antagonist LPS candidates. Here, we report the complete structural and biological activity analysis of the lipo‐oligosaccharide isolated from Thermomonas hydrothermalis, achieved by a multidisciplinary approach (chemical analysis, NMR, MALDI MS and cellular immunology). We demonstrate a tricky and interesting structure combined with a very interesting effect on human innate immunity.     

Xanthomonas citri pv. citri Pathotypes: LPS Structure and Function as Microbe‐Associated Molecular Patterns

Xanthomonas citri pv. citri is the pathogen responsible for Asiatic citrus canker, one of the most serious citrus diseases worldwide. The lipopolysaccharide (LPS) molecule has been demonstrated to be involved in X. citri pv. citri virulence. Despite enormous progress in investigations of the molecular mechanisms for bacterial pathogenicity, determination of the detailed LPS structure–activity relationship is limited, as the current knowledge is mainly based on structural determination of one X. citri pv. citri strain. As X. citri pv. citri strains are distinguished into three main pathogenicity groups, we characterized the full structure of the LPS from two pathotypes that differ in their host‐range specificity. This revealed an intriguing difference in LPS O‐chain structure. We also tested the LPSs and isolated lipid A moieties for their ability to act as microbe‐associated molecular patterns in Arabidopsis thaliana. Both LPS/lipid As induced ROS accumulation, but no difference was observed between the two pathotypes.     

The Multifaceted Inhibitory Effects of an Alkylquinolone on the Diatom Phaeodactylum tricornutum

The mechanisms underlying interactions between diatoms and bacteria are crucial to understand diatom behaviour and proliferation, and can result in far-reaching ecological consequences. Recently, 2-alkyl-4-quinolones have been isolated from marine bacteria, both of which (the bacterium and isolated chemical) inhibited growth of microalgae, suggesting these compounds could mediate diatom–bacteria interactions. The effects of several quinolones on three diatom species have been investigated. The growth of all three was inhibited, with half-maximal inhibitory concentrations reaching the sub-micromolar range. By using multiple techniques, dual inhibition mechanisms were uncovered for 2-heptyl-4-quinolone (HHQ) in Phaeodactylum tricornutum. Firstly, photosynthetic electron transport was obstructed, primarily through inhibition of the cytochrome b₆f complex. Secondly, respiration was inhibited, leading to repression of ATP supply to plastids from mitochondria through organelle energy coupling. These data clearly show how HHQ could modulate diatom proliferation in marine environments.     

The acylation and phosphorylation pattern of lipid A from Xanthomonas campestris strongly influence its ability to trigger the innate immune response in Arabidopsis

Lipopolysaccharides (LPSs) are major components of the cell surface of Gram-negative bacteria. LPSs comprise a hydrophilic heteropolysaccharide (formed by the core oligosaccharide and the O-specific polysaccharide) that is covalently linked to the glycolipid moiety lipid A, which anchors these macromolecules to the external membrane. LPSs are one of a group of molecules called pathogen-associated molecular patterns (PAMPs) that are indispensable for bacterial growth and viability, and act to trigger innate defense responses in eukaryotes. We have previously shown that LPS from the plant pathogen Xanthomonas campestris pv. campestris (Xcc) can elicit defense responses in the model plant Arabidopsis thaliana. Here we have extended these studies by analysis of the structure and biological activity of LPS from a nonpathogenic Xcc mutant, strain 8530. We show that this Xcc strain is defective in core completion and introduces significant modification in the lipid A region, which involves the degree of acylation and nonstoichiometric substitution of the phosphate groups with phosphoethanolamine. Lipid A that was isolated from Xcc strain 8530 did not have the ability to induce the defense-related gene PR1 in Arabidopsis, or to prevent the hypersensitive response (HR) that is caused by avirulent bacteria as the lipid A from the wild-type could. This suggests that Xcc has the capacity to modify the structure of the lipid A to reduce its activity as a PAMP. We speculate that such effects might occur in wild-type bacteria that are exposed to stresses such as those that might be encountered during plant colonization and disease.     

Solid-state NMR characterization of motion in flexible polyurethane foams

High-resolution solid-state ¹³C-NMR has been used to study the phase separation and molecular motion in two series of polyurethane foams. These two series differ by one possessing the additive of lithium chloride, LiCl. NMR relaxation times can map the motion throughout the polymer molecule and detect changes in that motion arising from either microseparation or phase mixing between the different segments. There are only slight changes in the soft segment T_1p(¹³C) values as well as an increase in the hard segment T_1p(¹H) values with increase in the hard segment content for the foams studied. The T_1p(¹H) and T_1p(¹³C) values do indicate that the phase separation of the hard and soft segments is similar for all foams. A decrease in the T_1p(¹H) and T_1p(¹³C) values with increasing LiCl content indicates that the motion of the soft segments is restricted more by the hard segments. This is explained by more phase mixing in the foams containing the LiCl additive. © 1994 John Wiley & Sons, Inc.     

Phase transition on the Si(001) clean surface prepared in UHV MBE chamber: a study by high-resolution STM and <Emphasis Type="Italic">in situ</Emphasis> RHEED

The Si(001) surface deoxidized by short annealing at T ~ 925°C in the ultrahigh vacuum molecuar beam epitaxy chamber has been in situ investigated using high-resolution scanning tunneling microscopy (STM)and redegreesected high-energy electron diffraction (RHEED. RHEED patterns corresponding to (2 × 1) and (4 × 4) structures were observed during sample treatment. The (4 × 4) reconstruction arose at T ≲ 600°C after annealing. The reconstruction was observed to be reversible: the (4 × 4) structure turned into the (2 × 1) one at T ≳ 600°C, the (4 × 4) structure appeared again at recurring cooling. The c (8 × 8) reconstruction was revealed by STM at room temperature on the same samples. A fraction of the surface area covered by the c (8 × 8) structure decreased, as the sample cooling rate was reduced. The (2 × 1) structure was observed on the surface free of the c (8 × 8) one. The c (8 × 8) structure has been evidenced to manifest itself as the (4 × 4) one in the RHEED patterns. A model of the c (8 × 8) structure formation has been built on the basis of the STM data. Origin of the high-order structure on the Si(001) surface and its connection with the epinucleation phenomenon are discussed.     

Roles of the Conserved Amino Acid Residues in Reduced Human Defensin 5: Cysteine and Arginine Are Indispensable for Its Antibacterial Action and LPS Neutralization

Antimicrobial peptides (AMPs) that are able to neutralize toxins are promising antibiotics. In this study we investigated the role of structurally conserved amino acids in reduced human defensin 5 (HD5_RED), which is an endogenous peptide with antibacterial action and the ability to neutralize lipopolysaccharide (LPS). Cys residues and high Arg content, rather than Gly¹⁸ and Arg⁶–Glu¹⁴, were found to be indispensable for HD5_RED binding to lipid A, for penetrating the bacterial outer and inner membranes, and for eliminating bacteria. Otherwise, all the conserved sites were requisite for HD5_RED to block the interaction between LPS and LPS-binding protein and to suppress the TLR4–NF-κB signaling pathway initiated by LPS. Accordingly, we designed the acetamidomethylated ^AcmCys-E21R-HD5_RED, which was much more potent than HD5_RED at eliminating bacteria and which can neutralize LPS. ^AcmCys-E21R-HD5_RED was also found to exhibit a synergistic effect with ciprofloxacin in killing multidrug-resistant Acinetobacter baumannii. The results of this study, in which multifunctional AMPs were designed based on structure–activity research, may help in the development of more peptide antibiotics.     

The Liquidus Temperature of Nuclear Waste Glasses: An International Round-Robin Study

Eight institutions from four countries participated in a round-robin study to determine the precision and bias of a liquidus temperature (T_L) procedure for waste glasses being adopted by ASTM International as ASTM C 1720-11. The participants of the round-robin study were asked to measure three different glasses with one or a combination of the following T_L measurement methods: a gradient temperature (GT) method, a uniform temperature (UT) method, and/or a crystal fraction extrapolation (CF) method. The T_L values reported by different institutions are generally consistent. The precision of T_L measurements with each method was evaluated and is presented herein. The round-robin glasses were all previously studied at Pacific Northwest National Laboratory and included ARG-1 (Glass A), Zr-9 (Glass B), and AmCm2-19 (Glass C), with measured T_L values spanning the temperature range of 960–1240°C. A precision (i.e., standard deviation) for T_L has been obtained from the data, even though the data were not acquired for all three glasses using all three methods from each participating organization. Also, the article provides a brief overview and the importance of the T_L measurement.     

Marine Phospholipids from Fishery By-Products Attenuate Atherosclerosis

In this study, the anti-atherosclerotic properties of three marine phospholipids (MPLs) extracts from fishery by-products including codfish roe, squid gonad, and shrimp head are verified. Their effects on key factors involved in atherosclerosis are examined and compared to explore whether the differences in their constitutions lead to the differences in the function. All three MPLs dampen oxidation of low- density lipoproteins (LDL) in vitro. Treating RAW264.7 macrophages and HUVECs endothelial cells with each MPLs ranging 10–100 µg mL⁻¹ does not decrease cell viability, yet ox-LDL caused cytotoxicity of both cells are alleviated by 50 or 100 µg mL⁻¹ MPLs treatment. In addition, the three MPLs reduce ox-LDL induced macrophage foam-like transition, mainly through inhibition of lipid uptake. Of the three MPLs, the one from squid gonad exhibits the best effect. On the other hand, all three MPLs modulate inflammatory responses, equally, by inhibiting the adhesion of monocytes to endothelial cells, and decreasing secretion of pro-inflammatory cytokines IL-6 and MCP-1. Using a high-cholesterol diet induced zebrafish model, it is found that all three MPLs, especially the one from squid gonad, alleviates cholesterol accumulation in early plaques, and decreases total cholesterol as well as lipid peroxide in vivo. Practical Applications: As a way of making the best of the increasingly scarce marine resources, valuable lipid components can be recovered from by-products and wastes from the fishery industry. Here, we tested the anti-atherosclerotic effects and the mechanisms of three MPLs extracted from codfish roe, squid gonad, and shrimp head. Our study provides further evidence that marine phospholipids extracted from fishery by-products could protect against atherosclerosis, and helps to elucidate the structure-function relationship of MPLs.     

Cure modeling and monitoring of epoxy/amine resin systems. II. Network formation and chemoviscosity modeling

The glass transition temperature (T_g) advancement and the chemoviscosity development under isothermal conditions have been investigated for four epoxy/amine systems, including commercial RTM6 and F934 resins. Differential scanning calorimetry (DSC) was the thermoanalytical technique used to determine the T_g advancement and rheometry the technique for the determination of the chemoviscosity profiles of these resin systems. The complex cure kinetics were correlated to the T_g advancement via an one‐to‐one relationship using Di Benedetto's formula. It was revealed that the three‐dimensional network formation follows a single activated mechanism independent of whether the cure kinetics follow a single or several activation mechanisms. The viscosity profiles showed the typical characteristics of epoxy/amine cure. A modified version of the Williams‐Landel‐Ferry equation (WLF) was adequate to model the viscosity profiles of all the resin systems, in the temperature range 130 to 170°C, with a very good degree of accuracy. The parameters of the WLF equation were found to vary in a systematic manner with cure temperature. Further correlation between T_g and viscosity showed that gelation, defined as the point where viscosity reaches 10⁴ Pas, occurs at a unique T_g value for each resin system, which is independent of the cure conditions. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 2178–2188, 2000     

Analysis of Pseudomonas aeruginosa PAO1 Lipid A Changes During the Interaction with Model Organism,Caenorhabditis elegans

Lipopolysaccharide (LPS) is the main surface constituent of Gram-negative bacteria. Lipid A, the hydrophobic moiety, outer monolayer of the outer cell membrane forms the major component of LPS. Immunogenic Lipid A is recognized by the innate immune system through the TLR 4/MD-2 complex. Pseudomonas aeruginosa PAO1, a Gram-negative bacterium is known to cause nosocomial infection and known for its adaptation to adverse environmental conditions. Pseudomonas aeruginosa can infect a broad host spectrum including Caenorhabditis elegans, a simple free living soil nematode. Here, we reveal that PAO1 modifies its Lipid A during the host interaction with C. elegans. The penta-acylated form of Lipid A was identified by using matrix assisted laser desorption ionization–time of flight analysis and the β-(1,6)-linked disaccharide of glucosamine with phosphate groups, 2 and 2′ amide linked fatty acid chain and 3 and 3′ ester linked fatty acids were investigated for the modification using the non destructive ¹H NMR, spin–lattice (T ₁) relaxation measurement, differential scanning calorimetry. T ₁ relaxation measurements showed that the 2 and 2′ amide linked fatty acid chain, –CH in the glucosamine disaccharide of PAO1 lipid A, in an exposed host had a different spin lattice relaxation time compared to an unexposed host and the findings were reconfirmed using in vitro human corneal epithelial cells cell lines. Furthermore, scanning electron microscope and confocal laser scanning microscopy analysis revealed that the P. aeruginosa PAO1 biofilm formation was disturbed in the exposed host condition. The daf-12, daf-16, tol-1, pmk-1, ins-7 and ilys3 immune genes of C. elegans were examined with live bacterial and isolated lipid moiety infection and the expression was found to be highly specific. Overall, the present study revealed that PAO1 modified its 2 and 2′ amide linked fatty acid chain in the lipid A of PAO1 LPS during the exposed host condition.     

Thermal Decomposition of NTO: An Explanation of the High Activation Energy

Burning rate characteristics of the low‐sensitivity explosive 5‐nitro‐1,2,4‐triazol‐3‐one (NTO) have been investigated in the pressure interval of 0.1–40 MPa. The temperature distribution in the combustion wave of NTO has been measured at pressures of 0.4–2.1 MPa. Based on burning rate and thermocouple measurements, rate constants of NTO decomposition in the molten layer at 370–425 °C have been derived from a condensed‐phase combustion model (k=8.08⋅10¹³⋅exp(−19420/T) s⁻¹. NTO vapor pressure above the liquid (ln P=−9914.4/T+14.82) and solid phases (ln P=−12984.4/T+20.48) has been calculated. Decomposition rates of NTO at low temperatures have been defined more exactly and it has been shown that in the interval of 180–230 °C the decomposition of solid NTO is described by the following expression: k=2.9⋅10¹²⋅exp(−20680/T). Taking into account the vapor pressure data obtained, the decomposition of NTO in the gas phase at 240–250 °C has been studied. Decomposition rate constants in the gaseous phase have been found to be comparable with rate constants in the solid state. Therefore, a partial decomposition in the gas cannot substantially increase the total rate. High values of the activation energy for solid‐state decomposition of NTO are not likely to be connected with a sub‐melting effect, because decomposition occurs at temperatures well below the melting point. It has been suggested that the abnormally high activation energy in the interval of 230–270 °C is a consequence of peculiarities of the NTO transitional process rather than strong bonds in the molecule. In this area, the NTO molecule undergoes isomerization into the aci‐form, followed by C3‐N2 heterocyclic bond rupture. Both processes depend on temperature, resulting in an abnormally high value of the observed activation energy.     

A generalized equation for the prediction of melting temperatures of homopolymers and copolymers

A generalized equation was derived to calculate the melting temperatures of homopolymers and copolymers. The Gibbs‐Thomson equation for homopolymers and a modified application to copolymers were derived using the proposed equation. The melting temperature T_m⁰ in the Flory equation corresponds to the melting temperature T_m^C,∞ of copolymer crystals with stems of infinite length. Also, T_m^C,n*, the melting temperature for copolymer crystals with stems containing the maximum possible number of structural units, n*, should be used instead of T_m⁰ as the basis of supercooling in crystallization. The proposed equation shows good agreement with experimental data for α‐alkene‐ethylene homogeneous copolymers.     

Studies on the Biosynthesis of the Antibiotic Moenomycin A

Feeding experiments ( ¹³C and ¹⁵N‐labeled precursors) shed light on the biosynthetic origin of the chromophore (unit A of 1 ), the N‐acetyl groups, the 4‐C‐methyl group of the moenuronamide unit (part F of 1 ), the sugar units, and the lipid part (unit I of 1 ) of the antibiotic moenomycin A( 1 ). The lipid part is completely isoprenoid and is constructed via the non‐mevalonate pathway. The central C ₁₀ part originates from a precursor like geranyl or linalyl diphosphate and is formed by a route involving ring formation between C‐2 and C‐6 of the monoterpene unit, two successive rearrangements to give a 7‐membered ring intermediate and cleavage of the ring between C‐5 and C‐11 (moenocinol numbering).     

An investigation of 1H and 13C longitudinal relaxation and relaxation in the rotating frame in solid poly(N‐vinylcarbazole)

¹H and ¹³C longitudinal relaxation times (T₁) and relaxation times in the rotating frame (T_1ρ) have been measured for poly(N‐vinylcarbazole) in the solid state in air and nitrogen atmospheres in an attempt to elucidate molecular motions. In air, the T₁ relaxation of both ¹H and ¹³C was dominated by interaction with absorbed paramagnetic oxygen. In nitrogen, the ¹³C T₁ relaxation times were long (>300 s) and were averaged by ¹³C–¹³C spin diffusion. The ¹³C T_1ρ relaxation times showed an exponential dependence on the strength of the rotating ¹³C magnetic field and were thus controlled by spin–spin processes rather than spin–lattice processes. © 2001 Society of Chemical Industry