Urinary neutrophil gelatinase-associated lipocalin identifies critically ill young children with acute kidney injury following intensive care admission

__Introduction__ Children admitted to a pediatric intensive care unit (ICU) are at high risk of developing acute kidney injury (AKI). Although serum creatinine (SCr) levels are used in clinical practice, they are insensitive for early diagnosis of AKI. Urinary neutrophil gelatinase-associated lipocalin (uNGAL) and kidney injury molecule-1 (KIM-1) are novel AKI biomarkers whose performance in pediatric ICU patients is largely unknown. In this study, we aimed to characterize uNGAL and KIM-1 patterns in children following ICU admission and to assess their properties in relation to identifying children at risk for AKI development. __Methods__ From June 2010 until January 2014, we conducted a prospective observational cohort study of term-born children ages 1day to 1year on mechanical ventilation. Blood and urine samples were obtained every 6 to 12hours up to 72hours post-admission. Blood samples were assayed for SCr, and urine samples were assayed for uNGAL and KIM-1. The RIFLE (risk, injury, failure, loss, end-stage renal disease) classification as 150%, 200% or 300% of median SCr reference values was used to define AKI. __Results__ A total of 100 children were included (80 survived). Their median age at admission was 27.7days (interquartile range (IQR), 1.5 to 85.5). The median duration of mechanical ventilation was 5.8days (IQR, 3.1 to 11.4). Thirty-five patients had evidence of AKI within the first 48hours post-admission, of whom 24 (69%) already had AKI when they entered the ICU. uNGAL and KIM-1 concentrations in AKI peaked between 6 to 12hours and between 12 to 24hours post-admission, respectively. The maximal area under the receiver operating characteristic curve (AUC) for uNGAL was 0.815 (95% confidence interval (CI), 0.685 to 0.945, P <0.001) at 0 to 6hours post-admission. The discriminative ability of KIM-1 was moderate, with a largest AUC of 0.737 (95% CI, 0.628 to 0.847; P <0.001) at 12 to 24hours post-admission. At the optimal cutoff point (126ng/ml), uNGAL concentration predicted AKI development correctly in 16 (84%) of 19 children, up to 24hours before a rise in SCr became apparent. __Conclusions__ Levels of uNGAL and KIM-1 increase in patients with AKI following ICU admission and peak at 6 to 12hours and 12 to 24hours post-admission, respectively. uNGAL seems to be a reliable marker for identifying children who will develop AKI 24hours later

MEK and PI3K-AKT inhibitors synergistically block activated IL7 receptor signaling in T-cell acute lymphoblastic leukemia

We identified mutations in the IL7Ra gene or in genes encoding the downstream signaling molecules JAK1, JAK3, STAT5B, N-RAS, K-RAS, NF1, AKT and PTEN in 49% of patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL). Strikingly, these mutations (except RAS/NF1) were mutually exclusive, suggesting that they each cause the aberrant activation of a common downstream target. Expressing these mutant signaling molecules—but not their wild-type counterparts—rendered Ba/F3 cells independent of IL3 by activating the RAS-MEK-ERK and PI3K-AKT pathways. Interestingly, cells expressing either IL7Ra or JAK mutants are sensitive to JAK inhibitors, but respond less robustly to inhibitors of the downstream RAS-MEK-ERK and PI3K-AKT-mTOR pathways, indicating that inhibiting only one downstream pathway is not sufficient. Here, we show that inhibiting both the MEK and PI3K-AKT pathways synergistically prevents the proliferation of BaF3 cells expressing mutant IL7Ra, JAK and RAS. Furthermore, combined inhibition of MEK and PI3K/AKT was cytotoxic to samples obtained from 6 out of 11 primary T-ALL patients, including 1 patient who had no mutations in the IL7R signaling pathway. Taken together, these results suggest that the potent cytotoxic effects of inhibiting both MEK and PI3K/AKT should be investigated further as a therapeutic option using leukemia xenograft models.Leukemia advance online publication, 13 May 2016; doi:10.1038/leu.2016.83

Quenched phosphorescence as alternative detection mode in the chiral separation of methotrexate by electrokinetic chromatography

Quenched phosphorescence was used, for the first time, as detection mode in the chiral separation of methotrexate (MTX) enantiomers by electrokinetic chromatography. The detection is based on dynamic quenching of the strong emission of the phosphorophore 1-bromo-4-naphthalene sulfonic acid (BrNS) by MTX under deoxygenated conditions. The use of a background electrolyte with 3 mg/mL 2-hydroxypropyl-β-cyclodextrin and 20% MeOH in 25 mM phosphate buffer (pH 7.0) and an applied voltage of 30 kV allowed the separation of l-MTX and its enantiomeric impurity d-MTX with sufficient resolution. In the presence of 1 mM BrNS, a detection limit of 3.2 × 10−7 M was achieved, about an order of magnitude better than published techniques based on UV absorption. The potential of the method was demonstrated with a degradation study and an enantiomeric purity assessment of l-MTX. Furthermore, l-MTX was determined in a cell culture extract as a proof-of-principle experiment to show the applicability of the method to biological samples

Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on-line biochemical assays

One way to profile complex mixtures for receptor affinity is to couple liquid chromatography (LC) on-line to biochemical detection (BCD). A drawback of this hyphenated screening approach is the relatively high consumption of sample, receptor protein and (fluorescently labeled) tracer ligand. Here, we worked toward minimization of sample and reagent consumption, by coupling nano-LC on-line to a light-emitting diode (LED) based capillary confocal fluorescence detection system capable of on-line BCD with low-flow rates. In this fluorescence detection system, a capillary with an extended light path (bubble cell) was used as a detection cell in order to enhance sensitivity. The technology was applied to a fluorescent enhancement bioassay for the acetylcholine binding protein, a structural analog of the extracellular ligand-binding domain of neuronal nicotinic acetylcholine receptors. In the miniaturized setup, the sensitive and low void volume LED-induced confocal fluorescence detection system operated in flow injection analysis mode allowing the measurement of IC(50) values, which were comparable with those measured by a conventional plate reader bioassay. The current setup uses 50 nL as injection volume with a carrier flow rate of 400 nL/min. Finally, coupling of the detection system to gradient reversed-phase nano-LC allowed analysis of mixtures in order to identify the bioactive compounds present by injecting 10 nL of each mixture

pH-Dependent Complexation of Histamine H₁ Receptor Antagonists and Human Serum Albumin Studied by UV Resonance Raman Spectroscopy

UV resonance Raman spectroscopy was used to characterize the binding of three first-generation histamine H₁ receptor antagoniststripelennamine (TRP), mepyramine (MEP), and brompheniramine (BPA)to human serum albumin (HSA) at pH 7.2 and pH 9.0. Binding constants differ at these pH values, which can be ascribed to the different extent of protonation of the ethylamino side chain of the ligands. We have recently shown [Tardioli et al. <i>J. Raman Spectrosc.</i> <b>2011</b>, <i>42</i>, 1016–1024] that for the solution conformation of TRP and MEP the side chain plays an important role by allowing an internal hydrogen bond with the aminopyridine nitrogen in TRP and MEP. Results presented in this paper suggest that the existence of such molecular structures has serious biological significance on the binding affinity of those ligands to HSA. At pH 7.2, only the stretched conformers of protonated TRP and MEP bind in HSA binding site I. Using UV absorption data, we derived binding constants for the neutral and protonated forms of TRP to HSA. The neutral species seems to be conjugated to a positive group of the protein, affecting both the tryptophan W214 and some of the tyrosine (Y) vibrations. BPA, for which the structure with an intramolecular hydrogen bonded side chain is not possible, is H bound to the indole ring nitrogen of W214, of which the side chain rotates over a certain angle to accommodate the drug in site I. We propose that the protonated BPA is also bound in site I, where the Y150 residue stabilizes the presence of this compound in the binding pocket. No spectroscopic evidence was found for conformational changes of the protein affecting the spectroscopic properties of W and Y in this pH range

Regioscan Zoetwatermaatregelen fase 2 : hoofdrapport

Dit hoofdrapport bevat de achtergronden bij de tweede versie van de Regioscan Zoetwatermaatregelen. Met Regioscan Zoetwatermaatregelen 2.0 kan het effect van lokale maatregelen aan de zoetwateropgave nog beter in beeld worden gebracht en is nu landsdekkend toepasbaar. De kansrijkheid van maatregelen kan zo snel worden ingeschat en gebruikt worden om met zoetwatergebruikers het gesprek aan te gaan