Controlling Liquid–Liquid Phase Separation of Cold-Adapted Crystallin Proteins from the Antarctic Toothfish

Liquid–liquid phase separation (LLPS) of proteins is important to a variety of biological processes both functional and deleterious, including the formation of membraneless organelles, molecular condensations that sequester or release molecules in response to stimuli, and the early stages of disease-related protein aggregation. In the protein-rich, crowded environment of the eye lens, LLPS manifests as cold cataract. We characterize the LLPS behavior of six structural γ-crystallins from the eye lens of the Antarctic toothfish Dissostichus mawsoni, whose intact lenses resist cold cataract in subzero waters. Phase separation of these proteins is not strongly correlated with thermal stability, aggregation propensity, or cross-species chaperone protection from heat denaturation. Instead, LLPS is driven by protein–protein interactions involving charged residues. The critical temperature of the phase transition can be tuned over a wide temperature range by selective substitution of surface residues, suggesting general principles for controlling this phenomenon, even in compactly folded proteins.     

Purification of Xylitol from Fermented Hemicellulosic Hydrolyzate Using Liquid–Liquid Extraction and Precipitation Techniques

Xylitol was produced by Candida guilliermondii by fermentation of sugarcane bagasse hemicellulosic hydrolysate. Undesirable impurities were extracted from the broth using either ethyl acetate, chloroform or dichloromethane. The best results on clarification of the broth without xylitol loss were obtained with ethyl acetate. When ethanol, acetone or tetrahydrofuran were used for precipitation of impurities, only tetrahydrofuran clarified the fermented broth, but a high xylitol loss (~30%) was observed.     

Destruction of Enteric Bacteria in Liquid Egg with β-Propiolactone

Liquid whole egg or egg white, inoculated with Escherichia coli 1485, Salmonella senftenberg ATCC 8400, or Salmonella typhimurium 84-I, was treated with concentrations of β-propiolactone ranging from 0.05 to 0.3%. Egg white containing 1 × 10³ to 1 × 10⁶ cells of E. coli 1485 per ml was sterilized in 1 hr at 27 C by lactone concentrations of 0.2 and 0.3%. Egg white containing 1 × 10⁵ cells of S. senftenberg ATCC 8400 per ml was sterilized in 12 hr at 10 C by 0.1% lactone and in 2 to 3 hr by 0.3% lactone at the same temperature.

Liquid whole egg inoculated with 1 × 10⁵ cells of either species of Salmonella was sterilized in 4 to 5 hr at 10 C with 0.2% lactone or in 2 to 3 hr by 0.3% lactone at this temperature. A mild heat treatment of either 15 min at 37 C or 1 min at 55 C markedly shortened the exposure times required for sterilization by β-propiolactone at 10 C.

After disinfection was complete, the lactone-treated liquid whole egg was reinoculated with low cell numbers of either species of Salmonella to determine the presence of residual lactone or toxic products. Liquid whole egg treated with 0.2% lactone would support the growth of salmonellae after 13 to 14 hr at 10 C. A heat treatment of 45 min at 37 C or 10 min at 55 C immediately after addition of 0.2% lactone allowed growth of the salmonellae in the lactone-treated liquid whole egg. No evidence of residual toxicity from the lactone treatment was found.

The amount of lactone needed to prevent the outgrowth of low cell numbers of either strain of Salmonella in liquid whole egg was quantitated. Liquid whole egg containing 0.06 to 0.07% lactone would not support salmonellae growth from inocula of 1 to 10 cells per ml of egg. Lactone concentrations above 0.08% prevented outgrowth of salmonellae inocula of 10 to 200 cells per ml of liquid whole egg.

     

Combined Liquid Chromatography–Tandem Mass Spectrometry Analysis of Progesterone Metabolites

Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography—tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5α-pregnane-3-one and (20S)-5α-pregnane-3α,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing.     

Surface Plasmon Enhancement at a Liquid–Metal–Liquid Interface

Herein, we report the first experimental demonstration of surface plasmon enhancement at a liquid–metal–liquid interface using a pseudo-Kretschmann geometry. Pumping gold nanoparticle clusters at the interface of a p-xylene–water mixture, we were able to measure a fluorescence enhancement of three orders of magnitude in Rose Bengal at an excitation wavelength of 532 nm. The observed increase is due to the local electric field enhancement and the reduction of the fluorescence lifetime of dye molecules in the close vicinity of the metal surface. Theoretical modeling using the T-matrix method of the electric field intensity enhancement of emulated surfaces supports the experimental results. This new approach will open a new road for the study of dynamic systems using plasmonics.     

Liquid chromatography–mass spectrometry-based metabolomics for authenticity assessment of fruit juices

High performance liquid chromatography?Cmass spectrometry (HPLC?CMS) technique, employing a hybrid triple quadrupole/linear ion trap (QqQ/LIT) mass analyzer, was used for comprehensive metabolomic fingerprinting of several fruit juices types, prepared from expensive (orange) or relatively low-priced (apple, grapefruit) fruits. Following the automated data mining and pre-treatment step, the suitability of the multivariate HPLC?CMS metabolomic data for authentication, i.e., classification of fruit juice and adulteration detection, was assessed with the use of advanced chemometric tools (principal component analysis, PCA, and linear discrimination analysis, LDA). The LDA classification model, constructed and validated employing a highly variable samples set, was able to reliably detect 15% addition of apple or grapefruit juice to orange juice. In the final stage of this study, high performance liquid chromatography?Cquadrupole?Cquadrupole-time-of-flight mass spectrometry (HPLC?CQqTOFMS) measurements were performed in order to obtain data for identification of pre-selected marker compounds using elemental formula calculation and online databases search.     

Liquid–vapour interface varying the softness and range of the interaction potential

Molecular dynamics simulations in a canonical ensemble were carried out for simple fluids. The inter-particles interaction law is described by the Morse function plus a repulsive term. This kind of combination allows to tune the repulsive term of the interaction function by fitting the range of the attractive well and vice versa. As a relevant result, we show that for an inhomogeneous system the particle softness affects the vapour pressure, the surface tension and also the equilibrium densities of a simple fluid. Lower numerical values for these same properties were obtained by using a more repulsive interaction potential. The differences among these same interfacial properties are bigger when the range of the attractive interaction is longer. The surface tension written in terms of the corresponding critical parameters, such as scaled surface tension, was plotted for different softness degrees. And from this comparison, a unique master curve was not found.     

Angularly Resolved Density Distributions–A Starting Point for Analysis of Liquid Structure

Abstract

We propose that the angular unfolding of atomic density distributions exposes some main features of liquid structure. Examples are the mass and the angular location of major maxima. Such structural features constitute a useful starting point for the analysis of liquid structure. To demonstrate this we have analyzed a molecular dynamics trajectory of an equimolar water-acetonitrile mixture. A new method to characterize the extrema of density distributions is used for the analysis. Using this method we draw some conclusions about different types of hydrogen bonds, their lifetimes, and their associated transition probabilities. We also draw some conclusions about recurrent molecular pair configurations.     

Comparative Evaluation of Three Supplements for Helicobacter pylori Growth in Liquid Culture

Helicobacter pylori, a microaerophilic fastidious bacterium, has been cultured on various plating and broth media since its discovery. Although the agar media can be sufficient for the identification, typing, and antibiotic resistance studies, no secretory antigen of H. pylori can be evaluated in such media. Thus, satisfactory growth of H. pylori in liquid culture which is needed for analysis of secretory proteins without the presence of interfering agents is in demand. We assessed the impact of β-cyclodextrin, Fetal Bovine Serum (FBS), and charcoal as supplements for H. pylori growth. Furthermore, we aimed to identify the most favorable supplement that supports the secretion of the dominant secretory protein, vacuolating cytotoxin (VacA). Five clinical strains were cultured on broth media and the growth, viability, morphology, and protein content of each strain were determined. Our results revealed that β-cyclodextrin supports the growth rate, viability, and cell lysate protein content to the extent similar to FBS. Application of β-cyclodextrin is found to postpone spiral to coccoid conversion up to 72 h of incubation. Although FBS supports a higher VacA protein content, presence of interfering macromolecules in FBS questions its utility particularly for purposes of studying extra cellular proteins such as VacA. This study recommends further application of β-cyclodextrin as a culture supplement with the potential capacity in neutralizing toxic compounds and flourishing the secretion of H. pylori proteins without addition of interfering proteins.     

Determination of Concentration of Methotrexate Enantiomers in Intracellular and Extracellular Fluids of HepG2 Cells by Liquid Chromatography–Tandem Mass Spectrometry

A rapid and sensitive liquid chromatography–tandem mass spectrometry assay (LC–MS/MS) with electrospray ionization was developed and validated for the quantitative determination of the concentration of methotrexate (MTX) enantiomers in intracellular and extracellular fluids of HepG₂ cells. The analytes were extracted from homogenates using organic solvent to precipitate proteins. The extracted samples were analyzed by LC–MS/MS, operating in multiple reactions monitoring (MRM) mode. The condition of HPLC included the following: Gemini column (3 μm, 3.0 × 75 mm) with chromatographic column was used, and the mobile phase consisting of gradient elution utilized 0.1 % formic acid as solvent A and acetonitrile as solvent B at a flow rate of 0.4 mL min^?1. The gradient was as follows: 0–7.0 min 10–90 % B, 7.0–10 min 90 % B followed by 3 min. The column temperature was maintained at 40 °C. The condition of MS included using electrospray ionization source; MRM mode with the transitions of m/z 455.2 → m/z 308.1 was used to quantify MTX enantiomers. The linear calibration curve was obtained in the concentration range of 10.0 to 10,000 ng mL^?1 for MTX enantiomers in intracellular and extracellular fluids. The inter- and intraday precision was less than 15 %. The mean recovery of (+)-MTX and (?)-MTX in the extracellular fluid of HepG₂ cells were 95.30 and 96.53 %, respectively, and the mean recovery of (+)-MTX and (?)-MTX in the intracellular fluid of HepG2 cells were 93.53 and 94.12 %, respectively. This method was successfully used to detect the concentration of MTX enantiomers in the intracellular and extracellular fluids of HepG₂ cells and that the concentration of (+)-MTX in intracellular fluid was twice higher than the concentration of (?)-MTX in intracellular fluid. The inhibitory effect of (+)-MTX and (?)-MTX was (+)-MTX > (?)-MTX. It is a simple, precise method that can effectively explain the difference in pharamocological effect of MTX enantiomers in vitro.     

Application of a Novel Oscillatory Flow Micro-bioreactor to the Production of γ-decalactone in a Two Immiscible Liquid Phase Medium

A novel micro-bioreactor based on the oscillatory flow technology was applied to the scale-down of the biotechnological production of γ-decalactone. A decrease up to 50% of the time required to obtain the maximum concentration of the compound was observed, when compared with other scaled-down platforms (stirred tank bioreactor or shake flask). A three-fold increase in γ-decalactone productivity was obtained by increasing oscillatory mixing intensity from Re_o ~482 to Re_o ~1447. This was presumably related to the effective contribution of the reactor geometry to enhanced mass transfer rates between the two immiscible liquid phases involved in the process by increasing the interfacial area. Revisions requested 11 October 2005; Revisions received 4 January 2006     

Study of Caprine β-casein using Reversed-phase High-performance Liquid Chromatography and Mass Spectroscopy: Identification of a New Genetic Variant

The genotypes and the main phosphorylation levels of β-casein of goat milk were studied using RP-HPLC/ESI-MS. A new variant of caprine β-casein named E has been characterized using RP-HPLC/ESI-MS, MALDI-MS and NanoESI MS/MS methods. Its sequence differed from that of variant A in the mono amino acid substitution D47 → Y47, which resulted in a 48 Da experimental mass difference between them. The calculated molecular mass of the new variant E 6 P was estimated as 23,869 Da. Its phosphorylation pattern was similar to that of variant A, the most abundant types being those with 5 and 6 P in similar quantities.     

Liquid chromatography–fluorescence and liquid chromatography–mass spectrometry detection of tryptophan degradation products of a recombinant monoclonal antibody

Light exposure is one of several conditions used to study the degradation pathways of recombinant monoclonal antibodies. Tryptophan is of particular interest among the 20 amino acids because it is the most photosensitive. Tryptophan degradation forms several products, including an even stronger photosensitizer and several reactive oxygen species. The current study reports a specific peptide mapping procedure to monitor tryptophan degradation. Instead of monitoring peptides using UV 214 nm, fluorescence detection with an excitation wavelength of 295 nm and an emission wavelength of 350 nm was used to enable specific detection of tryptophan-containing peptides. Peaks that decreased in area over time are likely to contain susceptible tryptophan residues. This observation can allow further liquid chromatography–mass spectrometry (LC–MS) analysis to focus only on those peaks to confirm tryptophan degradation products. After confirmation of tryptophan degradation, susceptibility of tryptophan residues can be compared based on the peak area decrease.     

Liquid chromatography–tandem mass spectrometry for the determination of low-molecular mass aldehydes in human urine

A simple and sensitive method is proposed for the determination of seven low-molecular mass aldehydes in human urine samples using liquid chromatography with tandem mass spectrometric detection. Urine samples diluted twofold with 0.3 M hydrochloric acid are aspirated into a LiChrolut EN solid-phase extraction column impregnated with 2,4-dinitrophenylhydrazine for cleanup, derivatization and preconcentration of the aldehydes. After elution of the hydrazones with acetonitrile, an aliquot is injected directly into the chromatograph. Identification and quantification of aldehydes was performed with electrospray in negative ion mode by selected reaction monitoring. By using synthetic urine samples, linearity is established over the concentration range 0.1–30 μg/l and limits of detection from 15 to 65 ng/l. The intra- and inter-day precision (RSD, %) of the aldehydes ranged from 2.9% to 6.4% and 3.6% to 9.3%, respectively, and specific uncertainties were ca. 5.0 ± 0.3 ng for all aldehydes. Average recoveries performed on two levels by enriching synthetic urine samples ranged between 92% and 100%. The method was also validated in terms of study sample stability including long-term and short-term analyte stability, freeze–thaw and extract stability. In summary, the method proposed surpasses other recent chromatographic alternatives in terms of the limit of detection and sample requirements for analysis.     

Taurine-reduction Powers Rapid Growth of Bilophila wadsworthia: A Liquid Minimal-salts Medium for Clinical Research?

Clinical isolates of Bilophila wadsworthia grew rapidly (1–3 days) in the liquid taurine-minimal-salts medium developed for B. wadsworthia RZATAU, whereas growth on Bacteroides Bile Esculin Agar takes up to 1 week. Though rapid growth of B. wadsworthia was achieved, and no other pure cultures grew, the medium was not selective for the organism in human faeces or in intra-abdominal specimens. We hope, however, that our understanding of the physiological and biochemical characteristics of the organism supplies a tool for further research.