Mucin glycosylation and sulphation in airway epithelial cells is not influenced by cystic fibrosis transmembrane conductance regulator expression

Abnormalities in mucus properties and clearance make a major contribution to the pathology of cystic fibrosis (CF). Our aim was to test the hypothesis that the defects in CF mucus are a direct result of mutations in the CF transmembrane conductance regulator (CFTR) protein. We evaluated a single mucin molecule MUC1F/5ACTR that carries tandem repeat sequence from MUC5AC, a major secreted airway mucin, in a MUC1 mucin vector. To establish whether the presence of mutant or normal CFTR directly influences the O-glycosylation and sulphation of mucins in airway epithelial cells, we used the CFT1-LC3 (DeltaF508 CFTR mutant) and CFT1-LCFSN (wild-type CFTR corrected) human airway epithelial cell lines. MUC1F/5ACTR mucin was immunoprecipitated, centricon purified, and O-glycosylation was evaluated by Matrix-assisted laser desorption ionization and electrospray tandem mass spectrometry to determine the composition of different carbohydrate structures. Mass spectrometry data showed the same O-glycans in both CFTR mutant and wild-type CFTR corrected cells. Metabolic labeling assays were performed to evaluate gross glycosylation and sulphation of the mucins and showed no significant difference in mucin synthesized in six independent clones of these cell lines. Our results show that the absence of functional CFTR protein causes neither an abnormality in mucin O-glycosylation nor an increase in mucin sulphation

Which electrospray-based ionization method best reflects protein-ligand interactions found in solution? A comparison of ESI, nanoESI, and ESSI for the determination of dissociation constants with mass spectrometry

We present a comparison of three different electrospray-based ionization techniques for the investigation of noncovalent complexes with mass spectrometry. The features and characteristics of standard electrospray ionization (ESI), chip-based nanoESI, and electrosonic spray ionization (ESSI) mounted onto a hybrid quadrupole time-of-flight mass spectrometer were compared in their performance to determine the dissociation constant (K D) of the model system hen egg white lysozyme (HEWL) binding to N,N′,N″-triacetylchitotriose (NAG3). The best K D value compared with solution data were found for ESSI, 19.4 ± 3.6 µM. Then, we determined the K Ds of the two nucleotide binding sites of adenylate kinase (AK), where we obtained K Ds of 2.2 ± 0.8 µM for the first and 19.5 ± 8.0 µM for the second binding site using ESSI. We found a weak charge state dependence of the K D for both protein-ligand systems, where for all ionization techniques the K D value decreases with increasing charge state. We demonstrate that ESSI is very gentle and insensitive to instrumental parameters, and the K D obtained is in good agreement with solution phase results from the literature. In addition, we tried to determine the K D for the lymphocyte-specific kinase LCK binding to a kinase inhibitor using nanoESI due to the very low amount of sample available. In this case, we found K D values with a strong charge state dependence, which were in no case close to literature values for solution phas

Shrinking droplets in electrospray ionization and their influence on chemical equilibria

We investigated how chemical equilibria are affected by the electrospray process, using simultaneous in situ measurements by laser-induced fluorescence (LIF) and phase Doppler anemometry (PDA). The motivation for this study was the increasing number of publications in which electrospray ionization mass spectrometry is used for binding constant determination. The PDA was used to monitor droplet size and velocity, whereas LIF was used to monitor fluorescent analytes within the electrospray droplets. Using acetonitrile as solvent, we found an average initial droplet diameter of 10 µm in the electrospray. The PDA allowed us to follow the evolution of these droplets down to a size of 1 µm. Rhodamine B-sulfonylchloride was used as a fluorescent analyte within the electrospray. By spatially resolved LIF it was possible to probe the dimerization equilibrium of this dye. Measurements at different spray positions showed no influence of the decreasing droplet size on the monomer-dimer equilibrium. However, with the fluorescent dye pair DCM and oxazine 1 it was shown that a concentration increase does occur within electrosprayed droplets, using fluorescence resonance energy transfer as a probe for the average pair distanc

Comparative Bioavailability Study of Two 81 mg Coated Tablet Formulations of Acetylsalicylic Acid in Fasting Healthy Volunteers

Introduction: Low-dose acetylsalicylic acid is used as antithrombotic agent and the enteric-coated formulations are widely used to minimize the gastrointestinal side effects. Aim: To compare the bioavailability of two acetylsalicylic acid formulations (Ecasil-81®, 81 mg coated tablet) in fasting healthy volunteers. Methods: Healthy volunteers (n=16) were recruited to a monocentric, open label, randomized, two-way crossover pharmacokinetic study, with seven days washout period between the treatments. They received a single 81 mg oral dose of a test (new formulation) or a standard reference formulation of acetylsalicylic acid (Ecasil-81®) after about 8 h fasting. Blood samples were collected over a period of 36 h. The salicylic acid plasma concentration was evaluated by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Noncompartmental pharmacokinetic analysis was performed using the WinNonlin program. Results: The maximum plasma concentration (Cmax) of salicylic acid was 5433 and 5719 ng/mL reached in 3.66 and 4.02 h (tmax) for the test and the reference formulation, respectively. The 90% confidence interval of the ratios of geometric means of Cmax and area under curve of plasma concentration until the last concentration observed (AUC0- last) were within the interval 80-125%. Conclusion: The new acetylsalicylic acid formulation has a bioavailability equivalent to the reference formulation for the rate and the extent of absorption

Measurement of multi-wall carbon nanotube penetration through a screen filter and single-fiber analysis

In this study, we carried out experiments to study penetration of airborne carbon nanotubes (CNTs) through a screen filter. An electrospray system was employed to aerosolize suspensions of multi-wall CNTs. The generated airborne CNTs were characterized by electron microscopy, and the length and diameter were measured. In the filtration experiments, the challenging CNTs are classified by a differential mobility analyzer. Monodisperse CNTs with the same electrical mobility were then employed to challenge the screen filter. Penetration was measured for CNTs in the range of 100-400nm mobility diameters. The results showed that the CNT penetration was less than the penetration for a sphere with the same mobility diameter, which was mainly due to the larger interception length of the CNTs. We compared the modeling results using single-fiber filtration efficiency theories with the experimental data, and found that the effective interception length can be approximated by the CNT aerodynamic diameter multiplying a scaling factor. A hypothesis is proposed to understand the observatio

Evolution of the solvent polarity in an electrospray plume

Solvent polarity plays an important role in electrospray ionization-mass spectrometry (ESI-MS), one of the most widely used analytical methods for biochemistry. To have a comprehensive understanding of how solvent polarity affects ESI-MS measurements, we systematically investigated the polarity change in the ESI plume formed from an ethanol solution using laser-induced fluorescence (LIF) spectroscopy. Two solvatochromic dyes (i.e., dyes whose fluorescence emission is sensitive to solvent polarity), Nile red and DCM (4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran), were used as probes. The peak emission wavelengths of these two dyes exhibited significant red shifts (8-12 nm) when the measuring spot was moved away from the spray tip and in radial direction in the plume, indicating a dramatic polarity change during shrinking of the droplets. The emission intensities were also measured with a polarity-insensitive dye as a reference. The results are consistent with the peak wavelength measurements. Two key mechanisms responsible for the change of solvent polarity in the plume were considered, water entrainment from the surrounding air and solvent evaporation. Furthermore, quantitative analysis of the solvent polarity change was performed by using the Lippert-Mataga polarity parameter Δf. The value of Δf reached 0.305-0.307 at the periphery of the ESI plume, which means that the solvent polarity in the smaller droplet is close to that of a mixture of 30% water and 70% ethanol (Δf = 0.307), even though the bulk solvent was ethanol containing less than 1% water as an impurit

Development and validation of a UPLC-MS/MS and UPLC-HR-MS method for the determination of fumonisin B1 and its hydrolysed metabolites and fumonisin B2 in broiler chicken plasma

A sensitive and specific method for the quantitative determination of Fumonisin B1 (FB1), its partially hydrolysed metabolites pHFB1a+b and hydrolysed metabolite HFB1, and Fumonisin B2 (FB2) in broiler chicken plasma using ultra-performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS) was developed. The sample preparation was rapid, straightforward and consisted of a deproteinization and phospholipid removal step using an Oasis((R)) Ostro(TM) 96-well plate. Chromatography was performed on an Acquity HSS-T3 column, using 0.3% formic acid and 10 mM ammonium formate in water, and acetonitrile as mobile phases. The MS/MS instrument was operated in the positive electrospray ionization mode and the two multiple reaction monitoring transitions were monitored for each component for quantification and identification, respectively. The method was validated in-house: matrix-matched calibration graphs were prepared and good linearity (r 0.99) was achieved over the concentration ranges tested (1-500 ng/mL for FB1 and FB2; 0.86-860 ng/mL for pHFB1a; 0.72-1430 ng/mL for pHFB1b and 2.5-2500 ng/mL for HFB1). Limits of quantification (LOQ) and detection (LOD) in plasma ranged between 0.72 to 2.5 ng/mL and 0.03 to 0.17 ng/mL, respectively. The results for the within-day and between-day precision and accuracy fell within the specified ranges. Moreover, the method was transferred to an UPLC high-resolution mass spectrometry (HR-MS) instrument in order to determine potential metabolites of HFB1, such as N-acyl-HFB1s and phase II metabolites. The method has been successfully applied to investigate the toxicokinetics and biotransformation of HFB1 in broiler chickens

Inhibition of cytochrome P450 activities by extracts of 'Hyptis verticillata' Jacq. : assessment for potential HERB-drug interactions

Understanding the potential for adverse drug reactions (ADRs), from herb-drug interactions, is a key aspect of medicinal plant safety, with particular relevance for public health in countries where medicinal plant use is highly prevalent. We undertook an in-depth assessment of extracts of Hyptis verticillata Jacq., via its impact on activities of key cytochrome P450 (CYP) enzymes (CYPs 1A1, 1A2, 1B1, 3A4 and 2D6), its antioxidant properties (determined by DPPH assays) and chemical characterisation (using LC-MS). The dried plant aqueous extract demonstrated potent inhibition of the activities of CYPs 1A1 (7.6 µg/mL), 1A2 (1.9 µg/mL), 1B1 (9.4 µg/mL) and 3A4 (6.8 µg/mL). Further analysis of other crude extracts demonstrated potent inhibition of CYP1A2 activity for a dried plant ethanol extract (1.5 µg/mL), fresh plant ethanol extract (3.9 µg/mL), and moderate activity for a fresh plant aqueous extract (27.8 µg/mL). All four extracts demonstrated strong antioxidant activity, compared to the positive control (ascorbic acid, 1.3 µg/mL), with the dried plant ethanol extract being the most potent (1.6 µg/mL). Analysis of the dried plant aqueous extract confirmed the identity of seven phytochemicals, five lignans and two triterpenes. Individual screening of these phytochemicals against the activity of CYP1A2 identified yatein as a moderate inhibitor (71.9 μM), likely to contribute to the plant extract’s potent bioactivity. Further analysis on the impact of this plant on key drug metabolizing enzymes in vivo appears warranted for likely ADRs, as well as furthering development as a potential chemopreventive agent