The Bionanoprobe: hard X-ray fluorescence nanoprobe with cryogenic capabilities.

Hard X-ray fluorescence microscopy is one of the most sensitive techniques for performing trace elemental analysis of biological samples such as whole cells and tissues. Conventional sample preparation methods usually involve dehydration, which removes cellular water and may consequently cause structural collapse, or invasive processes such as embedding. Radiation-induced artifacts may also become an issue, particularly as the spatial resolution increases beyond the sub-micrometer scale. To allow imaging under hydrated conditions, close to the `natural state', as well as to reduce structural radiation damage, the Bionanoprobe (BNP) has been developed, a hard X-ray fluorescence nanoprobe with cryogenic sample environment and cryo transfer capabilities, dedicated to studying trace elements in frozen-hydrated biological systems. The BNP is installed at an undulator beamline at sector 21 of the Advanced Photon Source. It provides a spatial resolution of 30 nm for two-dimensional fluorescence imaging. In this first demonstration the instrument design and motion control principles are described, the instrument performance is quantified, and the first results obtained with the BNP on frozen-hydrated whole cells are reported.

Cross linking to tissue transglutaminase and collagen favours gliadin toxicity in coeliac disease.

BACKGROUND AND AIMS: Intestinal inflammation in coeliac disease is driven by the gluten fraction of wheat proteins. Deamidation or cross linking of gluten peptides by tissue transglutaminase (tTG), the coeliac disease autoantigen, creates potent T cell stimulatory peptides. Therefore, our aim was to identify the reaction patterns of gluten peptides, intestinal extracellular matrix proteins, and tTG. METHODS: tTG activity was analysed by incorporation of monodansyl cadaverine into gliadins. Fluorescence labelled tTG reactive short gliadin peptides were used to demonstrate their deamidation and explore their cross linking patterns with tTG itself or extracellular matrix proteins. Patient sera and controls were checked for autoantibodies to matrix proteins. RESULTS: Gliadins alpha1-alpha11, gamma1-gamma6, omega1-omega3, and omega5 were substrates for tTG. tTG catalysed the cross linking of gliadin peptides with interstitial collagen types I, III, and VI. Coeliac patients showed increased antibody titres against the collagens I, III, V, and VI. CONCLUSIONS: tTG formed high molecular weight complexes with all tested gliadins. As all tested gliadins were substrates for tTG, the tTG catalysed modifications were not restricted to single gliadin types and epitopes. Furthermore, haptenisation and long term immobilisation of gliadin peptides by tTG catalysed binding to abundant extracellular matrix proteins could be instrumental in the perpetuation of intestinal inflammation and some associated autoimmune diseases in coeliac disease.

Autoantibodies of patients with coeliac disease are insufficient to block tissue transglutaminase activity.

BACKGROUND AND AIMS: Coeliac disease (CD) is characterised by the presence of autoantibodies against tissue transglutaminase (tTG), the endomysial autoantigen. This study was performed to determine the effect of purified autoantibodies on the enzymatic activity of tTG. METHODS: Total IgA and IgG class antibodies and purified anti-tTG autoantibodies were isolated from sera of untreated patients with CD and controls. The inhibitory capacity of the antibodies on tTG activity was checked by a fluorometric assay based on the incorporation of monodansyl cadaverine into casein and by tTG-catalysed cross linking of biotinylated cadaverine to gliadin. RESULTS: The enriched IgA and IgG fractions of five patients with CD and three controls resulted in no significantly different inhibition of enzymatic activity. In contrast, the use of affinity purified anti-tTG autoantibodies of 12 patients with CD led to a dose dependent reduction of tTG activity, compared to control immunoglobulins (n=6). However, the remaining activity was sufficient for cross linking of cadaverine into gliadin, and enzymatic tTG activity was only blocked completely by high concentrations of a monoclonal antibody, which is directed to the active centre of tTG. CONCLUSIONS: Despite a partial inhibitory effect of isolated anti-tTG autoantibodies from patients with CD, residual enzymatic activity remains sufficiently high to cast doubt on their in vivo relevance.

Volatile anaesthetics and the atmosphere: atmospheric lifetimes and atmospheric effects of halothane, enflurane, isoflurane, desflurane and sevoflurane.

The atmospheric lifetimes of the halogenated anaesthetics halothane, enflurane, isoflurane, desflurane and sevoflurane with respect to reaction with the hydroxyl radical (OH.) and UV photolysis have been determined from observations of OH. reaction kinetics and UV absorption spectra. Rate coefficients for the reaction with OH radicals for all halogenated anaesthetics investigated ranged from 0.44 to 2.7 x 10(-14) cm3 molec-1 s-1. Halothane, enflurane and isoflurane showed distinct UV absorption in the range 200-350 nm. In contrast, no absorption in this wavelength range was detected for desflurane or sevoflurane. The total atmospheric lifetimes, as derived from both OH. reactivity and photolysis, were 4.0-21.4 yr. It has been calculated that up to 20% of anaesthetics enter the stratosphere. As a result of chlorine and bromine content, the ozone depletion potential (ODP) relative to chlorofluorocarbon CFC-11 varies between 0 and 1.56, leading to a contribution to the total ozone depletion in the stratosphere of approximately 1% for halothane and 0.02% for enflurane and isoflurane. Estimates of the greenhouse warming potential (GWP) relative to CFC-12 yield values of 0.02-0.14, resulting in a relative contribution to global warming of all volatile anaesthetics of approximately 0.03%. The stratospheric impact of halothane, isoflurane and enflurane and their influence on ozone depletion is of increasing importance because of decreasing chlorofluorocarbons globally. However, the influence of volatile anaesthetics on greenhouse warming is small.

Current clinical practices in the assessment of velopharyngeal closure.

A questionnaire designed to survey methods of assessing velopharyngeal closure and the extent of training and experience in velopharyngeal assessment was distributed to 256 randomly selected American Cleft Palate Association members. There was a 50% response rate and 94% of those responding were associated with cleft palate teams. Findings included the following: (1) the respondents rely primarily upon the use of listener judgments of spontaneous speech samples, phonological analysis, and lateral view cine/videofluoroscopy; (2) the majority spend thirty minutes or less on an average diagnostic evaluation of velopharyngeal competency; and (3) 45% feel that they are inadequately trained in assessment of velopharyngeal closure.

Effect of vocal disguise on judgments of speakers' sex and race.

To determine the effect of speakers' attempts to disguise their voices on listeners' accuracy in judgments of speakers' sex and race, 26 speakers, 13 women and 13 men, recorded six sentences under three conditions: (a) in a normal manner, (b) in a manner in which they attempted to sound like a member of the opposite sex, and (c) in a manner in which they attempted to sound like a member of the black race. Three master tapes were constructed, one for each of the three conditions. A total of 40 judges, 20 in an experiment on sex identification and 20 in one on race identification, participated in two sessions, one for each of two tapes (control and disguise) in each experiment. In each session they were asked to judge the sex or race of the speaker of each sentence and, using a seven-point confidence rating scale, to indicate the over-all confidence in their judgments at the end of each session. Analysis indicated that, although listeners' accuracy for sex and race identification was greater under the control than disguised conditions for the majority of speakers, the differences between the two conditions were relatively small. Implications of these findings and suggestions for future research are discussed.