Quantitative studies are commonly realised in the biomedical research to compare RNA expression in different experimental or clinical conditions. These quantifications are performed through their comparison to the expression of the housekeeping gene transcripts like glyceraldehyde-3-phosphate dehydrogenase (G3PDH), albumin, actins, tubulins, cyclophilin, hypoxantine phosphoribosyltransferase (HRPT), L32. 28S and 18S rRNAs are also used as internal standards. In this paper, it is recalled that the commonly used internal standards can quantitatively vary in response to various factors. Possible variations are illustrated using three experimental examples. Preferred types of internal standards are then proposed for each of these samples and thereafter the general procedure concerning the choice of an internal standard and the way to manage its used are discussed. © 1999 Elsevier Science B.V. All rights reserved.Keywords: Internal standards; Housekeeping genes; RNase protection; RT-PCR www.elsevier.com/locate/jbiotec Quantitative assays widely use housekeeping gene transcripts as b-actin, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) or L32 whose presumed stable expression allows quantification of other expressions, for example those of cytokines, by comparison to this internal standard. In this paper, a series of in vivo and in vitro models are presented using housekeeping genes showing in certain cases the limits at the use of such internal standards. Different possible methods enabling the management of this problem will be discussed.The study of biological regulations is very often correlated to quantification assays, which can be related to proteins or RNA. This paper will discuss the problem of mRNA quantification.Abbre6iations: G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HPRT, hypoxantine phosphoribosyltransferase; PMA, phorbol 10-myristate 13-acetate.

Among its penicillin-binding proteins (PBPs), Enterococcus faecium possesses a low-affinity PBP5, PBP5fm, which is the main target involved in ␤-lactam resistance. A 7.7-kb EcoRI chromosomal fragment of E. faecium D63r containing the pbp5fm gene was cloned and sequenced. Two open reading frames (ORFs) were found. A 2,037-bp ORF encoded the deduced 73.8-kDa PBP5fm, the amino acid sequences of which were, respectively, 99.8, 78.5, and 62% homologous to those of the low-affinity plasmid-encoded PBP3r of Enterococcus hirae S185r and the chromosome-encoded PBP5 of E. hirae R40 and Enterococcus faecalis 56R. A second 597-bp ORF, designated psrfm, was found 2.3 kb upstream of pbp5fm. It appeared to be 285 bp shorter than and 74% homologous with the regulatory gene psr of E. hirae ATCC 9790. Different clinical isolates of E. faecium, for which a wide range of benzylpenicillin MICs were observed, showed that the increases in MICs were related to two mechanisms. For some strains of intermediate resistance (MICs of 16 to 64 g/ml), the increased level of resistance could be explained by the presence of larger quantities of PBP5fm which had an affinity for benzylpenicillin (second-order rate constant of protein acylation [k ؉2 /K] values of 17 to 25 M ؊1 s ؊1) that remained unchanged. For the two most highly resistant strains, EFM-1 (MIC, 90 g/ml) and H80721 (MIC, 512 g/ml), the resistance was related to different amino acid substitutions yielding very-low-affinity PBP5fm) which were synthesized in small quantities. More specifically, it appeared, with a three-dimensional model of the C-terminal domain of PBP5fm, that the substitutions of Met-485, located in the third position after the conserved SDN triad, by Thr in EFM-1 and by Ala in H80721 were the most likely cause of the decreasing affinity of PBP5fm observed in these strains.The genus Enterococcus, closely related to the genus Streptococcus, is involved in different clinical infections (37). Apart from their physiological properties, enterococci also differ from streptococci in that they generally are naturally 10-to 1,000-fold less susceptible to penicillins than streptococci (37). It was demonstrated that the natural low susceptibility of enterococci to penicillin is linked to the presence of at least one high-molecular-mass penicillin-binding protein (PBP) which has a low affinity for ␤-lactams (1, 17, 50). Enterococcus faecium appears to be the enterococcal species most resistant to ␤-lactam antibiotics, for which there are a wide range of benzylpenicillin MICs (0.5 to Ն 64 g/ml) for clinical isolates (22,30,50). Recently it became obvious that a new population of clinical E. faecium isolates for which the MICs of benzylpenicillin were very high (256 to 512 g/ml) had emerged in different countries (15,22,23,30).Among laboratory mutants and clinical isolates of E. faecium, two mechanisms have been shown to be involved in the high-level resistance to benzylpenicillin. These strains produced either an increased quantity of the essential low-affinity PBP5fm (15, 17) o...

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The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or shape determination. Up to now it has, however, been very difficult or impossible to study the catalytic properties of the HMM-PBPs in vitro. With simple substrates, we could demonstrate that several of these proteins could catalyse the hydrolysis of some thioesters or the transfer of their acyl moiety on the amino group of a suitable acceptor nucleophile. Many of the acyl-donor substrates were hippuric acid or benzoyl-D-alanine derivatives, and their spectroscopic properties enabled a direct monitoring of the enzymic reaction. In their presence, the binding of radioactive penicillin to the PBPs was also inhibited.

In recent years, various mass spectrometry procedures have been developed for bacterial identification. The accuracy and speed with which data can be obtained by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) could make this a powerful tool for environmental monitoring. However, minor variations in the sample preparation can influence the mass spectra significantly. Therefore, the first objectives of this study were the adjustment and the optimization of experimental parameters allowing a rapid identification of whole bacterial cells without laborious sample preparation. The tested experimental parameters were matrix, extraction solvent, salt content, deposition method, culture medium and incubation time. This standardized protocol was applied to identify reference and environmental bacterial strains of Escherichia coli, Salmonella and Acinetobacter. The environmental bacterial strains were isolated from sewage sludge using an original microextraction procedure based on repeated sonications and enzymatic treatments. The bacterial identification was realized by the observation of the respective genus‐, species‐ and strain‐specific biomarkers. This bacterial taxonomy could be completed within one hour, with minimal sample preparation, provided that sufficient bacteria had been collected prior to MALDI‐TOF analysis. Copyright © 2004 John Wiley & Sons, Ltd.

Tau proteins and amyloid-β (Aβ) peptides are the current recognized cerebrospinal fluid (CSF) biomarkers used as an aid in the diagnosis of Alzheimer's disease (AD). However, there is no consensus on their clinical use due to non-qualified cut-off values, probably related to the observed high pre-analytical and analytical variability. Standardized pre-analytical protocols have therefore been proposed. Importantly, these recommend the use of polypropylene collection/sampling tubes while, to date, no broad comparison of these types of tubes has been conducted. In this study, we first compared, as part of a real clinical workflow, the impact of four different collection tubes on the CSF concentration of Aβ peptides (Aβ42, Aβ40) and total (hTau) and phosphorylated (P-Tau181P) tau proteins measured using routine ELISA kits. We then extended this study to 11 polypropylene tubes used by different clinical laboratories, and investigated their plastic polymer composition using differential scanning calorimetry and Fourier Transformed Infrared spectroscopy. Significant concentration variations linked solely to the use of different types of tubes were observed. This was particularly marked for Aβ peptides, with >50% disparity occurring in less than five minutes. Polymer composition analysis revealed that most polypropylene tubes were in fact copolymers with at least polyethylene. There was no clear correlation between tube composition and pre-analytical behavior. Our results show that the use of polypropylene tubes does not guarantee satisfactory pre-analytical behavior. They also point to collection/sampling tubes being a major pre-analytical source of variability that could impact the significance of AD biological diagnosis.

IMPORTANCE Although typical forms of Alzheimer disease (AD) and Creutzfeldt-Jakob disease (CJD) are clinically distinguishable, atypical AD phenotypes may pose a diagnostic challenge. The major biological diagnostic biomarker for identifying CJD, 14-3-3 protein in cerebrospinal fluid (CSF), unfortunately lacks specificity when confronting a rapid dementia presentation. OBJECTIVE To assess the relevance of total CSF prion protein (t-PrP) levels in the differential biological diagnosis between atypical AD phenotypes and CJD. DESIGN, SETTING, AND PARTICIPANTS A retrospective study in an autopsy-confirmed cohort of 82 patients was performed to evaluate the relevance of CSF t-PrP to distinguish 30 definite cases of AD from 52 definite cases of CJD. Next, CSF t-PrP concentration was measured in a cohort of 104 patients including 55 patients with probable AD, 26 with probable sporadic CJD, and 23 control patients for whom 14-3-3 protein, total tau, phosphorylated tau 181 (P-tau 181), and Aβ1-42 were available. We investigated 46 patients diagnosed as having probable AD who presented atypical phenotypes. A diagnosis strategy was proposed to classify atypical AD phenotypes with suspicion of CJD based on a decision tree combining CSF biomarkers. MAIN OUTCOMES AND MEASURES We determined CSF t-PrP levels for all patients. We calculated the ratio of total tau and P-tau 181 and determined the diagnostic accuracy of each biomarker alone or in combination. We calculated the misclassification rate for each biomarker that corresponded to the percentage of patients within the group of atypical AD phenotypes wrongly classified as CJD. RESULTS In patients with CJD, CSF t-PrP concentrations were decreased compared with control participants and patients with AD. When considering the differential diagnosis of CJD compared with atypical AD phenotypes, CSF t-PrP determination reached 82.1% sensitivity and 91.3% specificity. The misclassification rate of atypical AD phenotypes decreased from 43.5%, obtained when using the CSF 14-3-3 protein determination alone, to only 4.3% when calculating the ratio total tau/(P-tau 181 × t-PrP). The proposed classification tree permitted correct classification of 98.4% of the patients. CONCLUSIONS AND RELEVANCE For unusual phenotypes of AD, especially cases presenting with a biological ambiguity suggesting CJD, determination of CSF t-PrP levels increased diagnostic accuracy. The use of CSF t-PrP levels may be beneficial in clinical practice in addition to the current classic biomarkers.

Abstract. Potential sterilization/disinfection of medical devices (MDs) is investigated using a specific plasma process developed at the Université de Montréal over the last decade. The inactivating medium of the microorganisms is the flowing afterglow of a reduced-pressure N2-O2 discharge, which provides, as the main biocidal agent, photons over a broad ultraviolet (UV) wavelength range. The flowing afterglow is considered less damaging to MDs than the discharge itself. Working at gas pressures in the 400-700 Pa range (a few torr) ensures, through species diffusion, the uniform filling of large volume chambers with the species outflowing from the discharge, possibly allowing batch processing within them. As a rule, bacterial endospores are used as bio-indicators (BI) to validate sterilization processes. Under the present operating conditions, Bacillus atrophaeus is found to be the most resistant one and is therefore utilized as BI. The current paper reviews the main experimental results concerning the operation and characterization of this sterilizer/disinfector, updating and completing some of our previously published papers. It uses modeling results as guidelines, which are particularly useful when the corresponding experimental data are not (yet) available, hopefully leading to more insight into this plasma afterglow system. The species flowing out of the N 2-O2 discharge can be divided into two groups, depending on the time elapsed after they left the discharge zone as they move toward the chamber, namely the early afterglow and the late afterglow. The early flowing afterglow from a pure N2 discharge (also called pink afterglow) is known to be comprised of N + 2 and N + 4 ions. In the present N2-O2 mixture discharge, NO + ions are additionally generated, with a lifetime that extends over a longer period than that of the nitrogen molecular ions. We shall suppose that the disappearance of the NO + ions marks the end of the early afterglow regime, thereby stressing our intent to work in an ion-free process chamber to minimize damage to MDs. Therefore, operating conditions should be set such that the sterilizer/disinfector chamber is predominantly filled by N and O atoms, possibly together with long-lived metastable-state O2( 1 Δg) (singlet-delta) molecules. Various aspects related to the observed survival curves are examined: the actual existence of two "phases" in the inactivation rate, the notion of UV irradiation dose (fluence) and its implications, the UV photon best wavelength range in terms of inactivation efficiency, the influence of substrate temperature and the reduction of UV intensity through surface recombination of N and O atoms on the object/packaging being processed. To preserve their on-shelf sterility, MDs are sealed/wrapped in packaging material. Porous packaging materials utilized in conventional sterilization systems (where MDs are packaged before being subjected to sterilization) were tested and found inadequate for the N 2-O2 afterglow system in contrast to a (non-porous) polyolefin polym...