In situ derivatization/solid-phase microextraction: determination of polar aromatic amines

A solid-phase microextraction GC/MS method for the trace determination of a wide variety of polar aromatic amines in aqueous samples was developed. Prior to extraction the analytes were derivatized directly in the aqueous solution by diazotation and subsequent iodination in a one-pot reaction. The derivatives were extracted by direct-SPME using a PDMS/DVB fiber and analyzed by GC/MS in the full-scan mode. By diazotation/iodination, the polarity of the analytes was significantly decreased and as a consequence extraction yields were dramatically improved. The derivatization proved to be suitable for strongly deactivated aromatic amines and even the very polar diamino compounds can efficiently be enriched after derivatization. We investigated 18 anilines comprising a wide range of functional groups, which could be determined simultaneously. The method was thoroughly validated, and the precision at a concentration of 0.5 microg/L was 3.8-11% relative standard deviation for nonnitrated analytes using aniline-d(5) as internal standard and 3.7-10% for nitroaromatic amines without internal standard. The in situ derivatization/SPME/GC/MS method was calibrated over the whole analytical procedure and was linear over 2 orders of magnitude. Using 10-mL samples, detection limits of 2-13 ng/L were achieved for 15 of the 18 analytes. For two aminodinitrotoluene isomers and a diaminonitrotoluene, detection limits ranged from 27 to 38 ng/L. By allowing quantification at the 0.1 microg/L level, analysis of all target compounds meets EU drinking water regulations. The method provides high sensitivity, robustness, and high sample throughput by automation. Finally, the method was applied to various real water samples and in wastewater from a former ammunition plant the contents of several aromatic amines were quantified.     

Exploring fluorescence antibunching in solution to determine the stoichiometry of molecular complexes

Fluorescence antibunching is a well-known technique for determining the number of independent emitters per molecule or molecular complex. It was rarely applied to autofluorescent proteins due to the necessity of collecting large numbers of fluorescence photons from a single molecule, which is usually impossible to achieve with rather photolabile autofluorescent proteins. Here, we measure fluorescence antibunching on molecules in solution, allowing us to accumulate data over a large number of molecules. We use that method for determining an average stoichiometry of molecular complexes. The proposed method is absolute in the sense that it does not need any calibration or referencing. We develop the necessary theoretical background and check the method on pure dye solutions and on molecular complexes with known stoichiometry.     

Selective control of gliding microtubule populations

First lab-on-chip devices based on active transport by biomolecular motors have been demonstrated for basic detection and sorting applications. However, to fully employ the advantages of such hybrid nanotechnology, versatile spatial and temporal control mechanisms are required. Using a thermo-responsive polymer, we demonstrate the selective starting and stopping of modified microtubules gliding on a kinesin-1-coated surface. This approach allows the self-organized separation of multiple microtubule populations and their respective cargoes.     

Highly permeable and selective pore-spanning biomimetic membrane embedded with aquaporin Z

A highly permeable yet highly selective pore-spanning biomimetic membrane embedded with aquaporin Z is molecularly designed and constructed via a combination of pressure-assisted vesicle adsorption and covalent-conjugation-driven vesicle fusion on a porous support. This approach represents a significant breakthrough in the architecture of biomimetic membranes embedded with aquaporin in a planar form.     

Nonsteady crack and craze behavior in PMMA under cyclical loading: III. Effect of load history on cohesive force distribution on the craze

Measurements of crack opening and craze profiles are made under a range of loading histories including cyclical deformations that lead to nonsteady crack propagation histories. Of particular interest is the comparison of the distribution of traction transmission of a newly formed craze relative to a cyclically stressed one as it approaches the slow-down phase. Real time, interferometric measurements provide precise and multiple craze profiles during individual cycles. Cyclic deformations reduce the stiffness of a craze in its center resulting in a stress drop as part of the craze strength evolution; also, its thickness changes nonuniformly during the acceleration/retardation phases of the advancing craze/crack. The implications are, that for quasi-statically formed crazes the craze material can be reasonably well characterized by a stress-strain relation, while that is no longer readily true for a cyclically deforming crack, since rate dependent characteristics of the craze and bulk material intervene. Cracks unloaded as part of cyclical deformation histories exhibit crack closure (compression) near the trailing end of the craze. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nonsteady crack and craze behavior in PMMA under cyclical loading: II. Effect of load history on growth rate and fracture morphology

This is the second of three papers devoted to time or frequency effects on crack propagation in PMMA under cyclic loading with high resolution measurements of crack and craze growth in the micron range and with subcycle temporal resolution. It is demonstrated that the breakdown rate of the craze leads to quasi-periodic spurts of propagation and growth retardation in dependence on both the magnitude of the stress intensity factor and on the imposed load frequency. The surface morphology is governed more by the time required to build and deteriorate the craze (process time) than by the amplitude of the stress intensity factor. Three different growth ranges are distinguished: At the highest load levels multiple crazes occur at various distances from the major crack plane which coalesce and give rise to an incoherent surface structure not characterizable in terms of a single craze, although the front of the craze/crack is still represented by a smooth curve; crack advance occurs over domains that are larger than a typical craze length. At lower stress levels the craze breaks at different levels within the craze structure, the spatial breakdown of the craze being limited to dimensions smaller than the craze length but with a correlation or memory effect that generates terraces (on the fracture surface) on a scale larger than the craze length. In a transition between this stage and the lowest stress level considered, both features may exist simultaneously on a fracture surface, one increasing or decreasing relative to the other. Increase/decrease of one phenomenon relative to the other does not occur at a fixed stress intensity range but depends on the past history. When a change in the loading produces a change in the fracture mode, it is possible to associate, during the transition, decreasing crack growth rates with increasing stress intensity factors. At the lowest loading a craze breaks essentially through its center leaving a submicroscopically smooth surface; the associated crack growth and craze break down occurs with more or less periodic increments that are each smaller than the full ‘equilibrium’ craze length. This revised version was published online in July 2006 with corrections to the Cover Date.     

PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae

Neisseria gonorrhoeae, the Gram-negative aetiological agent of gonorrhoeae, is one of many mucosal pathogens of man that expresses competence for natural transformation. Expression of this phenotype by gonococci appears to rely on the expression of type IV pili (Tfp), but the mechanistic basis for this relationship remains unknown. During studies of gonococcal pilus biogenesis, a homologue of the PilT family of proteins, required for Tfp-dependent twitching motility in Pseudomonas aeruginosa and social gliding motility in Myxococcus xanthus, was discovered. Like the findings in these other species, we show here that gonococcal PilT mutants constructed in vitro no longer display twitching motility. In addition, we demonstrate that they have concurrently lost the ability to undergo natural transformation, despite the expression of structurally and morphologically normal Tpf. These results were confirmed by the findings that two classes of spontaneous mutants that failed to express twitching motility and transformability carried mutations in PilT. Piliated PilT mutants and a panel of pilus assembly mutants were found to be deficient in sequence-specific DNA uptake into the cell, the earliest demonstrable step in neisserial competence. The PilT-deficient strains represent the first genetically defined mutants that are defective in DNA uptake but retain Tfp expression.     

Eye Movements and Behavioral Responses to Threatening and Nonthreatening Stimuli During Visual Search in Phobic and Nonphobic Subjects.

Spider-phobic and nonphobic subjects searched for a feared/fear-relevant (spider) or neutral target (mushroom) presented in visual matrices of neutral objects (flowers). In half of the displays, the mushroom target was paired with a spider distractor, or a spider target was paired with a mushroom distractor. Although all subjects responded faster to the neutral target than to the feared/fear-relevant target, phobics were slower to respond than nonphobics when a mushroom target was presented with a spider distractor. Their eyes appeared to be drawn to the feared distractor before fixating neutral targets. A further experiment indicated no group differences when subjects merely judged the homogeneity of matrices. Thus, threat seems to capture the attention of phobics only when it is part of a background that subjects are explicitly instructed to ignore. (PsycINFO Database Record (c) 2010 APA, all rights reserved)