Demonstration of 100 GHz electrically tunable liquid-crystal Bragg gratings for application in dynamic optical networks.

We demonstrate liquid-crystal-based integrated optical devices with >140 GHz electrical tuning for potential applications in dynamic optical networks. Bragg wavelength tuning covering five 25 GHz wavelength-division multiplexing channel spacing has been achieved with 170 V (peak-to-peak) sinusoidal voltages applied across electropatterned indium tin oxide-covered glass electrodes placed 60 microm apart. This tunability range was limited only by the initial grating strength and supply voltage level. We also observed two distinct threshold behaviors that manifest during increase of supply voltage, resulting in a hysteresis in the tuning curve for both TE and TM input light.

A multi-analytical platform approach to the metabonomic analysis of plasma from normal and Zucker (fa/fa) obese rats.

Plasma obtained from 20 week old normal Wistar-derived and Zucker (fa/fa) rats was analysed using a number of different analytical methodologies to obtain global metabolite profiles as part of metabonomic investigations of animal models of diabetes. Samples were analysed without sample pre-treatment using 1H NMR spectroscopy, after acetonitrile solvent protein precipitation by ultra-performance liquid chromatography-MS (UPLC-MS) and after acetonitrile protein precipitation and derivatisation for capillary gas chromatography-MS (GC-MS). Subsequent data analysis using principal components analysis revealed that all three analytical platforms readily detected differences between the plasma metabolite profiles of the two strains of rat. There was only limited overlap between the metabolites detected by the different methodologies and the combination of all three methods of metabolite profiling therefore provided a much more comprehensive profile than would have been provided by their use individually.

Metabonomics with 1H-NMR spectroscopy and liquid chromatography-mass spectrometry applied to the investigation of metabolic changes caused by gentamicin-induced nephrotoxicity in the rat.

The model nephrotoxin gentamicin was administered to male Wistar-derived rats daily, for 7 days, at 60 mg kg-1 day-1, subcutaneously, twice daily. Conventional clinical chemistry urinalysis showed a significant increase in N-acetyl-beta-D-glucosaminidase (NAG) activity from day 3. At necropsy on day 9, clear histological damage to the kidney was noted with all animals showing a generally severe nephropathy primarily focused on the proximal convoluted tubules. The urinary excretion pattern of endogenous metabolites over the time course of the study was studied using a combination of 1H-NMR spectroscopy and HPLC-TOF-MS/MS using electrospray ionization (ESI). Changes in the pattern of endogenous metabolites as a result of daily administration of gentamicin were readily detected by both techniques with significant perturbations of the urinary profile observed from day 7 onwards. The findings by 1H-NMR included raised glucose and reduced trimethylamine N-oxide (TMAO). Changes in metabonomic profiles were observed by HPLC-MS in both positive and negative ESI. The MS data showed reduced xanthurenic acid and kynurenic acid, whilst neutral loss experiments also revealed a changed pattern of sulphate conjugation on gentamicin administration.

D-Serine-induced nephrotoxicity: a HPLC-TOF/MS-based metabonomics approach.

HPLC-MS-based metabonomic analysis was used to investigate urinary metabolic perturbations associated with D-serine-induced nephrotoxicity. D-Serine causes selective necrosis of the proximal straight tubules in the rat kidney accompanied by aminoaciduria, proteinuria and glucosuria. Alderely Park (Wistar-derived) rats were dosed with either D-serine (250 mg/kg ip) or vehicle (deionised water) and urine was collected at 0-12, 12-24, 24-36 and 36-48 h post-dosing. Samples were analysed using a Waters Alliance HT 2795 HPLC system coupled to a Waters Micromass Q-ToF-micro equipped with an electrospray source operating in either positive or negative ion mode. Changes to the urinary profile were detected at all time points compared to control. In negative ion mode, increases were observed in serine (m/z=103.0077), m/z=104.0376 (proposed to be hydroxypyruvate) and glycerate (m/z=105.0215), the latter being metabolites of D-serine. Furthermore, an increase in tryptophan, phenylalanine and lactate and decreases in methylsuccinic acid and sebacic acid were observed. Positive ion analysis revealed a decrease in xanthurenic acid, which has previously been assigned and reported using HPLC-MS following exposure to mercuric chloride and cyclosporine A. A general aminoaciduria, including proline, methionine, leucine, tyrosine and valine was also observed as well as an increase in acetyl carnitine. Investigation of additional metabolites altered as a result of exposure to D-serine is on-going. Thus, HPLC-MS-based metabonomic analysis has provided information concerning the mechanism of D-serine-induced renal injury.

Cyclosporin A-induced changes in endogenous metabolites in rat urine: a metabonomic investigation using high field 1H NMR spectroscopy, HPLC-TOF/MS and chemometrics.

The model nephrotoxin cyclosporin A was administered to male Wistar-derived rats daily for 9 days at a dose level of 45 mg/kg per day. Urine samples were collected daily and the excretion pattern of low molecular mass organic molecules in the urine was studied using 1H NMR spectroscopy and HPLC-TOF/MS. Distinct changes in the pattern of endogenous metabolites, as a result of the daily administration of cyclosporin A, were observed by 1H NMR from day 7 onwards. The NMR-detected markers included raised concentrations of glucose, acetate, trimethylamine and succinate and reduced amounts of trimethylamine-N-oxide. In parallel studies by HPLC-TOF/MS a reduction in the quantities of kynurenic acid, xanthurenic acid, citric acid and riboflavin present in the urines was noted, together with reductions in a number of as yet unidentified compounds. In addition, signals resulting from the polyethylene glycol, present in the dosing vehicle, and cyclosporin A metabolites were detected by MS. However, these were excluded from the subsequent multivariate data analysis in order to highlight only changes to the endogenous metabolites. Analysis of both the 1H NMR and HPLC-MS spectroscopic data using pattern recognition techniques clearly identified the onset of changes due to nephrotoxicity.

A metabonomic investigation of the biochemical effects of mercuric chloride in the rat using 1H NMR and HPLC-TOF/MS: time dependent changes in the urinary profile of endogenous metabolites as a result of nephrotoxicity.

The effects of the administration of a single dose of the model nephrotoxin mercuric chloride (2.0 mg kg(-1), subcutaneous) to male Wistar-derived rats on the urinary metabolite profiles of a range of endogenous metabolites has been investigated using (1)H NMR and HPLC-MS. Urine samples were collected daily for 9 days from both dosed and control animals. Analysis of these samples revealed marked changes in the pattern of endogenous metabolites as a result of HgCl(2) toxicity. Peak disturbances in the urinary metabolite profiles were observed (using both NMR and HPLC-MS) at 3 days post dose. Thereafter the urinary metabolite profile gradually returned to a more normal composition. Markers of toxicity identified by (1)H NMR spectroscopy were raised concentrations of lactate, alanine, acetate, succinate, trimethylamine (TMA), and glucose. Reductions in the urinary excretion of citrate and alpha-ketoglutarate were also seen. Markers identified by HPLC-MS, in positive ion mode, were kynurenic acid, xanthurenic acid, pantothenic acid and 7-methylguanine which decreased after dosing. In addition an ion at m/z 188, probably 3-amino-2-naphthoic acid, was observed to increase after dosing. As well as these identified compounds other ions at m/z 297 and 267 decreased after dosing. In negative ion mode a range of sulfated compounds were observed, including phenol sulfate and benzene diol sulfate, which decreased after dosing. As well as the sulfated components an unidentified glucuronide at m/z 326 was also observed to decrease after dosing. The results of this study demonstrate the complementary nature of the NMR and MS-based techniques for metabonomic analysis.

Detection of mono- and di-hexoses as metabolites of 4-bromoaniline using HPLC-TOF-MS/MS.

1. The metabolic fate of 4-bromoaniline (4-BrA) was investigated in rat following intraperitoneal administration at 50 mg kg(-1) using HPLC-TOF-MS/MS. 2. The sensitivity provided by the use of TOF-MS/MS, aided by the distinctive isotope pattern resulting from the presence of the bromine substituent in the molecule, enabled the detection of many previously uncharacterized metabolites in the samples. 3. Several groups of minor metabolites were detected in the urine that corresponded to a number of isomeric hexose and di-hexose-containing conjugates (possibly glucosides and diglucosides) of 4-BrA. 4. As well as hexose and di-hexose conjugates of 4-BrA, several further groups of metabolites that also contained either a sulphamate or sulphate group in addition to the sugar moieties were also detected.

Screening and identification of unknown contaminants in water with liquid chromatography and quadrupole-orthogonal acceleration-time-of-flight tandem mass spectrometry.

In order to assess and maintain the quality of surface waters, target compound monitoring is often not sufficient. Many unknown micro-contaminants are present in water, originating in municipal, industrial or agricultural effluents. Some of these might pose a risk to drinking water production and consequently to human health. The possibilities of screening surface water and identification of these non-target water pollutants with modern data acquisition possibilities of hybrid quadrupole-orthogonal acceleration time of flight mass spectrometers (Q-TOF), such as data-dependent MS to MS/MS switching were investigated. Using model compounds, a procedure for the liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening of water extracts was developed, enabling the detection and identification of compounds at levels < or = 0.25 microg/l in surface water. Based on the accurate mass the elemental compositions for the precursor and product ions are calculated. The calculated chemical formulae are searched against the Merck index, the NIST library, an own database containing about 2,500 water pollutants (pesticides and other contaminants) as well as a CI-CID library containing tandem MS spectra of about 100 water contaminants. The developed approach was applied for the identification of unknown compounds, present in native surface water extract. For three of these compounds, structures were proposed. Confirmation of the proposed structures with standards was beyond the scope of this study.

Evaluation of quadrupole time-of-flight tandem mass spectrometry and ion-trap multiple-stage mass spectrometry for the differentiation of C-glycosidic flavonoid isomers.

LC-MS-MS is becoming a very important tool for the on-line identification of natural products in crude plant extracts. For an efficient use of this technique in the dereplication of natural products, a careful study of the parameters used to generate informative MS-MS spectra is needed. In this paper, the collision-induced dissociation (CID) MS-MS spectra of ubiquitous C-glycosidic flavonoids have been systematically studied using hybrid quadrupole time-of-flight and ion-trap (IT) mass analysers under various CID energy conditions. Efficient differentiation of flavonoid C-glycoside isomers was possible, based on the comparison of CID-MS-MS spectra of particular C-glycoside unit fragments. Striking differences between 6-C and 8-C flavonoid glycosides were especially observed in the product ion spectra of their 0.2X+ fragments ([M+H-120]+). Some guidelines for the on-line characterisation of C-glycosidic flavonoids by LC-MS-MS or LC-multiple-stage MS are given.

A promoter mutation in the tumor necrosis factor alpha gene is not associated with preeclampsia.

Preeclampsia is associated with increased plasma concentrations of tumor necrosis factor alpha (TNF alpha) and TNF receptors. A mutation in the promoter region of the TNF alpha gene (TNF T2) has been described which is associated with increased transcription of the gene. Due to the familial predisposition of preeclampsia, we hypothesized that this promoter mutation in the TNF alpha gene may contribute to the genetic etiology of preeclampsia. Our objective was to determine the allele frequency of this mutation in a population with well-characterized preeclampsia and with hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome as compared with normotensive controls. DNA was extracted from blood of 131 women with severe preeclampsia, 75 women with HELLP syndrome, and 41 normotensive gravid controls. Genotypes were determined using the polymerase chain reaction (PCR), allele-specific restriction with Nco1, and agarose gel electrophoresis. Results were analyzed with a chi 2 contingency table. No significant differences were found between patients with severe preeclampsia, HELLP syndrome, normotensive gravid controls, and previously published allele frequencies. The frequency of the TNF T2 allele is not increased in patients with preeclampsia or HELLP syndrome. Therefore, this promoter mutation is probably not a major genetic cause of preeclampsia. As more genes are cloned, sequenced and localized, this will enable investigators to take this 'candidate gene approach' to investigate potential genetic causes of preeclampsia.

A promoter mutation that increases transcription of the tumor necrosis factor-alpha gene is not associated with preterm delivery.

OBJECTIVE: Increased amniotic fluid concentrations of tumor necrosis factor-alpha are observed in women with preterm labor and subsequent preterm birth. We tested whether a mutation in the promoter region of tumor necrosis factor-alpha gene, TNF T2, which increases transcription of the gene, is more frequent in a preterm delivery cohort. STUDY DESIGN: Deoxyribonucleic acid was extracted from whole blood of 203 women and 44 fetuses delivered at < 37 weeks of estimated gestational age. The polymerase chain reaction was used to amplify the promoter region of the tumor necrosis factor-alpha gene. The resulting polymerase chain product was subjected to allele-specific enzymatic digestion with Nco I. Fragments were size fractionated on a 3% Metaphor agarose gel stained with ethidium bromide. Results were analyzed with use of a chi 2 contingency table. RESULTS: No statistically significant differences for either the TNF T1 or TNF T2 allele frequencies were found between women or fetuses delivered preterm compared with a control group or previously published allele frequencies. CONCLUSIONS: The frequency of this tumor necrosis factor-alpha promoter mutation, TNF T2, is not increased in either women or fetuses delivered at < 37 weeks' gestation. Basal levels of tumor necrosis factor-alpha are unlikely to affect a woman's risk of preterm delivery. Tumor necrosis factor-alpha variants should not be used as a predictive test for preterm delivery.

A rare case of a liveborn with free, de novo and partial trisomy 12 and an unusual phenotype.

Individuals with free and total trisomy 12 are rare and always mosaic. Incidences of partial trisomy 12 are more frequent and are classified into trisomy 12p and trisomy 12q. The phenotype of both trisomy 12p and trisomy 12q is well described in the literature. We report here, the rare occurrence of a liveborn with free and de novo trisomy 12, albeit not the whole chromosome. The clinical description of this infant includes characteristics of trisomy 12p and trisomy 12q syndromes. Few additional anomalies present in the infant are unaccounted for by both syndromes. We anticipate that these characteristics are caused by trisomy 12q13, which to our knowledge has not been reported in a trisomy before.

Analysis of a standard peptide mixture by capillary electrophoresis with mass spectrometry and with tandem mass spectrometry.

The combination of capillary electrophoresis with mass spectrometry allows efficient separation and identification of components in mixtures with greater specificity than can be obtained using capillary electrophoresis and ultra violet detection alone. For the mixture of peptides analysed in this application, molecular mass information was obtained on all of the components in the initial analysis. On-line capillary electrophoresis/tandem mass spectrometry was then used to generate amino acid sequence information for each of these peptides, at the pmole level, in the form of collision-induced production-ion spectra which were recorded only over the relevant time windows.

Analysis of the in vivo phosphorylation state of protein phosphatase inhibitor-2 from rabbit skeletal muscle by fast-atom bombardment mass spectrometry.

A new procedure has been developed for identifying phosphoserine residues in proteins, and is used to analyse the in vivo phosphorylation state of inhibitor-2. The method employs reverse-phase liquid chromatography to resolve phosphorylated and dephosphorylated forms of peptides and fast-atom bombardment mass spectrometry (FABMS) to identify phosphorylated derivatives. The positions of phosphorylation sites within peptides are located by gas-phase sequencer analysis after conversion of phosphoserine residues to S-ethylcysteine. The phosphorylation sites on inhibitor-2 were identified as serines-86, -120 and -121, the three residues phosphorylated in vitro by casein kinase-II. Serine-86 was phosphorylated to 0.7 mol/mol and serines-120 and -121 each to 0.3 mol/mol. These values were not altered significantly by intravenous injection of adrenalin or insulin. No phosphate was present in the region comprising residues 1-49, even after injection of adrenalin, demonstrating that inhibitor-2 is not a substrate for cyclic AMP-dependent protein kinase in vivo. The absence of phosphotyrosine also indicated that inhibitor-2 is not a physiological substrate for the insulin receptor. Surprisingly, no phosphate was present at threonine-72, the residue phosphorylated in vitro by glycogen synthase kinase-3, after injection of either propranolol, adrenalin or insulin. The implications of this finding for the in vivo activation of protein phosphatase 1I (the 1:1 complex between inhibitor-2 and the catalytic subunit of protein phosphatase-1) are discussed. FABMS analysis of inhibitor-2 confirmed the accuracy of the primary structure reported previously, and showed that the only post-translational modifications were an N-acetyl moiety and the three phosphoserine residues. FABMS also demonstrated the presence of an additional serine residue at the C-terminus, and showed that 50% of isolated inhibitor-2 molecules lack the C-terminal Ser-Ser dipeptide.

Prolonged outbreak of nosocomial urinary tract infection with a single strain of Pseudomonas aeruginosa.

Sixty-six hospitalized patients became infected with a single strain of multiply resistant Pseudomonas aeruginosa over a 22-month period. The catheterized urinary tract was the site of the infection in 59 patients (89%). The outbreak was confined to a urology ward until an infected patient from this ward spent 2 weeks in the surgical intensive care unit (SICU). Subsequently patients who acquired the infection in the SICU were discharged to surgical wards throughout the hospital. Urine measuring containers and urometers used in the SICU were the reservoir of the P. aeruginosa; daily sterilization of this equipment terminated the outbreak. Urometers appeared to be the reservoir of the epidemic strain in subsequent outbreaks. Five patients were still infected when they were readmitted 3 to 12 months after the first admission, and therefore represented an additional reservoir of infection.