Visualization of structural changes in cellulosic substrates during enzymatic hydrolysis using multimodal nonlinear microscopy

Enzymatic hydrolysis of cellulose provides a renewable source of monosaccharides for production of variety of biochemicals and biopolymers. Unfortunately, the enzymatic hydrolysis of cellulose is often incomplete, and the reasons are not fully understood. We have monitored enzymatic hydrolysis in terms of molecular density, ordering and autofluorescence of cellulose structures in real time using simultaneous CARS, SHG and MPEF microscopy with the aim of contributing to the understanding and optimization of the enzymatic hydrolysis of cellulose. Three cellulose-rich substrates with different supramolecular structures, pulp fibre, acid-treated pulp fibre and Avicel, were studied at microscopic level. The microscopy studies revealed that before enzymatic hydrolysis Avicel had the greatest carbon-hydrogen density, while pulp fibre and acid-treated fibre had similar density. Monitoring of the substrates during enzymatic hydrolysis revealed the double exponential SHG decay for pulp fibre and acid-treated fibre indicating two phases of the process. Acid-treated fibre was hydrolysed most rapidly and the hydrolysis of pulp fibre was spatially non-uniform leading to fractioning of the particles, while the hydrolysis of Avicel was more than an order of magnitude slower than that of both fibres.     

Yoctomole analysis of ganglioside metabolism in PC12 cellular homogenates

We report an ultrasensitive method for the analysis of glycosphingolipid catabolism. The substrate G(M1) and the set of seven metabolites into which it can be degraded (G(A1), G(M2), G(A2), G(M3), LacCer, GlcCer, and Cer) were labeled with the highly fluorescent dye tetramethylrhodamine. CE with LIF detection was used to assay these compounds with 150 +/- 80 yoctomole mass (1 ymol = 10(-24) mol = 0.6 copies) detection limits and 5 +/- 3 pM concentration detection limits. An alignment algorithm based on migration of two components was employed to correct for drift in the separation. The within-day and between-day precision in peak height was 20%, in peak width 15%, and in adjusted migration time 0.03%. After normalization to total sample injected, the RSD in peak height reduced to 2-6%, which approaches the limit set by molecular shot noise in the number of molecules taken for analysis. PC12 cells were incubated with the labeled G(M1). Fluorescent microscopy demonstrated uptake by the cells. CE was used to separate a cellular homogenate prepared from these cells. A set of peaks was observed, which were tentatively identified based on comigration with the standards. Roughly 120 pL of homogenate was injected, which contained a total of 150 zmol of labeled substrate and products. Metabolite that preserves the fluorescent label can be detected at the yoctomole level, which should allow characterization of this metabolic pathway in single cells.     

Coupled In/Te and Ni/vacancy ordering and the modulated crystal structure of a B8 type, Ni3±xIn1−yTe2+y solid solution phase

The commensurate superstructures of a NiAs/Ni₂In type parent structure, Ni_3.32InTe₂ and Ni_3.12In_0.86Te_2.14 (q=γ[0 0 1]^*, γ=2/3) as well as one dimensionally incommensurate structure of Ni₃InTe₂ (γ=0.71) were refined from neutron powder diffraction data (R_wp=4.77%, 4.53% and 4.91% for the three structures, respectively, at 298 K). The commensurate structures were refined in the P6₃/mmc space group (c=3c_NiAs). The stacking sequence at the hcp array is -In/Te/Te/- and the trigonal bipyramidal site within the In layer, Ni(2), is partially occupied while it is empty in the Te layers. The octahedral position in between the In and Te layers, Ni(1a), is fully occupied while the octahedral position in between two adjacent Te layers, Ni(1b), is partially occupied. With decreasing In and Ni content, the modulation wave vector, γ, was found to increase continuously until γ=1. From this, crenel functions to describe the whole homogeneity range of the solid solution were constructed with the length of the atomic domains Δ^Te=γ (and hence Δ^In=Δ^Ni=1−γ) and Δ^Ni(1b)=γ/2 (and hence Δ^Ni(1a)=1−γ/2) which were then used for the refinement of the incommensurate structure of Ni₃InTe₂. The corresponding effect in real space is that the single In layers separating double layers of Te occur less frequent when γ in increasing until at γ=1 the CdI₂ type structure of Ni_1+xTe₂ is reached.     

Mass spectrometric analysis of ceramide composition in mono-, di-, tri-, and tetraglycosylceramides from mouse kidney: an experimental model for uropathogenic Escherichia coli.

Many bacteria have been shown to bind to the carbohydrate part of glycosphingolipids, but also the lipid moieties of receptor-active glycolipids are of importance. To investigate the chemistry of the ceramides of kidney glycolipids to which the uropathogenic Escherichia coli bind, different mass spectrometric techniques were utilized. First, a mixture of glycolipids isolated from man and mice kidney was separated by thin-layer chromatography (TLC) and scanned by direct desorption from the plate by fast atom bombardment mass spectrometry (TLC/FAB-MS). Second, the glycolipids were purified by preparative TLC and analyzed by negative-ion FAB-MS. After methylation, further analyses were made with positive-ion FAB-MS, positive-ion electron ionization (EI)-MS, high-temperature capillary gas chromatography (GC/EI-MS) and positive-ion matrix-assisted laser desorption/ionization (MALDI)-MS. The ceramide compositions of the four glycolipids were determined using all these MS techniques and the reliability of the different methods for this type of analyses is discussed. Comparison of the mouse kidney glycolipids with the corresponding glycolipids from human kidney showed the same degree of hydroxylation of ceramides among mono- and disaccharide glycolipids, but a significantly higher degree of hydroxylation among mouse kidney glycolipids with three and four sugar residues. This result might be of relevance for the binding of P-fimbriated E. coli to the urinary tract tissues.     

Dissociative recombination of ammonia clusters studied by storage ring experiments

Dissociative recombination of ammonia cluster ions with free electrons has been studied at the heavy-ion storage ring CRYRING (Manne Siegbahn Laboratory, Stockholm University). The absolute cross sections for dissociative recombination of H+(NH3)2, H+(NH3)3, D+(ND3)2, and D+(ND3)3 in the collision energy range of 0.001-27 eV are reported, and thermal rate coefficients for the temperature interval from 10 to 1000 K are calculated from the experimental data and compared with earlier results. The fragmentation patterns for the two ions H+(NH3)2 and D+(ND3)2 show no clear isotope effect. Dissociative recombination of X+(NX3)2 (X=H or D) is dominated by the product channels 2NX3+X [0.95+/-0.02 for H+(NH3)2 and 1.00+/-0.02 for D+(ND3)2]. Dissociative recombination of D+(ND3)3 is dominated by the channels yielding three N-containing fragments (0.95+/-0.05).     

Simultaneous analysis of vocal fold vibration and transglottal airflow: exploring a new experimental setup

The purpose of this study was to develop an analysis system for studying the relationship between vocal fold vibration and the associated transglottal airflow. Recordings of airflow, electroglottography (EGG), oral air pressure, and acoustic signals were performed simultaneously with high-speed imaging at a rate of approximately 1900 frames/s. Inverse filtered airflow is compared with the simultaneous glottal area extracted from the high-speed image sequence. The accuracy of the synchronization between the camera images and the foot pedal synchronization pulse was examined, showing that potential synchronization errors increase with time distance to the synchronization pulse. Therefore, analysis was limited to material near the synchronization pulse. Results corroborate previous predictions that air flow lags behind area, but also they reveal that relationships between these two entities may be complex and apparently varying with phonation mode.     

Revealing Bell’s nonlocality for unstable systems in high energy physics

Entanglement and its consequences—in particular the violation of Bell inequalities, which defies our concepts of realism and locality—have been proven to play key roles in Nature by many experiments for various quantum systems. Entanglement can also be found in systems not consisting of ordinary matter and light, i.e. in massive meson–antimeson systems. Bell inequalities have been discussed for these systems, but up to date no direct experimental test to conclusively exclude local realism was found. This mainly stems from the fact that one only has access to a restricted class of observables and that these systems are also decaying. In this Letter we put forward a Bell inequality for unstable systems which can be tested at accelerator facilities with current technology. Herewith, the long awaited proof that such systems at different energy scales can reveal the sophisticated “dynamical” nonlocal feature of Nature in a direct experiment gets feasible. Moreover, the role of entanglement and CP\mathcal{CP} violation, an asymmetry between matter and antimatter, is explored, a special feature offered only by these meson–antimeson systems.     

Extremal dependence measure and extremogram: the regularly varying case

The dependence of large values in a stochastic process is an important topic in risk, insurance and finance. The idea of risk contagion is based on the idea of large value dependence. The Gaussian copula notoriously fails to capture this phenomenon. Two notions in a process or vector context which summarize extremal dependence in a function comparable to a correlation function are the extremal dependence measure (EDM) and the extremogram. We review these ideas and compare the two tools and end with a central limit theorem for a natural estimator of the EDM which allows drawing confidence bands comparable to those provided by Bartlett’s formula in a classical context of sample correlation functions.     

Radiative cooling of fullerene anions in a storage ring

Thermionic emission from hot fullerene anions, C_N ^-, has been measured in an electrostatic storage ring for even N values from 36 to 96. The decay is quenched by radiative cooling and hence the observations give information on the intensity of thermal radiation from fullerenes. The experiments are analysed by comparison with a simulation which includes the quantisation of photon energy and the statistics of emission. Experiments with heating of the molecules with a laser beam confirm the interpretation of the observations in terms of radiative cooling and give an independent estimate of the cooling rate for C₆₀ ^-. The measured cooling rates agree in general within a factor of two with the prediction from a classical dielectric model of a thermal radiation intensity of ∼ 300 eV/s for C₆₀ at 1 400 K, scaling approximately with the 6th power of the temperature and with the number of atoms in the molecule. Received 12 March 2001 and Received in final form 12 June 2001