Multicentre evaluation of a new point-of-care test for the determination of NT-proBNP in whole blood

Background: The Roche CARDIAC proBNP point-of-care (POC) test is the first test intended for the quantitative determination of N-terminal pro-brain natriuretic peptide (NT-proBNP) in whole blood as an aid in the diagnosis of suspected congestive heart failure, in the monitoring of patients with compensated left-ventricular dysfunction and in the risk stratification of patients with acute coronary syndromes. Methods: A multicentre evaluation was carried out to assess the analytical performance of the POC NT-proBNP test at seven different sites. Results: The majority of all coefficients of variation (CVs) obtained for within-series imprecision using native blood samples was below 10% for both 52 samples measured ten times and for 674 samples measured in duplicate. Using quality control material, the majority of CV values for day-to-day imprecision were below 14% for the low control level and below 13% for the high control level. In method comparisons for four lots of the POC NT-proBNP test with the laboratory reference method (Elecsys proBNP), the slope ranged from 0.93 to 1.10 and the intercept ranged from 1.8 to 6.9. The bias found between venous and arterial blood with the POC NT-proBNP method was ≤5%. All four lots of the POC NT-proBNP test investigated showed excellent agreement, with mean differences of between −5% and +4%. No significant interference was observed with lipaemic blood (triglyceride concentrations up to 6.3mmol/L), icteric blood (bilirubin concentrations up to 582μmol/L), haemolytic blood (haemoglobin concentrations up to 62mg/L), biotin (up to 10mg/L), rheumatoid factor (up to 42IU/mL), or with 50 out of 52 standard or cardiological drugs in therapeutic concentrations. With bisoprolol and BNP, somewhat higher bias in the low NT-proBNP concentration range (<175ng/L) was found. Haematocrit values between 28% and 58% had no influence on the test result. Interference may be caused by human anti-mouse antibodies (HAMA) types 1 and 2. No significant influence on the results with POC NT-proBNP was found using volumes of 140-165μL. High NT-proBNP concentrations above the measuring range of the POC NT-proBNP test did not lead to false low results due to a potential high-dose hook effect. Conclusions: The POC NT-proBNP test showed good analytical performance and excellent agreement with the laboratory method. The POC NT-proBNP assay is therefore suitable in the POC setting. Clin Chem Lab Med 2006;44:1269-7

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Regulation der Stickstoffixieung in Klebsiella pneumoniae: NilL, ein Protein, das zwei Umweltsignale auf den nif-Transkriptionsaktivator NifA überträgt

Klebsiella pneumonia moduliert NifL die Aktivität des Transkriptionsaktivators NifA in Abhängigkeit der Umweltsignale Stickstoff und Sauerstoff. NifL ist ein Flavoprotein, welches das Sauerstoffsignal wahrscheinlich durch einen redoxsensitiven Konformationswechsel transduziert. Wir haben die Redoxeigenschaften von NifL, welches heterolog in Escherichia coli in Anwesenheit von molekularem Sauerstoff und unterschiedlicher Stickstoffquellen exprimiert wurde, untersucht. FAD Analysen haben gezeigt, dass gereinigtes NifL den FAD-Kofaktor unabhängig von der Sauertoff-und Stickstoffquelle während der Synthese trägt. Das Redoxpotential von NifL, welches in Anwesenheit von Ammonium als Stickstoffquelle synthetisiert wurde, betrug 277 ±5 mV bei pH 8.0 und 25 °C. Wurde NifL unter stickstofflimitierenden Bedingungen synthetisiert, betrug das Redoxpotential 274 ±6 mV bei pH 8.0 und 25 °C. Vollständig reduziertes NifL, ob unter stickstofflimitierenden oder nichtlimitierenden Bedingungen synthetisiert, reoxidierte schnell in Anwesenheit von molekularem Sauerstoff. Diese Resultate lassen den Schluss zu, dass die Stickstoffquelle während der Synthese das Redoxpotential des NifL gebundenen FADs nicht beeinflusst. Die beiden NifL Fraktionen wiesen allerdings den Unterschied auf, dass eine nicht flavinspezifische Absorption im uv/vis-Spektrum bei 420 nm nur bei NifL, welches in Anwesenheit von Ammonium synthetisiert wurde, auftrat. Durch die Beobachtung der NifA abhängigen Expression von K. pneumoniae ø(nifH-lacZ)-Fusionen in verschiedenen genetischen Hintergründen haben wir die Sauerstoffregulation der NifL Aktivität in E. coli and K. pneumoniae Stämmen untersucht. In Stämmen beider Organismen mit fnr-Deletionen konnte die Inhibierung von NifA durch NifL nicht durch Abwesenheit von Sauerstoff dereprimiert werden. Diese Phänotypen konnten durch die Synthese von Fnr in trans komplementiert werden. Fnr scheint also, direkt oder indirekt, an der NifL abhängigen Sauerstoffkontrolle der nif-Gene beteiligt zu sein. Die Rolle von Fnr in der nif-Regulation könnte in der transkriptionellen Kontrolle von Genen der Atmungskette liegen. Deshalb untersuchten wir die Lokalisation von NifL. 55 % des NifL wurde in K. pneumoniae, aerob und stickstofflimitiert gewachsen, in der Membranfraktion gefunden. Wenn die K. pneumoniae Zellen anaerob angezogen wurden und von stickstofflimitierenden Bedingungen auf nichtlimitierende Bedingungen gewechselt wurden, fand sich nur 10 % des NifL in der Membranfraktion. Die unter dereprimierende Bedingungen höhere Membranaffinitaet von NifL scheint also, eine räumliche Trennung von NifL und dem Transkriptionsaktivator NifA herzustellen. Lokalisationsexperimente mit dem primären Stickstoffsensor GlnK zeigten außerdem, dass unter stickstofflimitierenden Wachstumsbedingungen unabhängig von der Anwesenheit von molekularem Sauerstoff, 15- 20 % des GlnK membranassoziiert vorliegt. Ein Wechsel zu Stickstoffsättigung resultierte in der raschen Degradation des cytosolischen GlnK. Das membranassoziierte GlnK zeigte eine sehr viel längere Halbwertszeit. Daraus resultiert, dass ein proteolytischer Abbau von GlnK der Mechanismus für die Aktivierung von NifL beim Wechsel zu Stickstoffsättigung sein könnte. Die inhibitorische Aktivität von NifL konnte in vitro durch die Anwesenheit von ATP und ADP gesteigert werden, wenn NifL unter Stickstoffsättigung synthetisiert wurde (NifLNH4). Interessanterweise war NifLNH4 in der Lage ATP zu hydrolisierten (2500 mU/mg). Im Gegensatz dazu wurde die inhibitorische Aktivität von NifL, welches unter stickstofflimitierenden Bedingungen synthetisiert wurde, nicht von Adeninnukleotiden beeinflusst und zeigte auch keine ATP-Hydrolyseaktivität. Dies lässt den Schluss zu, dass die Stimulation der inhibitorischen Aktivität von NifL auf einer bestimmten Konformation beruht, die durch Ammonium induziert wird. Unsere Hypothese ist, dass die Anwesenheit von Ammonium die Konformation von NifL dahingegen verändert, dass NifL die Energie der ATP-Hydrolyse zur Bildung von NifL-NifA-Komplexen nutzen kann

In-Depth Profiling of Lysine-Producing Corynebacterium glutamicum by Combined Analysis of the Transcriptome, Metabolome, and Fluxome

An in-depth analysis of the intracellular metabolite concentrations, metabolic fluxes, and gene expression (metabolome, fluxome, and transcriptome, respectively) of lysine-producing Corynebacterium glutamicum ATCC 13287 was performed at different stages of batch culture and revealed distinct phases of growth and lysine production. For this purpose, (13)C flux analysis with gas chromatography-mass spectrometry-labeling measurement of free intracellular amino acids, metabolite balancing, and isotopomer modeling were combined with expression profiling via DNA microarrays and with intracellular metabolite quantification. The phase shift from growth to lysine production was accompanied by a decrease in glucose uptake flux, the redirection of flux from the tricarboxylic acid (TCA) cycle towards anaplerotic carboxylation and lysine biosynthesis, transient dynamics of intracellular metabolite pools, such as an increase of lysine up to 40 mM prior to its excretion, and complex changes in the expression of genes for central metabolism. The integrated approach was valuable for the identification of correlations between gene expression and in vivo activity for numerous enzymes. The glucose uptake flux closely corresponded to the expression of glucose phosphotransferase genes. A correlation between flux and expression was also observed for glucose-6-phosphate dehydrogenase, transaldolase, and transketolase and for most TCA cycle genes. In contrast, cytoplasmic malate dehydrogenase expression increased despite a reduction of the TCA cycle flux, probably related to its contribution to NADH regeneration under conditions of reduced growth. Most genes for lysine biosynthesis showed a constant expression level, despite a marked change of the metabolic flux, indicating that they are strongly regulated at the metabolic level. Glyoxylate cycle genes were continuously expressed, but the pathway exhibited in vivo activity only in the later stage. The most pronounced changes in gene expression during cultivation were found for enzymes at entry points into glycolysis, the pentose phosphate pathway, the TCA cycle, and lysine biosynthesis, indicating that these might be of special importance for transcriptional control in C. glutamicum

Fnr Is Required for NifL-Dependent Oxygen Control of nif Gene Expression in Klebsiella pneumoniae

In Klebsiella pneumoniae, NifA-dependent transcription of nitrogen fixation (nif) genes is inhibited by NifL in response to molecular oxygen and combined nitrogen. We recently showed that K. pneumoniae NifL is a flavoprotein, which apparently senses oxygen through a redox-sensitive, conformational change. We have now studied the oxygen regulation of NifL activity in Escherichia coli and K. pneumoniae strains by monitoring its inhibition of NifA-mediated expression of K. pneumoniae ø(nifH′-′lacZ) fusions in different genetic backgrounds. Strains of both organisms carrying fnr null mutations failed to release NifL inhibition of NifA transcriptional activity under oxygen limitation: nif induction was similar to the induction under aerobic conditions. When the transcriptional regulator Fnr was synthesized from a plasmid, it was able to complement, i.e., to relieve NifL inhibition in the fnr mutant backgrounds. Hence, Fnr appears to be involved, directly or indirectly, in NifL-dependent oxygen regulation of nif gene expression in K. pneumoniae. The data indicate that in the absence of Fnr, NifL apparently does not receive the signal for anaerobiosis. We therefore hypothesize that in the absence of oxygen, Fnr, as the primary oxygen sensor, activates transcription of a gene or genes whose product or products function to relieve NifL inhibition by reducing the flavin adenine dinucleotide cofactor under oxygen-limiting conditions