Fix the problem - A practical guide to whole-mount immunohistochemistry of teased nerve fibres.

BACKGROUND: Immunohistochemical staining of entire nerve fibres allows for studying the molecular composition of functional fibre subunits and may add to the diagnostic value of nerve fibre teasing. NEW METHOD: In this study, we established a sealed-slide method for reproducible immunostaining of deep axoplasmic proteins in permanently straightened nerve fibres. RESULTS: Immunostaining of teased nerve fibres very much is facilitated by tip-fixation with biocompatible glass adhesives. Antibody penetration in fresh nerves can be achieved by thermic and chemical permeabilisation while enzymatic digestion allows for sufficient permeability after aldehyde fixation. COMPARISON WITH EXISTING METHODS: The methods recommended herein are easy to perform and represent a reliable and reproducible way to whole mount immunostaining. CONCLUSIONS: Sealed-slide immunostaining of tip-fixed and permeabilised nerve biopsies will help to validate neurophysiological abnormalities and to screen for target molecules and predictive markers of peripheral nerve disorders such as in inherited neuropathies and Guillain-Barré syndrome.

Nodo-paranodopathy, internodopathy and cleftopathy: Target-based reclassification of Guillain-Barré-like immune-mediated polyradiculoneuropathies in dogs and cats.

Recent views on Guillain-Barré syndrome (GBS) question the accuracy of classification into axonal and demyelinating subtypes that represent convergent neurophysiological phenotypes rather than immunological targets. Instead it has been proposed to clarify the primarily affected fibre subunit in nerve biopsies. As nerve biopsies rarely are part of routine work-up in human patients we evaluated tissues taken from companion animals affected by GBS-like polyradiculoneuropathy to screen for distribution of immune cells, targeted fibre components and segregating non-inflammatory lesions. We identified that immune responses were directed either at Schmidt-Lanterman clefts, the paranode-node complex or both. Based on infiltrative and non-inflammatory changes, four subtypes and/or stages were distinguished, some of which indicate localisation of primary target antigens while others represent convergent late stage pictures, as a consequence to epitope spreading. The impact of histological subtyping onto clinical management and prognosis remains to be evaluated in future clinical trials. Natural development and clinical manifestation of large animal dysimmune neuropathy may reflect human Guillain-Barré syndrome more accurately than experimental models and therefore provide complementary clues for translational research.

Reactive uptake studies of NO3 and N2O5 on alkenoic acid, alkanoate, and polyalcohol substrates to probe nighttime aerosol chemistry.

Heterogeneous reactions between NO3 and N2O5 and diethyl sebacate (DES), glycerol, oleic acid (OA), linoleic acid (LA), and conjugated linoleic acid (CLA) were studied to understand better nighttime aerosol chemistry. The reactive uptake coefficient of NO3 on the liquid alkenoic acids (OA, LA, and CLA) was found to be >0.07, which is higher than previous results for unsaturated organics, including alkenoic acids. This reaction could potentially be an important loss process of particle-phase unsaturated organic compounds in the atmosphere and in laboratory secondary organic aerosol studies. The reactive uptake coefficient of N2O5 on liquid glycerol was also found to be relatively large with a value of (3.2-8.5)x10(-4), suggesting that N2O5 heterogeneous reactions with alcohols may also be atmospherically relevant. For all measurements with OA, CLA, and DES, the reactive uptake coefficients decreased significantly upon freezing. One possible explanation is that the liquid reaction is due to both a surface reaction and a bulk reaction and that the freezing process significantly decreases the importance of any bulk reactions. NO3 reactive uptake coefficients for liquid-phase compounds decreased in magnitude in the order: alkenoic acids>DES>glycerol. This is different compared to previous gas-phase studies and the difference may be due to the large viscosity of glycerol compared to the other organic compounds studied. N2O5 reactive uptake coefficients for liquid-phase compounds decreased in magnitude in the order: glycerol>LA>DES congruent with OA congruent with CLA.

Reactive uptake of NO3, N2O5, NO2, HNO3, and O3 on three types of polycyclic aromatic hydrocarbon surfaces.

We investigated the reactive uptake of NO3, N2O5, NO2, HNO3, and O3 on three types of solid polycyclic aromatic hydrocarbons (PAHs) using a coated wall flow tube reactor coupled to a chemical ionization mass spectrometer. The PAH surfaces studied were the 4-ring systems pyrene, benz[a]anthracene, and fluoranthene. Reaction of NO3 radicals with all three PAHs was observed to be very fast with the reactive uptake coefficient, gamma, ranging from 0.059 (+0.11/-0.049) for benz[a]anthracene at 273 K to 0.79 (+0.21/-0.67) for pyrene at room temperature. In contrast to the NO3 reactions, reactions of the different PAHs with the other gas-phase species (N2O5, NO2, HNO3, and O3) were at or below the detection limit (gamma

Compound-specific delta13C analysis of individual amino sugars--a tool to quantify timing and amount of soil microbial residue stabilization.

There is strong scientific evidence that microbial residues such as amino sugars may be stabilized in soil. However, up to now, no investigation has been carried out to quantify both the amount and timing of such stabilization. This is primarily due to methodological constraints, because it is not possible to differentiate between stabilized (old) and recently produced (new) amino sugars when these biomarkers are conventionally analyzed, e.g. by means of gas chromatography and flame ionization detection. Therefore, the aim of the present study was to test whether compound-specific isotope analysis (delta13C) of amino sugars extracted from soil could be used to differentiate between old and new microbial residues. For this aim a method for the delta13C analysis of individual amino sugars was developed and optimized. First results of delta13C values of glucosamine, galactosamine, mannosamine, and muramic acid in soil samples from two different ecological studies are presented, clearly indicating that discrimination between soil inherent and newly formed amino sugars is possible in stable isotope labeling experiments. Our results further showed that, in the short term (within 1 month), only few amino sugars were built, thus making highly 13C-enriched substrates necessary for the quantification of new amino sugar production and for the determination of amino sugar turnover rates.

Minimization of carbon addition during derivatization of monosaccharides for compound-specific delta13C analysis in environmental research.

Little is known about the delta13C composition of monosaccharides representing the largest carbon reservoir in the biosphere. The main reason for this might be that monosaccharides have to be derivatized prior to gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analyses and that a large isotopic correction is necessary for the carbon that has to be added to the original molecule during derivatization, resulting in large uncertainty of the calculated delta13C values of individual monosaccharides. The amount of added derivatization carbon is twice (alditol acetates) or even three times (trimethylsilyl (TMS) derivatives) as high as the amount of the original monosaccharide carbon. In addition, isotope fractionation occurs during acetylation. Therefore, the objectives of this study were (i) to minimize carbon addition during derivatization for GC/C/IRMS measurements of monosaccharides in soil and sediment samples and (ii) to quantify improvements in accuracy and precision of the final results. Minimization of carbon addition was accomplished by derivatization with methylboronic acid (MBA) and TMS thereafter (MBA method). Monosaccharides derivatized with the MBA method instead of TMS reduced the number of added carbon atoms from 2.2-2.7 to 0.3-0.8 per sugar carbon atom. Although the precision of GC/C/IRMS measurements with both methods is comparable (about 0.3 per thousand), delta13C values of an internal standard indicated that the newly developed MBA method is about 2 per thousand more accurate than the TMS method. delta13C comparison between soil samples that differed only slightly in their bulk carbon isotope signature showed that the MBA method is better in proving these small differences on a significant level. Total precision of the whole MBA method including all analytical and calculation steps is better by a factor of almost three than the TMS method.