The Effect of Mutation in Lipopolysaccharide Biosynthesis on Bacterial Fitness.

This paper presents the genome sequence of a mutant strain ( 4351) and the effect of mutation in lipopolysaccharide biosynthesis on bacterial fitness. Lipopolysaccharides are the major component of the outer leaflet of the Gram-negative outer membrane. We report here a frameshift mutation of the gene in the genome of 4351.

The effect of pH adjusted electrolytes on capillary isoelectric focusing assessed by high-resolution dynamic computer simulation.

The effect of the composition of electrolytes on capillary IEF is assessed for systems with carrier ampholytes covering two pH units and with catholytes of decreased pH, anolytes of increased pH, and both electrode solutions with adjusted pH values. For electrolytes composed of formic acid as anolyte and ammonium hydroxide as catholyte, simulation is demonstrated to provide the expected IEF system in which analytes with pI values within the formed pH gradient are focused and become immobile. Addition of formic acid to the catholyte results in the formation of an isotachophoretic zone structure that migrates toward the cathode.

Capillary zone electrophoresis of proteins applying ionic liquids for dynamic coating and as background electrolyte component.

The use of ionic liquids in capillary electrophoresis, either as coating material or as components of the background electrolyte needs systematic standardization to set up optimal conditions. Excellent separation of the proteins was achieved using 1-ethyl-3-methylimidazolium tetrafluoroborate ([emim][BF]) or 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF]) ionic liquids using the properly made ionic-liquid-water binary mixtures for the experiments. The binary mixture has a distinctly stable and well perceptible low pH, which depends on the concentration of the ionic liquid, and on the preparation time of the mixture.

NACE-ESI-MS/MS method for separation and characterization of phosphorylation and acylation isomers of lipid A.

Lipid A represents a heterogeneous group of bacterial outer membrane phosphoglycolipids, which play a major role in the pathogenesis of Gram-negative sepsis. The number and position of phosphoryl and acyl groups in lipid A molecules are key structural determinants in their bioactivities. In this study, a NACE-ESI-MS/MS method was developed for the simultaneous analysis of lipid A isomers possessing a different degree of phosphorylation and acylation.

Microchip gel electrophoretic analysis of perchloric acid-soluble serum proteins in systemic inflammatory disorders.

Perchloric acid (PCA) precipitation is a well-known method for the separation of heavily glycosylated proteins and for reducing the masking effect of major serum proteins. The aim of this study is to characterize PCA-soluble serum proteins in healthy individuals and in patients with systemic inflammatory diseases, such as Crohn's disease and sepsis. A PCA precipitation protocol was prepared and adapted to the analytical methods.

Genome sequence of 4303.

Background: spp. are Gram-negative intracellular pathogenic bacteria belonging to the family and can cause bacterial dysentery, a severe diarrheal disease. The pathophysiological impact of the Gram-negative bacteria is highly related to the composition and structural variability of lipopolysaccharides, the major lipoid components of the outer membrane.

Fast determination of lactic, succinic, malic, tartaric, shikimic, and citric acids in red Vranec wines by CZE-ESI-QTOF-MS.

A fast and simple method with CZE coupled to ESI/QTOF-MS was optimized and validated for quantitative determination of organic acids (lactic acid, succinic acid, malic acid, tartaric acid, shikimic acid, and citric acid) in red wines. The BGE was ammonium acetate and the separation of the analytes was performed in a polybrene-coated capillary in the presence of EOF. The sample preparation included dilution and filtration of the wine.

Characterization of complex, heterogeneous lipid A samples using HPLC-MS/MS technique III. Positive-ion mode tandem mass spectrometry to reveal phosphorylation and acylation patterns of lipid A.

In this study, we report the detailed analysis of the fragmentation patterns of positively charged lipid A species based on their tandem mass spectra obtained under low-energy collision-induced dissociation conditions of an electrospray quadrupole time-of-flight mass spectrometer. The tandem mass spectrometry experiments were performed after the separation of the compounds with a reversed-phase high performance liquid chromatography method. We found that both, phosphorylated and nonphosphorylated lipid A molecules can be readily ionized in the positive-ion mode by adduct formation with triethylamine added to the eluent.

Advanced online mass spectrometry detection of proteins separated by capillary isoelectric focusing after sequential injection.

Capillary isoelectric focusing hyphenated with mass spectrometry detection, following the sequential injection of the carrier ampholytes and the sample zone, is highly efficient for the characterization of proteins. The main advantage of the sequential injection protocol is that ampholytes, with pH ranges, which are not supposed to cover the isoelectric points of the sample components, can be used for separation. The method then allows online mass spectrometry detection of separated analytes either in the absence (substances that have left the pH gradient) or in the presence of low-level ampholytes (substances that are migrating within the pH gradient).

Mass Spectrometry for Profiling LOS and Lipid A Structures from Whole-Cell Lysates: Directly from a Few Bacterial Colonies or from Liquid Broth Cultures.

Lipopolysaccharides (LPSs, endotoxins) are components of the outer cell membrane of most Gram-negative bacteria and can play an important role in a number of diseases of bacteria, including Gram-negative sepsis. The hydrophilic carbohydrate part of LPSs consists of a core oligosaccharide (in the case of an R-type LPS or lipooligosaccharide, LOS) linked to an O-polysaccharide chain (in the case of an S-type LPS), which is responsible for O-specific immunogenicity. The hydrophobic lipid A anchor is composed of a phosphorylated diglucosamine backbone to which varying numbers of ester- and amide-linked fatty acids are attached and this part of the LPSs is associated with endotoxicity.

Capillary Electrophoresis Chips for Fingerprinting Endotoxin Chemotypes and Subclasses.

Endotoxins (lipopolysaccharides, LPS; lipooligosaccharides, LOS) are components of the envelope of Gram-negative bacteria. These molecules, responsible for both advantageous and harmful biological activity of these microorganisms, are highly immunogenic and directly involved in numerous bacterial diseases in humans, such as Gram-negative sepsis. The characterization of endotoxins is of importance, since their physiological and pathophysiological effects depend on their chemical structure.

Characterization of complex, heterogeneous lipid A samples using HPLC-MS/MS technique II. Structural elucidation of non-phosphorylated lipid A by negative-ion mode tandem mass spectrometry.

Non-phosphorylated lipid A species confer reduced inflammatory potential for the bacteria. Knowledge on their chemical structure and presence in bacterial pathogens may contribute to the understanding of bacterial resistance and activation of the host innate immune system. In this study, we report the fragmentation pathways of negatively charged, non-phosphorylated lipid A species under low-energy collision-induced dissociation conditions of an electrospray ionization quadrupole time-of-flight instrument.

Availability and quality of illegitimate somatropin products obtained from the Internet.

Background Growth hormones are widely available on the Internet for those who want to enhance their physical performance and improve body satisfaction. Illegitimate websites market somatropin injections without medical prescription and encourage misuse. Customers potentially put their health at risk when purchasing parenteral medications online.

Characterization of complex, heterogeneous lipid A samples using HPLC-MS/MS technique I. Overall analysis with respect to acylation, phosphorylation and isobaric distribution.

We established a new reversed phase-high performance liquid chromatography method combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry for the simultaneous determination and structural characterization of different lipid A types in bacteria (Escherichia coli O111, Salmonella adelaide O35 and Proteus morganii O34) showing serological cross-reactivity. The complex lipid A mixtures (obtained by simple extraction and acid hydrolysis of the outer membrane lipopolysaccharides) were separated and detected without phosphate derivatization. Several previously unidentified ions were detected, which differed in the number and type of acyl chains and number of phosphate groups.

Modeling of formation and prevention of a pure water zone in capillary isoelectric focusing with narrow pH range carrier ampholytes.

This paper comprises a continuation of computer simulation studies dealing with carrier ampholyte based CIEF in presence of narrow pH gradients. With this technique, amphoteric sample components with pI values outside the pH gradient are migrating isotachophoretically toward the cathode or anode whereas components with pI values within the gradient become focused. In order to understand the processes occurring in presence of the electric field, the behavior of both carrier ampholytes and amphoteric sample components is investigated by computer modeling.

Investigation of retention mechanism of resorcinarene based cavitands by linear and nonlinear chromatography.

Cavitands are cavity-shaped cyclic oligomers and they can create host-guest interactions with various analytes, therefore they have applications in supramolecular chemistry, nanoscale reactions, chromatographic separations, drug encapsulation and delivery, biochemistry. The investigation of the chromatographic behavior of large molecules, such as resorcinarenes and cavitands is meager up to now. To understand the retention of resorcinarenes and cavitands in liquid chromatography, we studied their retention mechanism by the thermodynamic parameters calculated from the van't Hoff equation and by generation of an adsorption isotherm, which can describe the adsorption of the solute on the stationary phase surface.

Retention behavior of resorcinarene-based cavitands on C8 and C18 stationary phases.

The understanding of the retention behavior of large molecules is an area of interest in liquid chromatography. Resorcinarene-based cavitands are cavity-shaped cyclic oligomers that can create host-guest interactions. We have investigated the chromatographic behavior of two types of cyclic tetramers as analytes in high-performance liquid chromatography.

Structural background for serological cross-reactivity between bacteria of different enterobacterial serotypes.

The structure of the oligosaccharide repeating units of endotoxins from Gram-negative bacteria is characteristic for the different serogroups and serotypes of bacteria. Detailed examination of the cross-reactions of three enterobacterial serotypes, Proteus morganii O34, Escherichia coli O111, and Salmonella enterica sv. Adelaide O35, was performed using sensitive tests (ELISA, immunoblotting).

Analgesic topical capsaicinoid therapy increases somatostatin-like immunoreactivity in the human plasma.

The aim of the present study was to evaluate the therapeutic potential of local capsaicinoid (EMSPOMA(®) cream) treatment on chronic low back pain in patients with degenerative spine diseases and to investigate the possible mechanism of action of the therapy. The qualitative and quantitative analyses of capsaicinoids in EMSPOMA(®) cream were performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the clinical study 20 patients with degenerative spine diseases were involved in a self-controlled examination.

Phytochemical evaluation of Lythrum salicaria extracts and their effects on guinea-pig ileum.

n-Hexane, chloroform, ethyl acetate and 50% ethanol in water extracts prepared from the air-dried flowering parts of Lythrum salicaria L. were tested for in vitro pharmacological properties on Guinea-pig ileum, which is suitable for detecting a whole range of neuronal and smooth muscle effects. UHPLC-MS was used to evaluate polyphenol components of the extracts.

Antifungal activity of fused Mannich ketones triggers an oxidative stress response and is Cap1-dependent in Candida albicans.

We investigated the antifungal activity of fused Mannich ketone (FMK) congeners and two of their aminoalcohol derivatives. In particular, FMKs with five-membered saturated rings were shown to have minimum inhibitory concentration (MIC90s) ranging from 0.8 to 6 µg/mL toward C.

Application of chip-based flow cytometry for amphotericin B and fluconazole susceptibility testing on Candida strains.

Chip-based flow cytometry is a rather new method that offers an easy, fast opportunity for examination of yeasts, such as Candida cells. In our study cell-chip technology was tested with ATCC Candida strains to determine their viability and susceptibility against antifungal agents, amphotericin B and fluconazole. We found this technology to be suitable for the detection of Candida cells, for the differentiation between dead and living cells, and for the determination of amphotericin B and fluconazole susceptibility of different Candida strains (Bouquet et al.

High-resolution dynamic computer simulation analysis of the behavior of sample components with pI values outside the pH gradient established by carrier ampholyte CIEF.

The behavior of sample components whose pI values are outside the pH gradient established by 101 hypothetical biprotic carrier ampholytes covering a pH 6-8 range was investigated by computer simulation under constant current conditions with concomitant constant electroosmosis toward the cathode. Data obtained with the sample being applied between zones of carrier ampholytes and on the anodic side of the carrier ampholytes were studied and found to evolve into zone structures comprising three regions between anolyte and catholyte. The focusing region with the pH gradient is bracketed by two isotachopheretic zone structures comprising selected sample and carrier components as isotachophoretic zones.

Quantitative microfluidic analysis of S- and R-type endotoxin components with chip capillary electrophoresis.

A novel, fast, and sensitive ME method was developed to analyze and differentiate the smooth (S) and rough (R) type bacterial endotoxin components labeled covalently with a fluorescent dye. The quantitative analysis of purified lipopolysaccharides, or partially purified samples from whole-cell lysates becomes possible with this method. Two groups with three sub-groups in the first group of S-type lipopolysaccharides can be classified based on the electrophoretic profiles.