Rapid enzymatic test for phenotypic HIV protease drug resistance

A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid onestep procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIVinfected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for highthroughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (K-i). The Michaelis constant (K-m) and the inhibition constant (K-i) of recombinant wildtype protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIVpositive patients scored from sensitive to highly resistant according to mutations detected by genotypic analysis. The measured K-i values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The noninfectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays

Performance Assessment in Fingerprinting and Multi Component Quantitative NMR Analyses

An interlaboratory comparison (ILC) was organized with the aim to set up quality control indicators suitable for multicomponent quantitative analysis by nuclear magnetic resonance (NMR) spectroscopy. A total of 36 NMR data sets (corresponding to 1260 NMR spectra) were produced by 30 participants using 34 NMR spectrometers. The calibration line method was chosen for the quantification of a five-component model mixture. Results show that quantitative NMR is a robust quantification tool and that 26 out of 36 data sets resulted in statistically equivalent calibration lines for all considered NMR signals. The performance of each laboratory was assessed by means of a new performance index (named Qp-score) which is related to the difference between the experimental and the consensus values of the slope of the calibration lines. Laboratories endowed with a Qp-score falling within the suitable acceptability range are qualified to produce NMR spectra that can be considered statistically equivalent in terms of relative intensities of the signals. In addition, the specific response of nuclei to the experimental excitation/relaxation conditions was addressed by means of the parameter named NR. NR is related to the difference between the theoretical and the consensus slopes of the calibration lines and is specific for each signal produced by a well-defined set of acquisition parameters

Rapid enzymatic test for phenotypic HIV protease drug resistance

A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid onestep procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIVinfected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for highthroughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (K-i). The Michaelis constant (K-m) and the inhibition constant (K-i) of recombinant wildtype protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIVpositive patients scored from sensitive to highly resistant according to mutations detected by genotypic analysis. The measured K-i values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The noninfectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays

Body Fat Free Mass Is Associated with the Serum Metabolite Profile in a Population-Based Study

To characterise the influence of the fat free mass on the metabolite profile in serum samples from participants of the population-based KORA (Cooperative Health Research in the Region of Augsburg) S4 study. Analyses were based on metabolite profile from 965 participants of the S4 and 890 weight-stable subjects of its seven-year follow-up study (KORA F4). 190 different serum metabolites were quantified in a targeted approach including amino acids, acylcarnitines, phosphatidylcholines (PCs), sphingomyelins and hexose. Associations between metabolite concentrations and the fat free mass index (FFMI) were analysed using adjusted linear regression models. To draw conclusions on enzymatic reactions, intra-metabolite class ratios were explored. Pairwise relationships among metabolites were investigated and illustrated by means of Gaussian graphical models (GGMs). We found 339 significant associations between FFMI and various metabolites in KORA S4. Among the most prominent associations (p-values 4.75 × 10(-16)-8.95 × 10(-06)) with higher FFMI were increasing concentrations of the branched chained amino acids (BCAAs), ratios of BCAAs to glucogenic amino acids, and carnitine concentrations. For various PCs, a decrease in chain length or in saturation of the fatty acid moieties could be observed with increasing FFMI, as well as an overall shift from acyl-alkyl PCs to diacyl PCs. These findings were reproduced in KORA F4. The established GGMs supported the regression results and provided a comprehensive picture of the relationships between metabolites. In a sub-analysis, most of the discovered associations did not exist in obese subjects in contrast to non-obese subjects, possibly indicating derangements in skeletal muscle metabolism. A set of serum metabolites strongly associated with FFMI was identified and a network explaining the relationships among metabolites was established. These results offer a novel and more complete picture of the FFMI effects on serum metabolites in a data-driven network

Simplivariate Models: Uncovering the Underlying Biology in Functional Genomics Data

One of the first steps in analyzing high-dimensional functional genomics data is an exploratory analysis of such data. Cluster Analysis and Principal Component Analysis are then usually the method of choice. Despite their versatility they also have a severe drawback: they do not always generate simple and interpretable solutions. On the basis of the observation that functional genomics data often contain both informative and non-informative variation, we propose a method that finds sets of variables containing informative variation. This informative variation is subsequently expressed in easily interpretable simplivariate components

Human Cysteine Cathepsins Are Not Reliable Markers of Infection by Pseudomonas aeruginosa in Cystic Fibrosis

Cysteine cathepsins have emerged as new players in inflammatory lung disorders. Their activities are dramatically increased in the sputum of cystic fibrosis (CF) patients, suggesting that they are involved in the pathophysiology of CF. We have characterized the cathepsins in CF expectorations and evaluated their use as markers of colonization by Pseudomonas aeruginosa. The concentrations of active cathepsins B, H, K, L and S were the same in P. aeruginosa-positive (19 Ps+) and P. aeruginosa-negative (6 Ps−) samples, unlike those of human neutrophil elastase. Also the cathepsin inhibitory potential and the cathepsins/cathepsin inhibitors imbalance remained unchanged and similar (∼2-fold) in the Ps+ and Ps− groups (p<0.001), which correlated with the breakdown of their circulating cystatin-like inhibitors (kininogens). Procathepsins, which may be activated autocatalytically, are a potential proteolytic reservoir. Immunoblotting and active-site labeling identified the double-chain cathepsin B, the major cathepsin in CF sputum, as the main molecular form in both Ps+ and Ps− samples, despite the possible release of the ∼31 kDa single-chain form from procathepsin B by sputum elastase. Thus, the hydrolytic activity of cysteine cathepsins was not correlated with bacterial colonization, indicating that cathepsins, unlike human neutrophil elastase, are not suitable markers of P. aeruginosa infection