5-lipoxygenase-dependent biosynthesis of novel 20:4 n-3 metabolites with anti-inflammatory activity.

5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) into pro-inflammatory leukotrienes. N-3 PUFA like eicosapentaenoic acid are subject to a similar metabolism and are precursors of pro-resolving mediators. Stearidonic acid (18:4 n-3, SDA) is a plant source of n-3 PUFA that is elongated to 20:4 n-3, an analogue of AA. However, no 5-LO metabolites of 20:4 n-3 have been reported. In this study, control and 5-LO-expressing HEK293 cells were stimulated in the presence of 20:4 n-3. Metabolites were characterized by LC-MS/MS and their anti-inflammatory properties assessed using AA-induced autocrine neutrophil stimulation and leukotriene B4-mediated chemotaxis. 8­hydroxy­9,11,14,17-eicosatetraenoic acid (Δ17-8-HETE) and 8,15-dihydroxy-9,11,13,17-eicosatetraenoic acid (Δ17-8,15-diHETE) were identified as novel metabolites. Δ17-8,15-diHETE production was inhibited by the leukotriene A4 hydrolase inhibitor SC 57461A. Autocrine neutrophil leukotriene stimulation and neutrophil chemotaxis, both BLT1-dependent processes, were inhibited by Δ17-8,15-diHETE at low nM concentrations. These data support an anti-inflammatory role for Δ17-8,15-diHETE, a novel 5-LO product.

Elongated conformers of charge states +11 to +15 of bovine ubiquitin studied using ESI-FAIMS-MS.

Recent advancements in high-field asymmetric waveform ion mobility spectrometry (FAIMS) have led to significant improvements in the application of this technology to the study of protein conformers. Compared with previous work, the maximum value of the separation voltage (i.e., the dispersion voltage) has increased, thereby enabling multiple, elongated conformers of individual charge states of bovine ubiquitin to be separated in the gas phase (e.g., four conformers of each of the +11 and +12 charge states were separated). The use of a carrier gas mixture of 40% nitrogen and 60% helium changed the separation selectivity compared with pure nitrogen and enhanced the signal intensity, especially for the +14 and +15 charge states (the latter was not detected in a nitrogen carrier gas). Conformer cross sections were determined using the FAIMS/energy-loss method and found to be similar within a given charge state. The cross sections for conformers of charge states +13, + 14, and +15 plateau at about 2000 A2 suggesting that the structure of bovine ubiquitin is essentially unfolded after the addition of the 13th proton.

Gas-phase conformers of the [M + 2H](2+) ion of bradykinin investigated by combining high-field asymmetric waveform ion mobility spectrometry, hydrogen/deuterium exchange, and energy-loss measurements.

The continuous separation capability of high-field asymmetric waveform ion mobility spectrometry (FAIMS) was used in combination with complementary techniques for probing biomolecular ions in the gas phase. Gas-phase conformers of the [M + 2H](2+) ion of bradykinin were examined using a combination of FAIMS, H/D exchange, and energy-loss measurements. When FAIMS data and H/D exchange data were analyzed separately, the presence of only two conformers of the [M + 2H](2+) ion of bradykinin could be detected. However, in an experiment in which FAIMS and H/D exchange were combined, at least four different conformers of the gas-phase [M + 2H](2+) ion of bradykinin were detected, including one of very low abundance. Cross sections calculated for the four conformers, based on energy-loss measurements, were 250, 240, 250, and 244 A(2), in order of decreasing abundance.

Quantitation of morphine and codeine in human urine using high-filed asymmetric waveform ion mobility spectrometry (FAIMS) with mass spectrometric detection.

Morphine and codeine have been identified and measured in a human urine matrix using high-field asymmetric waveform ion mobility spectrometry (FAIMS) in a tandem combination with electrospray ionization (ESI) and mass spectrometric (MS) detection. The addition of helium to the nitrogen carrier gas resulted in a substantial improvement in the sensitivity of the ESI-FAIMS-MS instrument for the determination of morphine and codeine. Limits of detection in human urine were 60 ng/mL for morphine and 20 ng/mL for codeine with no clean-up, derivatization, or chromatographic separation of the sample prior to analysis.

Atmospheric pressure ion trapping in a tandem FAIMS-FAIMS coupled to a TOFMS: studies with electrospray generated gramicidin S ions.

A tandem FAIMS-FAIMS system for ion trapping at room temperature and atmospheric pressure is described. The first FAIMS device consisted of a side-to-side configuration (sFAIMS) suitable for ion separation, whereas the second FAIMS device was appropriate for ion trapping (tFAIMS). Ions pre-selected by the sFAIMS entered the tFAIMS and were captured by virtual trapping fields at the hemispherical tip of the inner electrode. The use of the sFAIMS, with wider electrode diameters, and consequently better ion separation efficiency than the tFAIMS, lowered the number of background ions captured in the trapping region of tFAIMS, and thus reduced the space charge effects in the trap. This tandem device was coupled to a laboratory built time-of-flight mass spectrometer and was evaluated using the electrospray generated [M + 2H]2+ ion of gramicidin S. The half-time (t1/2) of the exponential decay of the ion cloud in tFAIMS, determined by monitoring the residual intensity of ions extracted from the ion trapping region of tFAIMS after various delay times, was about 2 s.

Analysis of a tryptic digest of pig hemoglobin using ESI-FAIMS-MS.

The continuous gas-phase ion separation and atmospheric pressure focusing properties of high-field asymmetric waveform ion mobility spectrometry (FAIMS) offer significant advantages for the mass spectrometric analysis of tryptic digests of proteins. In this study, tryptic peptides of pig hemoglobin were examined by ESI-FAIMS-MS using a newly designed FAIMS device. The new, hemispherical geometry of the inner electrode served to deliver the ions, via the gas flows, to the center axis of the FAIMS analyzer, improving the sensitivity relative to previous prototypes. Mass spectra collected using this new FAIMS showed significantly less chemical background noise than conventional ESI-MS, while maintaining approximately the same absolute sensitivity as that observed with ESI-MS. As a consequence of the ion separation in FAIMS, the identification of the tryptic fragments was simplified and some peptides, such as the triply protonated WAGVANALAHK3+, that were obscured by the intense background of ESI-MS, were readily detected using ESI-FAIMS-MS. In addition, the FAIMS device was shown to separate isobaric ions at m/z 532.4. Correlations between CV and mass-to-charge ratio, as well as CV and ionic collision cross section, were evaluated for 38 peptide ions identified in the tryptic digest. The correlation between the CV of the peptide and the mass-to-charge ratio is very poor, indicating good orthogonality between the separation by FAIMS and the separation by mass spectrometry.

Investigation of bovine ubiquitin conformers separated by high-field asymmetric waveform ion mobility spectrometry: cross section measurements using energy-loss experiments with a triple quadrupole mass spectrometer.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) was used to separate gas-phase conformers of bovine ubiquitin produced by electrospray ionization. These conformers were sampled by a triple quadrupole mass spectrometer where energy-loss experiments, following the work of Douglas and co-workers, were used to determine their cross sections. The measured cross sections for some conformers were readily altered by the voltages applied to the interface ion optics, therefore very gentle mass spectrometer interface conditions were required to preserve gas-phase conformers separated by FAIMS. Cross sections for 19 conformers (charge states +5 through +13) were measured. Two conformers for the +12 charge state, which were readily separated in FAIMS, were found to have similar cross sections. Based on a method to calibrate the collision gas thickness, the cross sections measured using the FAIMS/energy-loss method were compared with literature values determined using drift tube ion mobility spectrometry. The comparison illustrated that the conformers of bovine ubiquitin that were identified using drift tube ion mobility spectrometry were also observed using the FAIMS device.

Detection of microcystins using electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry/mass spectrometry.

A combination of electrospray ionization, high-field asymmetric waveform ion mobility spectrometry, and mass spectrometry (ESI-FAIMS/MS) was used to analyze standard solutions of microcystins-LR, -RR, and -YR. The ability of FAIMS to separate ions in the gas phase reduced the amount of background in the mass spectrum without compromising the absolute signal for these microcystins. This reduction in background resulted in a ten-fold improvement in the signal-to-background ratio over conventional ESI-MS. Detection limits, using direct infusion, were determined to be 4, 2, and 1 nM for microcystins-LR, -RR, and -YR, respectively.

Evaluation of carrier gases for use in high-field asymmetric waveform ion mobility spectrometry.

Effects of carrier gas type (N2, O2, CO2, N2O, and SF6) on changes in the ratio of high- to low-field ion mobility, Kh/K, of cesium, gramicidin S, tetrahexylammonium, heptadecanoic acid, and aspartic acid in fields of up to 67 Td are presented. The theory of the mobility of ions at high E/N in different gases is discussed. Plots of Kh/K as a function of the ionic energy parameter, E/N, for the five ions in each of the gases were derived from experimental data collected using a high-field asymmetric waveform ion mobility spectrometer. The change in the ratio of high- to low-field ion mobility of cesium in carrier gases of O2 and N2 showed excellent agreement with literature values. The behavior of cesium in O2 and N2 is used to illustrate that the ratio Kh/K as a function of effective temperature is invariant with gas type as long as the well depth of the interaction potential significantly exceeds thermal energy. From these results, it appears that the well depth of the interaction potential of the heavier ions studied here, including gramicidin S, tetrahexylammonium, and heptadecanoic acid, with bath gases such as N2 and O2, is shallow relative to thermal energy.

Trace level determination of perchlorate in water matrices and human urine using ESI-FAIMS-MS.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) separates perchlorate from interfering isobaric ions of bisulfate and dihydrogenphosphate in the gas-phase. The use of a new FAIMS prototype and waveform generator, along with the use of a mixed carrier gas, in this electrospray ionisation (ESI)-FAIMS-mass spectrometry (MS) study gave a detection limit for perchlorate in a relatively "clean" matrix of tap water of 0.050 ppb. Flow injection analysis (FIA) of dilutions of fortified waste water, a fortified river water certified reference material (CRM; SLRS-4, National Research Council of Canada), and a fortified human urine Standard Reference Material (SRM; 2381, National Institute of Standards and Technology) gave detection limits of 0.37 ppb, 0.50 ppb, and 4.8 ppb, respectively, in the undiluted matrices.

Detection of chlorinated and brominated byproducts of drinking water disinfection using electrospray ionization-high-field asymmetric waveform ion mobility spectrometry-mass spectrometry.

The lower limit of detection for low molecular weight polar and ionic analytes using electrospray ionization-mass spectrometry (ESI-MS) is often severely compromised by an intense background that obscures ions of trace components in solution. Recently, a new technique, referred to as high-field asymmetric waveform ion mobility spectrometry (FAIMS), has been shown to separate gas-phase ions at atmospheric pressure and room temperature. A FAIMS instrument is an ion filter that may be tuned, by control of electrical voltages, to continuously transmit selected ions from a complex mixture. This capability offers significant advantages when FAIMS is coupled with ESI, a source that generates a wide variety of ions, including solvent clusters and salt adducts. In this report, the tandem arrangement of ESI-FAIMS-MS is used for the analysis of haloacetic acids, a class of disinfection byproducts regulated by the US EPA. FAIMS is shown to effectively discriminate against background ions resulting from the electrospray of tap water solutions containing the haloacetic acids. Consequently, mass spectra are simplified, the selectivity of the method is improved, and the limits of detection are lowered compared with conventional ESI-MS. The detection limits of ESI-FAIMS-MS for six haloacetic acids ranged between 0.5 and 4 ng/mL in 9:1 methanol/tap water (5 and 40 ng/mL in the original tap water samples) with no preconcentration, derivatization, or chromatographic separation prior to analysis.

Radiolucencies with indistinct or ragged borders.

The diagnostic challenge will always be present when encountering an ill-defined radiolucency. Is it an artifact, normal anatomy, or one of these inflammatory, proliferative, or neoplastic disorders? If it cannot be explained as an artifact or anatomic variant, then consideration certainly should be given for a short-term follow-up radiograph or immediate biopsy. Understanding the clinical, radiographic, and histopathologic features, as well as the treatment protocols for these various disorders should help the dentist make the proper diagnostic decisions and provide the appropriate patient management.

Cross-sectional tomography. A diagnostic technique for determining the buccolingual relationship of impacted mandibular third molars and the inferior alveolar neurovascular bundle.

Twenty-two patients with 31 impacted mandibular third molars were examined with a new, precise, cross-sectional tomographic technique to assess the radiographic size, shape, branching pattern, location, and degree of cortication of the mandibular canal, and the inclination of impacted mandibular third molars in the buccolingual plane. The mandibular canal, including bifid canals, was accurately identified in 30 cases (96.8%). The cross-sectional appearance of the canal was an uncorticated, or partially corticated, radiolucent oval that measured on average (+/- SD) 2.9 +/- 0.7 x 2.5 +/- 0.6 mm in diameter. It was located more frequently (45.2%) on the buccal aspect of the impacted mandibular third molar. About 60% of the mandibular canals notched the inner cortical plate of the mandible or the third molar root surface. Cystic expansion and quantification of cortical bone destruction were readily assessed by this technique. It was concluded that diagnostic information obtained from cross-sectional tomograms significantly aids the oral and maxillofacial surgeon during the preoperative diagnostic workup and that the radiation risks are comparable to those of other accepted localization techniques.

Identification of nocardiae and streptomycetes of medical importance.

During a taxonomic investigation of Nocardia spp. and Streptomyces spp., we received 658 isolates from laboratories of both human and veterinary medicine. Our procedure leading to the identification of 92% of these isolates, the species that they represented, and a characteristic pattern of properties of the strains of these species are presented. A key devised for the tentative identification of species of nocardiae and streptomycetes that are believed to be of medical importance is included to assist clinical microbiologists in identifying their strains.

Some bits and pieces of the genus Nocardia: N. carnea, N. vaccinii, N. transvalensis, N. orientalis and N. aerocolonigenes.

Since 1951 when our taxonomic study of the genus Nocardia began, only eight strains of N. carnea (Rossi-Doria) Castellani & Chalmers, four strains of N. vaccinii Demaree & Smith, five of N. transvalensis Pijper & Pullinger, 21 or N. orientalis (Pittenger & Brigham) Pridham and 14 of N. aerocolonigenes (Shinobu & Kawato) Pridham have been found. These five groups of strains are described and are compared with strains of nine accepted species of Nocardia. With the possible exception of N. orientalis, more strains of these five taxa must be assembled and examined to establish the reliability of our descriptions and identifications.