A sensitized RNA interference screen identifies a novel role for the PI3K p110γ isoform in medulloblastoma cell proliferation and chemoresistance.

Medulloblastoma is the most common malignant brain tumor in children and is associated with a poor outcome. We were interested in gaining further insight into the potential of targeting the human kinome as a novel approach to sensitize medulloblastoma to chemotherapeutic agents. A library of small interfering RNA (siRNA) was used to downregulate the known human protein and lipid kinases in medulloblastoma cell lines. The analysis of cell proliferation, in the presence or absence of a low dose of cisplatin after siRNA transfection, identified new protein and lipid kinases involved in medulloblastoma chemoresistance. PLK1 (polo-like kinase 1) was identified as a kinase involved in proliferation in medulloblastoma cell lines. Moreover, a set of 6 genes comprising ATR, LYK5, MPP2, PIK3CG, PIK4CA, and WNK4 were identified as contributing to both cell proliferation and resistance to cisplatin treatment in medulloblastoma cells. An analysis of the expression of the 6 target genes in primary medulloblastoma tumor samples and cell lines revealed overexpression of LYK5 and PIK3CG. The results of the siRNA screen were validated by target inhibition with specific pharmacological inhibitors. A pharmacological inhibitor of p110γ (encoded by PIK3CG) impaired cell proliferation in medulloblastoma cell lines and sensitized the cells to cisplatin treatment. Together, our data show that the p110γ phosphoinositide 3-kinase isoform is a novel target for combinatorial therapies in medulloblastoma.

Eliminating heterophilic antibody interference for ferritin detection using Olympus F(ab')2 based reagent.

Interferences with the Olympus immunoturbidimetric assay for ferritin have been reported because the antibodies used in the immunoassay are derived from rabbits. Rabbits are familiar pets known to be a risk factor for developing heterophilic (or interfering) antibodies. This report shows how the current Olympus Ferritin assay has been improved to eliminate the interference from heterophilic antibodies.

A validated assay for measuring doxorubicin in biological fluids and tissues in an isolated lung perfusion model: matrix effect and heparin interference strongly influence doxorubicin measurements.

Doxorubicin is an antineoplasic agent active against sarcoma pulmonary metastasis, but its clinical use is hampered by its myelotoxicity and its cumulative cardiotoxicity, when administered systemically. This limitation may be circumvented using the isolated lung perfusion (ILP) approach, wherein a therapeutic agent is infused locoregionally after vascular isolation of the lung. The influence of the mode of infusion (anterograde (AG): through the pulmonary artery (PA); retrograde (RG): through the pulmonary vein (PV)) on doxorubicin pharmacokinetics and lung distribution was unknown. Therefore, a simple, rapid and sensitive high-performance liquid chromatography method has been developed to quantify doxorubicin in four different biological matrices (infusion effluent, serum, tissues with low or high levels of doxorubicin). The related compound daunorubicin was used as internal standard (I.S.). Following a single-step protein precipitation of 500 microl samples with 250 microl acetone and 50 microl zinc sulfate 70% aqueous solution, the obtained supernatant was evaporated to dryness at 60 degrees C for exactly 45 min under a stream of nitrogen and the solid residue was solubilized in 200 microl of purified water. A 100 microl-volume was subjected to HPLC analysis onto a Nucleosil 100-5 microm C18 AB column equipped with a guard column (Nucleosil 100-5 microm C(6)H(5) (phenyl) end-capped) using a gradient elution of acetonitrile and 1-heptanesulfonic acid 0.2% pH 4: 15/85 at 0 min-->50/50 at 20 min-->100/0 at 22 min-->15/85 at 24 min-->15/85 at 26 min, delivered at 1 ml/min. The analytes were detected by fluorescence detection with excitation and emission wavelength set at 480 and 550 nm, respectively. The calibration curves were linear over the range of 2-1000 ng/ml for effluent and plasma matrices, and 0.1 microg/g-750 microg/g for tissues matrices. The method is precise with inter-day and intra-day relative standard deviation within 0.5 and 6.7% and accurate with inter-day and intra-day deviations between -5.4 and +7.7%. The in vitro stability in all matrices and in processed samples has been studied at -80 degrees C for 1 month, and at 4 degrees C for 48 h, respectively. During initial studies, heparin used as anticoagulant was found to profoundly influence the measurements of doxorubicin in effluents collected from animals under ILP. Moreover, the strong matrix effect observed with tissues samples indicate that it is mandatory to prepare doxorubicin calibration standard samples in biological matrices which would reflect at best the composition of samples to be analyzed. This method was successfully applied in animal studies for the analysis of effluent, serum and tissue samples collected from pigs and rats undergoing ILP.

Alteration of glucose metabolism in cultured astrocytes after AQP9-small interference RNA application.

Aquaglyceroporin-9 (AQP9) facilitates diffusion of water and energy substrates such as glycerol and monocarboxylates. AQP9 is present in plasma membrane and mitochondria of astrocytes and catecholaminergic neurons, suggesting that it plays a role in the energetic status of these cells. Using specific small interference RNA directed against AQP9 in astrocyte cultures, we showed that glycerol uptake is decreased which is associated with an increase in glucose uptake and oxidative metabolism. Our results not only confirm the presence of AQP9 in astrocytes but also suggest that changes in AQP9 expression alter glial energy metabolism.

Enhanced sensitivity detection of protein immobilization by fluorescent interference on oxidized silicon.

We present a silicon chip-based approach for the enhanced sensitivity detection of surface-immobilized fluorescent molecules. Green fluorescent protein (GFP) is bound to the silicon substrate by a disuccinimidyl terephtalate-aminosilane immobilization procedure. The immobilized organic layers are characterized by surface analysis techniques, like ellipsometry, atomic force microscopy (AFM) and X-ray induced photoelectron spectroscopy. We obtain a 20-fold enhancement of the fluorescent signal, using constructive interference effects in a fused silica dielectric layer, deposited before immobilization onto the silicon. Our method opens perspectives to increase by an order of magnitude the fluorescent response of surface immobilized DNA- or protein-based layers for a variety of biosensor applications.

Interference of Native Language in English Language Acquisition in Santa Cruz and Calheta Secondary schools

This study analyzes the interference of native language when students are learning a foreign language, in this case English. According to this study, interferences from the native language such as lexical-semantic and phonological are not infrequent. It is one of the main barriers that create difficulties when learning English. The outcome of this study is presented at the end of the Monografia: to provide English-language teachers and students the most significant linguistic interferences that occur during the learning process of English language. Furthermore, some teaching strategies are discussed to avoid the biases that appear due to the interferences of the native language. As a result of this study, I hope to contribute to the learning success among English-language students.

Weighted metric multidimensional scaling

This paper establishes a general framework for metric scaling of any distance measure between individuals based on a rectangular individuals-by-variables data matrix. The method allows visualization of both individuals and variables as well as preserving all the good properties of principal axis methods such as principal components and correspondence analysis, based on the singular-value decomposition, including the decomposition of variance into components along principal axes which provide the numerical diagnostics known as contributions. The idea is inspired from the chi-square distance in correspondence analysis which weights each coordinate by an amount calculated from the margins of the data table. In weighted metric multidimensional scaling (WMDS) we allow these weights to be unknown parameters which are estimated from the data to maximize the fit to the original distances. Once this extra weight-estimation step is accomplished, the procedure follows the classical path in decomposing a matrix and displaying its rows and columns in biplots.

Informing the judgments of fingerprint analysts using quality metric and statistical assessment tools

The aim of this research was to evaluate how fingerprint analysts would incorporate information from newly developed tools into their decision making processes. Specifically, we assessed effects using the following: (1) a quality tool to aid in the assessment of the clarity of the friction ridge details, (2) a statistical tool to provide likelihood ratios representing the strength of the corresponding features between compared fingerprints, and (3) consensus information from a group of trained fingerprint experts. The measured variables for the effect on examiner performance were the accuracy and reproducibility of the conclusions against the ground truth (including the impact on error rates) and the analyst accuracy and variation for feature selection and comparison.¦The results showed that participants using the consensus information from other fingerprint experts demonstrated more consistency and accuracy in minutiae selection. They also demonstrated higher accuracy, sensitivity, and specificity in the decisions reported. The quality tool also affected minutiae selection (which, in turn, had limited influence on the reported decisions); the statistical tool did not appear to influence the reported decisions.

Identifying the best body mass index metric to assess adiposity change in children.

OBJECTIVE: Although dual-energy X-ray absorptiometry (DEXA) is the preferred method to estimate adiposity, body mass index (BMI) is often used as a proxy. However, the ability of BMI to measure adiposity change among youth is poorly evidenced. This study explored which metrics of BMI change have the highest correlations with different metrics of DEXA change. METHODS: Data were from the Quebec Adipose and Lifestyle Investigation in Youth cohort, a prospective cohort of children (8-10 years at recruitment) from Québec, Canada (n=557). Height and weight were measured by trained nurses at baseline (2008) and follow-up (2010). Metrics of BMI change were raw (ΔBMIkg/m(2) ), adjusted for median BMI (ΔBMIpercentage) and age-sex-adjusted with the Centers for Disease Control and Prevention growth curves expressed as centiles (ΔBMIcentile) or z-scores (ΔBMIz-score). Metrics of DEXA change were raw (total fat mass; ΔFMkg), per cent (ΔFMpercentage), height-adjusted (fat mass index; ΔFMI) and age-sex-adjusted z-scores (ΔFMz-score). Spearman's rank correlations were derived. RESULTS: Correlations ranged from modest (0.60) to strong (0.86). ΔFMkg correlated most highly with ΔBMIkg/m(2) (r = 0.86), ΔFMI with ΔBMIkg/m(2) and ΔBMIpercentage (r = 0.83-0.84), ΔFMz-score with ΔBMIz-score (r = 0.78), and ΔFMpercentage with ΔBMIpercentage (r = 0.68). Correlations with ΔBMIcentile were consistently among the lowest. CONCLUSIONS: In 8-10-year-old children, absolute or per cent change in BMI is a good proxy for change in fat mass or FMI, and BMI z-score change is a good proxy for FM z-score change. However change in BMI centile and change in per cent fat mass perform less well and are not recommended.

Quantum metric spaces as a model for pregeometry

A new arena for the dynamics of spacetime is proposed, in which the basic quantum variable is the two-point distance on a metric space. The scaling dimension (that is, the Kolmogorov capacity) in the neighborhood of each point then defines in a natural way a local concept of dimension. We study our model in the region of parameter space in which the resulting spacetime is not too different from a smooth manifold.

RNA interference against aquaporin-4 decreases the apparent diffusion coefficient in the normal brain

Aim: Diffusion weighted magnetic resonance imaging (MRI) is now widely used in human brain diagnosis.1 To date molecular mechanisms underlying changes in Apparent Diffusion Coefficient (ADC) signals remain poorly understood. AQP4, localized to astrocytes, is one of the most highly expressed cerebral AQPs.2 AQP4 is involved in water movement within the cell membrane of cultured astrocytes.3 We hypothesize that AQP4 contributes to water diffusion and underlying ADC values in normal brain. Methods: We used an RNA interference (RNAi) protocol in vivo, to acutely knockdown expression of AQP4 in rat brain and to determine whether this was associated with changes in brain ADC values using MRI protocols as previously described.4 RNAi was performed using specific small interference RNA (siRNA) against AQP4 (siAQP4) and a non-targeted-siRNA (siGLO) as a control. The specificity and efficiency of the siAQP4 were first tested in vitro in astrocyte and hippocampal slice cultures. In vivo, siRNAs were injected into the rat cortex 3d prior to MRI acquisition and AQP4 was assessed by western blot (n=4) and immunohistochemistry (n=6). Histology was performed on adjacent slices. Results: siAQP4 application on primary astrocyte cultures induced a 76% decrease in AQP4 expression after 4 days. In hippocampal slice cultures; we also found a significant decrease in AQP4 expression in astrocytes after siAQP4. In vivo, injection of non-targeted siRNA (siGLO) tagged with CY3 allowed us to show that GFAP positive cells (astrocytes) were positively stained with CY3-siGLO, showing efficient transfection. Western blot and immunohistochemical analysis showed that siAQP4 induced a ~30% decrease in AQP4 expression without modification of tissue properties or cell death. After siAQP4 treatment, a significant decrease in ADC values (~50%) were observed without altered of T2 values. Conclusions: Together these results suggest that AQP4 reduces water diffusion through the astrocytic plasma membrane and decreases ADC values. Our findings demonstrate for the first time that astrocytic AQP4 contributes significantly to brain water diffusion and ADC values in normal brain. These results open new avenues to interpretation of ADC values under normal physiological conditions and in acute and chronic brain injuries.

Self-interference of charge carriers in ferromagnetic SrRuO3

We report a systematic study of the low-temperature electrical conductivity in a series of SrRuO3 epitaxial thin films. At relatively high temperature the films display the conventional metallic behavior. However, a well-defined resistivity minimum appears at low temperature. This temperature dependence can be well described in a weak localization scenario: the resistivity minimum arising from the competition of electronic self-interference effects and the normal metallic character. By appropriate selection of the film growth conditions, we have been able to modify the mean-free path of itinerant carriers and thus to tune the relative strength of the quantum effects. We show that data can be quantitatively described by available theoretical models.

Induced quantum metric fluctuations and the validity of semiclassical gravity

We propose a criterion for the validity of semiclassical gravity (SCG) which is based on the stability of the solutions of SCG with respect to quantum metric fluctuations. We pay special attention to the two-point quantum correlation functions for the metric perturbations, which contain both intrinsic and induced fluctuations. These fluctuations can be described by the Einstein-Langevin equation obtained in the framework of stochastic gravity. Specifically, the Einstein-Langevin equation yields stochastic correlation functions for the metric perturbations which agree, to leading order in the large N limit, with the quantum correlation functions of the theory of gravity interacting with N matter fields. The homogeneous solutions of the Einstein-Langevin equation are equivalent to the solutions of the perturbed semiclassical equation, which describe the evolution of the expectation value of the quantum metric perturbations. The information on the intrinsic fluctuations, which are connected to the initial fluctuations of the metric perturbations, can also be retrieved entirely from the homogeneous solutions. However, the induced metric fluctuations proportional to the noise kernel can only be obtained from the Einstein-Langevin equation (the inhomogeneous term). These equations exhibit runaway solutions with exponential instabilities. A detailed discussion about different methods to deal with these instabilities is given. We illustrate our criterion by showing explicitly that flat space is stable and a description based on SCG is a valid approximation in that case.

Quantum metric spaces as a model for pregeometry

A new arena for the dynamics of spacetime is proposed, in which the basic quantum variable is the two-point distance on a metric space. The scaling dimension (that is, the Kolmogorov capacity) in the neighborhood of each point then defines in a natural way a local concept of dimension. We study our model in the region of parameter space in which the resulting spacetime is not too different from a smooth manifold.