FLT3 Mutations in Early T-Cell Precursor ALL Characterize a Stem Cell Like Leukemia and Imply the Clinical Use of Tyrosine Kinase Inhibitors

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.     

PKC-beta1 isoform activation is required for EGF-induced NF-kappaB inactivation and IkappaBalpha stabilization and protection of F-actin assembly and barrier function in enterocyte monolayers

Using monolayers of intestinal Caco-2 cells, we reported that activation of NF-kappaB is required for oxidative disruption and that EGF protects against this injury but the mechanism remains unclear. Activation of the PKC-beta1 isoform is key to monolayer barrier integrity. We hypothesized that EGF-induced activation of PKC-beta1 prevents oxidant-induced activation of NF-kappaB and the consequences of NF-kappaB activation, F-actin, and barrier dysfunction. We used wild-type (WT) and transfected cells. The latter were transfected with varying levels of cDNA to overexpress or underexpress PKC-beta1. Cells were pretreated with EGF or PKC modulators +/- oxidant. Pretreatment with EGF protected monolayers by increasing native PKC-beta1 activity, decreasing IkappaBalpha phosphorylation/degradation, suppressing NF-kappaB activation (p50/p65 subunit nuclear translocation/activity), enhancing stable actin (increased F-actin-to-G-actin ratio), increasing stability of actin cytoskeleton, and reducing barrier hyperpermeability. Cells stably overexpressing PKC-beta1 were protected by low, previously nonprotective doses of EGF or modulators. In these clones, we found enhanced IkappaBalpha stabilization, NF-kappaB inactivation, actin stability, and barrier function. Low doses of the modulators led to increases in PKC-beta1 in the particulate fractions, indicating activation. Stably inhibiting endogenous PKC-beta1 substantially prevented all measures of EGF's protection against NF-kappaB activation. We conclude that EGF-mediated protection against oxidant disruption of the intestinal barrier function requires PKC-beta1 activation and NF-kappaB suppression. The molecular event underlying this unique effect of PKC-beta1 involves inhibition of phosphorylation and increases in stabilization of IkappaBalpha. The ability to inhibit the dynamics of NF-kappaB/IkappaBalpha and F-actin disassembly is a novel mechanism not previously attributed to the classic subfamily of PKC isoforms.     

Theta-isoform of PKC is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-theta

Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the -isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC- is crucial for changes in cytoskeletal and barrier dynamics. We have created the first multiple sets of gastrointestinal cell clones transfected with varying levels of cDNA to stably inhibit native PKC- (antisense, AS; dominant negative, DN) or to express its activity (sense). We studied transfected and WT Caco-2 cells. First, relative to WT cells, AS clones underexpressing PKC- showed monolayer injury as indicated by decreased native PKC- activity, reduced tubulin phosphorylation, increased tubulin disassembly (decreased polymerized and increased monomeric pools), reduced architectural integrity of microtubules, reduced stability of occludin, and increased barrier hyperpermeability. In these AS clones, PKC- was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC- was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-/tubulin complex. Second, DN transfection to inhibit the endogenous PKC- led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC- led to a mostly cytosolic distribution of -isoform (<10% in particulate fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased barrier dysfunction. In conclusion, 1) PKC- isoform is required for changes in the cytoskeletal assembly and barrier permeability in intestinal monolayers, and 2) the molecular event underlying this novel biological effect of PKC- involves changes in phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the cytoskeletal and barrier dynamics is a unique function not previously attributed to PKC-. microtubules; tubulin; occludin; epithelial barrier permeability; protein kinase C isoform     

Gas chromatographic method for detection of urinary sucralose: application to the assessment of intestinal permeability

We developed a capillary column gas chromatography (CCGC) method for the measurement of urinary sucralose (S) and three other sugar probes including, sucrose, lactulose (L) and mannitol (M) for use in in vivo studies of intestinal permeability. We compared the capillary method with a packed column gas chromatography (PCGC) method. We also investigated a possible role for sucralose as a probe for the measurement of whole gut permeability. Sample preparation was rapid and simple. The above four sugars were detected precisely, without interference. We measured intestinal permeability using 5- and 24-h urine collections in 14 healthy volunteers. The metabolism of sugars was evaluated by incubating the intestinal bacteria with an iso-osmolar mixture of mannitol, lactulose and sucralose at 37 degrees C for 19 h. Sugar concentrations and the pH of the mixture were monitored. The use of the CCGC method improved the detection of sucralose as compared to PCGC. The average coefficient of variation decreased from 15% to 4%. It also increased the sensitivity of detection by 200-2000-fold. The GC assay was linear between sucralose concentrations of 0.2 and 40 g/l (r=1.000). Intestinal bacteria metabolized lactulose and acidified the media but did not metabolize sucralose or mannitol. The new method for the measurement of urinary sucralose permits the simultaneous quantitation of sucrose, mannitol and lactulose, and is rapid, simple, sensitive, accurate and reproducible. Because neither S nor M is metabolized by intestinal bacteria, and because only a tiny fraction of either sugar is absorbed, this pair of sugar probes appears to be available for absorption throughout the GI tract. Thus, the 24-h urinary concentrations of S and M, or the urinary S/M ratio following an oral dose of a sugar mixture, might be good markers for whole gut permeability.     

The Efficacy of Salicylic Acid Levels on Photosynthetic Activity,Growth, and Essential Oil Content and Composition of Pennyroyal Plants Under Salt Stress

Journal of Plant Growth Regulation - This research was conducted to find out whether the foliar application of salicylic acid (SA) at 0.5, 1, and 1.5&nbsp;mM in comparison with water spray...     

Fiber-based liquid-phase micro-extraction of mebeverine enantiomers followed by chiral high-performance liquid chromatography analysis and its application to pharmacokinetics study in rat plasma

The applicability of two-phase liquid-phase micro-extraction (LPME) in porous hollow polypropylene fiber for the sample preparation and the stereoselective pharmacokinetics of mebeverine (MEB) enantiomers (an antispasmodic drug) in rat after intramuscular administration were studied. Plasma was assayed for MEB enantiomer concentrations using stereospecific high-performance liquid chromatography with ultraviolet detection after a simple, inexpensive, and efficient preconcentration and clean-up hollow fiber-based LPME. Under optimized micro-extraction conditions, MEB enantiomers were extracted with 25 μl of 1-octanol within a lumen of a hollow fiber from 0.5 ml of plasma previously diluted with 4.5 ml alkalized water (pH 10). The chromatographic analysis was carried out through chiral liquid chromatography using a DELTA S column and hexane-isopropyl alcohol (85:15 v/v) containing 0.2% triethylamine as mobile phase. The mean recoveries of (+)-MEB and (-)-MEB were 75.5% and 71.0%, respectively. The limit of detection (LOD) was 3.0 ng/ml with linear response over the concentration range of 10-2500 ng/ml with correlation coefficient higher than 0.993 for both enantiomers. The pharmacokinetic studies showed that the mean plasma levels of (+)-MEB were higher than those of (-)-MEB at almost all time points. Also, (+)-MEB exhibited greater t(max) (peak time in concentration-time profile), C(max) (peak concentration in concentration-time profile), t(1/2) (elimination half-life), and AUC(0-240 min) (area under the curve for concentration versus time) and smaller CL (clearance) and V(d) (apparent distribution volume) than its antipode. The obtained results implied that the absorption, distribution, and elimination of (-)-MEB were more rapid than those of (+)-MEB and there were stereoselective differences in pharmacokinetics.     

Emotion Recognition from Physiological Signals Using Parallel Stacked Autoencoders

Neurophysiology - The problem of recognition of emotions in humans is still open from a few aspects. In our study, we used a deep learning method named stacked autoencoder to classify regions of...     

In silico designing of peptide inhibitors against pregnane X receptor: the novel candidates to control drug metabolism

Drug-induced pregnane X receptor (PXR) activation may be unavoidable with some treatments. Besides, drug-induced PXR activation may lead to drug interactions with a number of treatments. Human PXR antagonists represent a means to counteract such interactions. Indeed, the ideal small molecule would have good drug-like properties, but be devoid of nonspecific cell-target effects and ability to induce cytotoxicity. Researchers assess on the possibility of deriving inhibitory peptides, which are derived from interfaces of “protein–protein interactions” (PPIs) could inhibit interactions of their origin domain by mimicking its mode of binding to cognate partners. Such peptides could serve as promising leads for rational design of inhibitory drugs. In this study, In silico designing of peptide inhibitors and peptidomimetics against PPIs was done to prevent unwanted drug metabolisms caused by PXR activation. In sum, no designed peptides had considerable higher energy of interactions and more H-bonds with the receptor compared to the SRC-1 peptides. Hence, none of the designed peptides are predicted to be more active against the target receptor. However, compared to the SRC-1 peptide, a designed peptidomimetic (mimetic3) had considerable higher energy of interactions with the receptor. It formed five H-bonds with PXR that was equal to the number of H-bonds that SRC-1 formed with the receptor. Therefore, by designing such mimetics, it would be probable to have a realistic chance of achieving peptides with improved binding affinity compared to SRC-1 and consequently prevent the unwanted drug metabolisms caused by PXR activation.