Ions formed from lipids during electrospray ionization of crude lipid extracts have been mass-selected within a quadrupole linear ion trap mass spectrometer and allowed to react with ozone vapor. Gas-phase ion-molecule reactions between unsaturated lipid ions and ozone are found to yield two primary product ions for each carbon-carbon double bond within the molecule. The mass-to-charge ratios of these chemically induced fragments are diagnostic of the position of unsaturation within the precursor ion. This novel analytical technique, dubbed ozone-induced dissociation (OzID), can be applied both in series and in parallel with conventional collision-induced dissociation (CID) to provide near-complete structural assignment of unknown lipids within complex mixtures without prior fractionation or derivatization. In this study, OzID is applied to a suite of complex lipid extracts from sources including human lens, bovine kidney, and commercial olive oil, thus demonstrating the technique to be applicable to a broad range of lipid classes including both neutral and acidic glycerophospholipids, sphingomyelins, and triacylglycerols. Gas-phase ozonolysis reactions are also observed with different types of precursor ions including [M+H]+, [M+Li]+, [M+Na]+, and [M-H]-: in each case yielding fragmentation data that allow double bond position to be unambiguously assigned. Within the human lens lipid extract, three sphingomyelin regioisomers, namely SM(d18:0/15Z-24:1), SM(d18:0/17Z-24:1), and SM(d18:0/19Z-24:1), and a novel phosphatidylethanolamine alkyl ether, GPEtn(11Z-18:1e/9Z-18:1), are identified using a combination of CID and OzID. These discoveries demonstrate that lipid identification based on CID alone belies the natural structural diversity in lipid biochemistry and illustrate the potential of OzID as a complementary approach within automated, high-throughput lipid analysis protocols.

The position(s) of carbon-carbon double bonds within lipids can dramatically affect their structure and reactivity and thus has a direct bearing on biological function. Commonly employed mass spectrometric approaches to the characterization of complex lipids, however, fail to localize sites of unsaturation within the molecular structure and thus cannot distinguish naturally occurring regioisomers. In a recent communication [Thomas, M. C.; Mitchell, T. W.; Blanksby, S. J. J. Am. Chem. Soc. 2006, 128, 58-59], we have presented a new technique for the elucidation of double bond position in glycerophospholipids using ozone-induced fragmentation within the source of a conventional electrospray ionization mass spectrometer. Here we report the on-line analysis, using ozone electrospray mass spectrometry (OzESI-MS), of a broad range of common unsaturated lipids including acidic and neutral glycerophospholipids, sphingomyelins, and triacylglycerols. All lipids analyzed are found to form a pair of chemically induced fragment ions diagnostic of the position of each double bond(s) regardless of the polarity, the number of charges, or the adduct ion (e.g., [M - H](-), [M - 2H](2-), [M + H](+), [M + Na](+), [M + NH(4)](+)). The ability of OzESI-MS to distinguish lipids that differ only in the position of the double bonds is demonstrated using the glycerophosphocholine standards, GPCho(9Z-18:1/9Z-18:1) and GPCho(6Z-18:1/6Z-18:1). While these regioisomers cannot be differentiated by their conventional tandem mass spectra, the OzESI-MS spectra reveal abundant fragment ions of distinctive mass-to-charge ratio (m/z). The approach is found to be sufficiently robust to be used in conjunction with the m/z 184 precursor ion scans commonly employed for the identification of phosphocholine-containing lipids in shotgun lipidomic analyses. This tandem OzESI-MS approach was used, in conjunction with conventional tandem mass spectral analysis, for the structural characterization of an unknown sphingolipid in a crude lipid extract obtained from a human lens. The OzESI-MS data confirm the presence of two regioisomers, namely, SM(d18:0/15Z-24:1) and SM(d18:0/17Z-24:1), and suggest the possible presence of a third isomer, SM(d18:0/19Z-24:1), in lower abundance. The data presented herein demonstrate that OzESI-MS is a broadly applicable, on-line approach for structure determination and, when used in conjunction with established tandem mass spectrometric methods, can provide near complete structural characterization of a range of important lipid classes. As such, OzESI-MS may provide important new insight into the molecular diversity of naturally occurring lipids.

The gas phase reactions of the bridgehead 3-carboxylato-1-adamantyl radical anion were observed with a series of neutral reagents using a modified electrospray ionisation linear ion trap mass spectrometer. This distonic radical anion was observed to undergo processes suggestive of radical reactivity including radicalradical combination reactions, substitution reactions and addition to carbon-carbon double bonds. The rate constants for reactions of the 3-carboxylato-1-adamantyl radical anion with the following reagents were measured (in units 10 12 cm3 molecule 1 s 1): 18O2 (85±4), NO (38.4±0.4), I2 (50±50), Br2 (8±2), CH3SSCH3 (12±2), styrene (1.20±0.03), CHCl3 (H abstraction 0.41±0.06, Cl abstraction 0.65±0.1), CDCl3 (D abstraction 0.035±0.01, Cl abstraction 0.723±0.005), allyl bromide (Br abstraction 0.53±0.04, allylation 0.25±0.01). Collision rates were calculated and reaction efficiencies are also reported. This study represents the first quantitative measurement of the gas phase reactivity of a bridgehead radical and suggests that distonic radical anions are good models for the study of their elusive uncharged analogues. SummaryThe gas phase reactions of the bridgehead 3-carboxylato-1-adamantyl radical anion were observed with a series of neutral reagents using a modified electrospray ionisation linear ion trap mass spectrometer. This distonic radical anion was observed to undergo processes suggestive of radical reactivity including radical-radical combination reactions, substitution reactions and addition to carbon-carbon double bonds. The rate constants for reactions of the 3-carboxylato-1-adamantyl radical anion with the following reagents were measured (in units 10

Chronic inflammation is a contributing factor in many age-related diseases. In a previous study, we have shown that Sri Lankan cinnamon (C. zeylanicum) was one of the most potent anti-inflammatory foods out of 115 foods tested. However, knowledge about the exact nature of the anti-inflammatory compounds and their distribution in the two major cinnamon species used for human consumption is limited. The aim of this investigation was to determine the anti-inflammatory activity of C. zeylanicum and C. cassia and elucidate their main phytochemical compounds. When extracts were tested in LPS and IFN-γ activated RAW 264.7 macrophages, most of the anti-inflammatory activity, measured by down-regulation of nitric oxide and TNF-α production, was observed in the organic extracts. The most abundant compounds in these extracts were E-cinnamaldehyde and o-methoxycinnamaldehyde. The highest concentration of E-cinnamaldehyde was found in the DCM extract of C. zeylanicum or C. cassia (31 and 34 mg g(-1) of cinnamon, respectively). When these and other constituents were tested for their anti-inflammatory activity in RAW 264.7 and J774A.1 macrophages, the most potent compounds were E-cinnamaldehyde and o-methoxycinnamaldehyde, which exhibited IC₅₀ values for NO with RAW 264.7 cells of 55 ± 9 μM (7.3 ± 1.2 μg mL(-1)) and 35 ± 9 μM (5.7 ± 1.5 μg mL(-1)), respectively; and IC₅₀ values for TNF-α of 63 ± 9 μM (8.3 ± 1.2 μg mL(-1)) and 78 ± 16 μM (12.6 ± 2.6 μg mL(-1)), respectively. If therapeutic concentrations can be achieved in target tissues, cinnamon and its components may be useful in the treatment of age-related inflammatory conditions.

A novel water-dispersible conducting polymer analogous to poly(3,4-dioxythiophene):polystyrene sulfonate (PEDOT:PSS) has been chemically synthesized in a single reaction in high yield. PEDOT:DS, a new member of the polythiophene family, is composed of a complex between PEDOT and the sulfonated polysaccharide polyanion dextran sulfate. Drop-cast films of aqueous suspensions of the material display a native conductivity of up to 7 ± 1 S cm -1 , increasing to 20 ± 2 S cm -1 after treatment with ethylene glycol and thermal annealing. Mass ratios of the precursors NaDS and EDOT were varied from 5:1 to 2:1 and a decrease in the NaDS:EDOT ratio produces tougher, less hygroscopic films of higher conductivity. Ultraviolet-visible spectroelectrochemistry yields spectra typical of PEDOT complexes. Cyclic voltammetry reveals that PEDOT:DS is electrochemically active from -1.0 to 0.8V vs. Ag/Ag + in acetonitrile, with similar characteristics to PEDOT:PSS. Water dispersions of PEDOT:DS are successfully processed by drop casting, spray coating, inkjet printing and extrusion printing. Furthermore, laser etching of dried films allows the creation of patterns with excellent definition. To assess the cytotoxicity of PEDOT:DS, L-929 cells were cultured with a polymer complex concentration range of 0.002 to 0.2 g l -1 in cell culture medium. No significant difference is found between the proliferation rates of L-929 cells exposed to PEDOT:DS and those in plain medium after 96h. However, PEDOT:PSS shows around 25% less cell growth after 4days, even at the lowest concentration. Taken together, these results suggest PEDOT:DS has exceptional potential as an electromaterial for the biointerface. Corresponding author:Professor Gordon G Wallace However, PEDOT:PSS shows around 25% less cell growth after 4 days, even at the lowest concentration. Taken together, these results suggest PEDOT:DS has exceptional potential as a electromaterial for the biointerface.

Cataracts cause vision loss and blindness by impairing the ability of the ocular lens to focus light onto the retina. Various cataract risk factors have been identified, including drug treatments, age, smoking and diabetes. However, the molecular events responsible for these different forms of cataract are ill-defined, and the advent of modern cataract surgery in the 1960s virtually eliminated access to human lenses for research. Here, we demonstrate large-scale production of light-focusing human micro-lenses from spheroidal masses of human lens epithelial cells purified from differentiating pluripotent stem cells. The purified lens cells and micro-lenses display similar morphology, cellular arrangement, mRNA expression and protein expression to human lens cells and lenses. Exposing the micro-lenses to the emergent cystic fibrosis drug Vx-770 reduces micro-lens transparency and focusing ability. These human micro-lenses provide a powerful and large-scale platform for defining molecular disease mechanisms caused by cataract risk factors, for anti-cataract drug screening and for clinically relevant toxicity assays.

Non-erosive reflux disease (NERD) and esophageal adenocarcinoma (EAC) are often regarded as bookends in the gastroesophageal reflux disease spectrum. However, there is limited clinical evidence to support this disease paradigm while the underlying mechanisms of disease progression remain unclear. In this study, we used 16S rRNA sequencing and mass-spectrometer-based proteomics to characterize the esophageal microbiota and host mucosa proteome, respectively. A total of 70 participants from four patient groups (NERD, reflux esophagitis, Barrett’s esophagus, and EAC) and a control group were analyzed. Our results showed a unique NERD microbiota composition, distinct to control and other groups. We speculate that an increase in sulfate-reducing Proteobacteria and Bacteroidetes along with hydrogen producer Dorea are associated with a mechanistic role in visceral hypersensitivity. We also observed a distinct EAC microbiota consisting of a high abundance of lactic acid-producing bacteria (Staphylococcus, Lactobacillus, Bifidobacterium, and Streptococcus), which may contribute towards carcinogenesis through dysregulated lactate metabolism. This study suggests the close relationship between esophageal mucosal microbiota and the appearance of pathologies of this organ.

The phenylperoxyl radical has long been accepted as a critical intermediate in the oxidation of benzene and an archetype for arylperoxyl radicals in combustion and atmospheric chemistry. Despite being central to many contemporary mechanisms underpinning these chemistries, reports of the direct detection or isolation of phenylperoxyl radicals are rare and there is little experimental evidence connecting this intermediate with expected product channels. We have prepared and isolated two charge-tagged phenyl radical models in the gas phase [i.e., 4-(N,N,N-trimethylammonium)phenyl radical cation and 4-carboxylatophenyl radical anion] and observed their reactions with dioxygen by ion-trap mass spectrometry. Measured reaction rates show good agreement with prior reports for the neutral system (k(2)[(Me(3)N(+))C(6)H(4)˙ + O(2)] = 2.8 × 10(-11) cm(3) molecule(-1) s(-1), Φ = 4.9%; k(2)[((-)O(2)C)C(6)H(4)˙ + O(2)] = 5.4 × 10(-11) cm(3) molecule(-1) s(-1), Φ = 9.2%) and the resulting mass spectra provide unequivocal evidence for the formation of phenylperoxyl radicals. Collisional activation of isolated phenylperoxyl radicals reveals unimolecular decomposition by three pathways: (i) loss of dioxygen to reform the initial phenyl radical; (ii) loss of atomic oxygen yielding a phenoxyl radical; and (iii) ejection of the formyl radical to give cyclopentadienone. Stable isotope labeling confirms these assignments. Quantum chemical calculations for both charge-tagged and neutral phenylperoxyl radicals confirm that loss of formyl radical is accessible both thermodynamically and entropically and competitive with direct loss of both hydrogen atom and carbon dioxide.