Detection of small amounts of human adenoviruses in stools: comparison of a new immuno real-time PCR assay with classical tools.

The detection of low virus concentrations in biological matrices, especially stool samples, is facing significant limitations as far as common diagnostic methods (enzyme-linked-immunosorbent assay (ELISA) or quantitative real-time PCR (qPCR)) are considered. Here the development of a new immuno real-time PCR (iPCR) is described and its performance in the detection of human adenoviruses (HAdVs) in spiked stools is compared with those of ELISA and qPCR assays. For the iPCR, detection of the sandwich formed by the complexation of capture antibody-antigen-detection antibody was performed by qPCR thanks to the substitution of peroxydase by a chimeric DNA. This modification increased the detection sensitivity 200-fold compared to ELISA. The direct qPCR results revealed that only 0.3-9.5% of the spiked HAdV were detectable, resulting from important losses of DNA occurring at the extraction step. This step was not necessary in the iPCR workflow, avoiding this drawback. The losses of viral particles occurred at the elution step from the stool only. The recovery rate of the iPCR was thus better and ranged between 21 and 54%. As a result, iPCR enabled the detection of lower virus concentrations in stool samples compared to those detected by ELISA and qPCR. The iPCR could be considered as a 'hyper sensitive ELISA' for early detection of HAdV infections, especially in the case of immunocompromised patients after haematopoietic stem cell transplant.

How to control the recombinant prion protein adhesion for successful storage through modification of surface properties.

Depletion of neuroproteins on the inner walls of storage tubes influences the accuracy of tests used for identification of various neurodegenerative disorders. In this paper, a strategy is described for surface modification of Eppendorf tubes leading to non-adhesive properties towards the recombinant human prion proteins (PrPrec(hum)). Tubes were pre-activated by helium plasma and grafted with three diverse coatings: pure poly(N-isopropylacrylamide) (PNIPAM), PNIPAM admixed with either neutral PEG(20)sorbitan monolaurate (PEG(20)) or positively charged cetyl trimethylammonium bromide (CTAB) at varying plasma activation times and polymer to surfactant ratios. New functionalized surfaces were analyzed by goniometry, streaming potential measurement and X-ray photoelectron spectroscopy, whereas the protein adhesion was monitored by enzyme linked immunosorbent assays and confocal microscopy. The mapping of PrPrec(hum) adhesion associated with surface analyses enabled us to determine that no or negligible depletion of PrPrec(hum) can be obtained by surfaces possessing basic component in the range between 50 and 60 mJ m(-2) and streaming potential ζ(7.4) - -50 mV.

Improvement of the detection of neurodegenerative Alzheimer's disease through a specific surface chemistry applied onto the inner surface of the titration well.

The main objective of this paper was to illustrate the enhancement of the sensitivity of the ELISA titration of Tau proteins while reducing other non-specific adsorptions that could increase the optical densities and could lead to false positives. This goal was obtained thanks to the association of cold plasma and wet chemistries of the inner surface of the titration well. The PP surface was cold plasma-activated, then coated with different amphiphilic molecules bearing either ionic charges and/or long hydrocarbon chains. The support treated and coated with hexatrimethylammonium bromide improves the signal detection of proteins while reducing the background due to non-specific associations of biomolecules such as hyperphosphorylated Tau protein. However, coating with 3-butenylamine hydrochloride could also be suitable.

Study of the adhesion of neurodegenerative proteins on plasma-modified and coated polypropylene surfaces.

The inner polymeric surface of an ELISA titration well is plasma-modified and coated with different surfactant molecules. The titration of neurodegenerative proteins markers (prion, Tau and ß-synuclein), previously demonstrated as more efficient with such modified tubes, is related to the adhesion behaviour of these proteins and their corresponding capture antibodies. The adhesion process is studied in terms of anchoring and specific mechanisms. The proteins and antibodies binding onto such modified surfaces is related to the substrate hydrophilic character calculated from the angle contact measure, to the polymer surface charge measured through the streaming potential determination at different pH and the inner surface roughness determined from AFM images. Furthermore, the influence of the blocking agent used during the ELISA titration is also studied.

Transport of arginine and ornithine into isolated mitochondria of Saccharomyces cerevisiae.

In this work we have characterised the transport of L-arginine and L-ornithine into mitochondria isolated from a wild-type Saccharomyces cerevisiae strain and an isogenic arg11 knock-out mutant. The Arg11 protein (Arg11p) is a mitochondrial carrier required for arginine biosynthesis [Crabeel, M., Soetens, O., De Rijcke, M., Pratiwi, R. & Pankiewicz, R. (1996) J. Biol. Chem. 271, 25011-25019]. Reconstitution experiments have confirmed that it is an L-ornithine carrier also transporting L-arginine and L-lysine by order of decreasing affinity, but not L-histidine [Palmieri, L., De Marco, V., Iacobazzi, V., Palmieri, F., Runswick, M. & Walker, J. (1997) FEBS Lett. 410, 447-451]. Evidence is presented here that the mitochondrial inner membrane contains an L-arginine and L-ornithine transporting system distinct from Arg11p, in keeping with the arginine leaky phenotype of arg11 knock-out mutants. The newly characterised carrier, which we propose to name Bac1p (basic amino acid carrier), behaves as an antiporter catalysing the electroneutral exchange of the basic amino acids L-arginine, L-lysine, L-ornithine and L-histidine and displays the highest affinity for L-arginine (Km of 30 microM). L-Arginine uptake has a pH optimum in the range of 7.5-9 and is inhibited by several sulphydryl reagents, by pyridoxal 5'-phosphate and by cations.

Phylogenetic classification of the mitochondrial carrier family of Saccharomyces cerevisiae.

The screening of the open reading frames identified in the whole yeast genome has allowed us to discover 34 proteins belonging to the mitochondrial carrier family. By phylogenetic study, they can be divided into 27 subfamilies including ADP/ATP, phosphate and citrate carriers, putative oxoglutarate and GDC carriers and 22 new subfamilies. Topology predictions using the 'positive inside rule' approach have shown that the yeast carriers are similarly oriented with both extremities exposed to the cytosol. In each subfamily, a strict conservation of the charged residues in the six transmembrane alpha-helices is observed, suggesting a functional role for these residues and the existence of 27 functionally distinct carriers.