Incidence and prognostic value of tumour cells detected by RT–PCR in peripheral blood stem cell collections from patients with Ewing tumour

To retrospectively evaluate the incidence of tumour cell contamination of peripheral blood stem cell (PBSC) collections and to correlate these data with the clinical outcome after high-dose chemotherapy (HDCT) with stem cell rescue in patients with a high-risk Ewing tumour. Peripheral blood stem cell collections obtained from 171 patients were analysed. Tumour contamination was assessed by reverse transcriptase–polymerase chain reaction (RT–PCR). The files of 88 patients who underwent HDCT followed by PBSC reinfusion were reviewed in detail, and their outcome compared to the PBSC RT–PCR results. Seven of 88 PBSC collections (8%) contained tumour cells as detected by RT–PCR. Peripheral blood stem cells were collected after a median of five cycles of chemotherapy. No clinical factor predictive of tumour cell contamination of PBSC harvest could be identified. Event-free survival (EFS) and overall survival (OS) of the whole study population were 45.3 % and 51.8 % at 3 years from the date of the graft, respectively. Forty-five patients relapsed with a median time of 15 months after graft, only four of whom had tumour cell contamination of the PBSC harvest. Tumour cell contamination of PBSC collection is rare and does not seem to be associated with a significantly poorer EFS or OS in this high-risk population

Cortical inhibitory but not excitatory synaptic transmission and circuit refinement are altered after the deletion of NMDA receptors during early development

Abstract Neurons in the cerebral cortex form excitatory and inhibitory circuits with specific laminar locations. The mechanisms underlying the development of these spatially specific circuits is not fully understood. To test if postsynaptic N-methyl-D-aspartate (NMDA) receptors on excitatory neurons are required for the development of specific circuits to these neurons, we genetically ablated NMDA receptors from a subset of excitatory neurons in the temporal association cortex (TeA) through in utero electroporation and assessed the intracortical circuits connecting to L5 neurons through in vitro whole-cell patch clamp recordings coupled with laser-scanning photostimulation (LSPS). In NMDAR knockout neurons, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated connections were largely intact. In contrast both LSPS and mini-IPSC recordings revealed that γ-aminobutyric acid type A (GABAA) receptor-mediated connections were impaired in NMDAR knockout neurons. These results suggest that postsynaptic NMDA receptors are important for the development of GABAergic circuits

Ncm, a Photolabile Group for Preparation of Caged Molecules: Synthesis and Biological Application

<div><p>Ncm, 6-nitrocoumarin-7-ylmethyl, is a photolabile protective group useful for making “caged” molecules. Ncm marries the reliable photochemistry of 2-nitrobenzyl systems with the excellent stability and spectroscopic properties of the coumarin chromophore. From simple, commercially available starting materials, preparation of Ncm and its caged derivatives is both quick and easy. Photorelease of Ncm-caged molecules occurs on the microsecond time scale, with quantum efficiencies of 0.05–0.08. We report the synthesis and physical properties of Ncm and its caged derivatives. The utility of Ncm-caged glutamate for neuronal photostimulation is demonstrated in cultured hippocampal neurons and in brain slice preparations.</p></div

Physical chemical characterization of <i>N</i>-Ncm amino acids.

<p>(A) UV-visible absorption spectra of a solution of <i>N</i>-Ncm-Glu in 0.1 M phosphate buffer (pH 7.4) recorded before (blue solid line, labeled “‒UV”) and after (red solid line, labeled “+UV”) photolysis with 166 mW of UV light from an argon ion laser for 2 min. The blue dashed curve shows the long-wavelength (360–500 nm) region of the ‒UV curve on an expanded scale (refer to the <i>y</i>-axis scale on the right). (B) Transient absorbance change of a solution of <i>N</i>-Ncm-Glu in DMSO following photolysis with an 8.6-nsec pulse of 355-nm light delivered at time zero. In the upper panel, gray points are data and solid curve is a least-squares single-exponential fit to the data. The residuals of the fit are shown in the lower panel. (C) Dependence of photolysis kinetics on mole-fraction of water. The <i>t</i>_1/2 values for transient spectral decay are plotted against mole fraction of water in DMSO for <i>N</i>-Ncm-Glu and <i>N</i>-Ncm-Gly. The <i>t</i>_1/2 for <i>N</i>-Ncm-GABA in DMSO is also shown. Each point is the average of 3 replicate measurements; error bars represent standard deviations (where not seen, error bar is smaller than the symbol).</p

Nitroso photoproduct of the Ncm cage and its reaction with glutathione.

<p>(A) UV-visible spectrum of a 30 μM solution of 7-formyl-6-nitrosocoumarin (<b>14</b>) in 10 mM sodium phosphate buffer (pH 7). (B) Schematic representation of the reaction of <b>14</b> with a thiolate (RS^‒), which adds nucleophilically to the nitroso group, and concomitantly disrupts the chromophore. The bimolecular rate constant is <i>k</i>. (C) Spectrophotometrically monitored time course of the reaction of <b>14</b> (25 μM) with glutathione (50 μM) in 10 mM sodium phosphate buffer (pH 7.0, 24°C). Grey circles are experimental data acquired at 20 Hz; red curve is the nonlinear least-squares fit to the function, , where <i>k</i>_app = 0.5[GSH]₀<i>k</i>, (<i>k</i> is the bimolecular rate constant indicated in panel B, and the initial glutathione concentration is [GSH]₀ = 50 μM), and <i>y</i>₀ and <i>S</i> are adjustable parameters (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163937#sec002" target="_blank">Materials and Methods</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163937#pone.0163937.s005" target="_blank">S1 Text</a> for details). For the data set shown, <i>k</i> = 3.74 × 10³ M^-1s^-1.</p

Summary of Ncm derivatives.

<p>Summary of Ncm derivatives.</p