Effect of RED knock-down on the subcellular localization of NP in influenza virus infected cells.

<p>A549 cells were transfected with control non-target (NT) or RED siRNAs, and were subsequently infected with WSN influenza virus at a m.o.i. of 5 pfu/cell. At 5 hpi, cells were fixed, permeabilized, and stained with an antibody specific for the NP protein and with Hoechst 33342. Samples were analyzed under a fluorescence microscope (Inverted Zeiss Observer Z1). <b>A</b>. Representative images of NP localization. <b>B</b>. Percentage of cells with different NP localization. The results of two independent experiments is shown, in which 107 and 94 cells (exp #a), and 164 and 115 cells (exp #b), were scored for the NT and RED siRNA experimental condition, respectively.</p

Detection of ternary PB2-RED-SMU1 complexes.

<p><b>A</b>. The <i>Gaussia princeps</i> luciferase-based complementation assay was performed with the PB2-Gluc1 and Gluc2-SMU1 fusion proteins, in the absence or presence of over-expressed RED protein. The Normalized Luminescence Ratios (NLR) were determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004164#ppat-1004164-g001" target="_blank">Figure 1A</a>. The dashed line indicates the NLR cut-off that reduces false positive background below 2.5%, as determined using a random reference set of human proteins. The data are expressed as the mean +/− SD of triplicates and are representative of two independent experiments. <b>B</b>. The Gluc2-RED and Gluc1-SMU1 fusion proteins were co-expressed in HEK-293T cells, together with the wild-type PB2 protein or PB2-Strep fusion protein (transfection). Alternatively, HEK-293T cells co-expressing Gluc2-RED and Gluc1-SMU1 were superinfected with the rWSN or rWSN-PB2-Strep viruses (infection). Cell lysates were subjected to purification using StrepTactin beads. Control samples (cells co-expressing Gluc2-RED+Gluc1 or Gluc2+Gluc1-SMU1) were processed in parallel. Luciferase activities and NLRs were determined on an aliquot of the lysates (upper graph) and on the StrepTactin eluates (lower graph) as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004164#ppat-1004164-g001" target="_blank">Figure 1A</a>. The data are expressed as the mean +/− SD of triplicates and are representative of two independent experiments (infection) or a single experiment (transfection).</p

Steady-state levels and subcellular localisation of endogenous RED and SMU1 in infected cells.

<p><b>A</b>. A549 cells were infected at a m.o.i. of 5 pfu/cell with the rWSN virus (+) or mock-infected (−). Total cell extracts were prepared at 0, 3, 6 and 9 hours post-infection (hpi), loaded on a 4–12% SDS-polyacrylamide gel and analyzed by western blotting using antibodies specific for RED, SMU1, NP and GAPDH. <b>B</b>. A549-GFP1-10 cells on coverslips were infected with the rWSN-PB2-GFP11 recombinant virus at a m.o.i. of 5 pfu/cell (WSN), or mock-infected. These conditions allow direct visualization of PB2 in infected cells by trans-complementation of GFP fragments GFP1-10 and GFP11 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004164#ppat.1004164-Avilov1" target="_blank">[38]</a>. At 6 hpi, cells were fixed, permeabilized, and stained with an antibody specific for the RED or SMU1 protein and with Hoechst 33342. Samples were analyzed under a fluorescence microscope (Inverted Zeiss Observer Z1). Merge views corresponding to RED (red) and SMU1 (cyan), or RED and PB2-GFPcomp (green), are shown. A scale bar is shown.</p

Effect of RED or SMU1 knock-down on the accumulation of influenza virus mRNAs.

<p>A549 cells were transfected with RED (R), SMU1 (S) or control non-target (NT) siRNAs, and were subsequently infected with the WSN virus at a m.o.i. of 5 pfu/cell. Polyadenylated RNAs were purified at 3, 6 and 9 hpi, and RT-qPCR was performed to detect specifically the viral NS1, NS2, M1 and M2 mRNAs, as well as the cellular GAPDH mRNAs. <b>A</b>. Schematic representation of the primers and probes used for RT-qPCR. The NS1, NS2, M1 and M2 mRNAs are depicted. The positions of primers and probes are indicated by black and white arrowheads, respectively, that are oriented according to the sense or antisense orientation of the oligonucleotides. <b>B</b>. The NS1, NS2, M1 and M2 mRNA copy numbers, as determined using the standard curve method and normalized with respect to GAPDH mRNA copy numbers, are shown. The data are expressed as the mean +/− SD of two independent experiments in triplicates, except for the SMU1-siRNA-3 hpi and SMU1-siRNA-6 hpi conditions (one experiment in triplicates). For each mRNA at each time-point, the ratio (in percent) of mRNA copy number in RED- and SMU1-silenced cells to mRNA copy number in NT siRNA-treated cells are indicated below the graphs. Two-way ANOVA was performed to evaluate the effects of RED siRNA treatment. *<b>*</b>: p<0.0001; *: p<0.001; ns: non significant (p>0.05). <b>C</b>. The ratios of NS2/NS1 and M2/M1 mRNA copy numbers in RED- and SMU1-silenced cells are expressed as percentages of the ratios measured in control cells (black bars: 100%). When indicated, two-way ANOVA was performed to evaluate the effect of siRNA treatment. *: p<0.001; ns: non significant (p>0.05).</p

Mapping of the interaction domain of RED with the viral polymerase.

<p><b>A</b>. Schematic representation of the full-length RED, N-terminal domain (Nt-RED) and C-terminal domain (Ct-RED) of RED that were transiently expressed in the experiment shown in B. The domain corresponding to the truncated, secreted form of RED (sec-RED) is also represented. <b>B</b>. For each indicated pair of Gluc1 and Gluc2 fusion proteins, the NLR was determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004164#ppat-1004164-g001" target="_blank">Figure 1A</a>. The NLR are expressed as percentages (100%: the NLR measured in the presence of Gluc2-RED). The data are expressed as the mean +/− SD and are representative of two independent experiments (PB1, PB2) or a single experiment (SMU1) in triplicates. <b>C</b>. Steady-state levels of the recombinant Gluc2-RED, Gluc2-Nt-RED and Gluc2-Ct-RED proteins. HEK-293T cells were transfected with the Gluc2-RED, Gluc2-Nt-RED, Gluc2-Ct-RED plasmids, or mock-transfected with the empty pCI plasmid (control). Total cell extracts were prepared 24 hours post-transfection, loaded on a 4–12% SDS-polyacrylamide gel and analyzed by western blotting using a polyclonal antibody specific for <i>Gaussia princeps</i> luciferase. <b>D</b>. Co-purification of the viral polymerase and recombinant, HA-tagged polypeptides corresponding to full-length or truncated forms of the RED protein. HEK-293T cells were transfected with the HA-RED, HA-Nt-RED or HA-Ct-RED expression plasmids, or mock-transfected with the pCI plasmid (−). 24 hours post-transfection, they were infected at a m.o.i. of 5 with recombinant WSN (W) or WSN-PB2-Strep (S) viruses and incubated at 37°C for 6 hours. Whole-cell lysates were prepared and a fraction was incubated with StrepTactin beads as described in Material and Methods. Protein complexes were eluted, loaded on a 4–12% SDS-polyacrylamide gel and analyzed by western blotting using either StrepTactin to detect the PB2-Strep protein (upper panel) or a monoclonal antibody specific for the HA tag (lower panel).</p

Interaction of RED with influenza virus polymerase in human cells.

<p><b>A</b>. Schematic representation of the <i>Gaussia princeps</i> luciferase-based complementation assay and Normalized Luminescence Ratios (NLRs) calculation method. The luminescence activity measured in cells co-transfected with the plasmids encoding the P-Gluc1 and P′-Gluc2 fusion proteins is divided by the sum of the luminescence activities measured in control samples co-transfected with either the P-Gluc1 and Gluc2 plasmids or the Gluc1 and P′-Gluc2 plasmids. For details, see the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004164#s4" target="_blank">Materials and Methods</a> section, and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004164#ppat.1004164-Cassonnet1" target="_blank">[27]</a>. <b>B</b>. For each indicated pair of Gluc1 and Gluc2 fusion proteins, the NLR was determined as described in A. The data are expressed as the mean +/− SD of triplicates and are representative of two independent experiments. The dashed line indicates the NLR cut-off value that reduces false positive background below 2.5%, as determined using a random reference set of human proteins. <b>C</b>. Co-purification of the endogenous RED and SMU1 proteins with the viral polymerase in infected cells. HEK-293T cells were infected at a m.o.i. of 5 with recombinant wild-type rWSN (W) or rWSN-PB2-Strep (S) viruses and incubated at 37°C for 6 hours. Whole-cell lysates were prepared and a fraction was incubated with StrepTactin beads as described in Material and Methods. Protein complexes were eluted, loaded on a 4–12% SDS-polyacrylamide gel and analyzed by western blotting using either StrepTactin to detect the PB2-Strep protein (upper panel) or an antibody specific for the RED or SMU1 protein (middle and lower panels). * and **: non-specific detection of the PB2 and NP protein, respectively, as inferred from previous experiments <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004164#ppat.1004164-Ge1" target="_blank">[33]</a>. The bands detected in the PB2-Strep panel at 70 kD and the faster migrating band (present only in lysates) were also detected in mock-infected cells (data not shown).</p

Effect of RED or SMU1 knock-down on influenza virus multicycle growth.

<p><b>A</b>. A549 cells were transfected with control non-target siRNAs (NT), a pool of four RED siRNAs (Rp), individual RED siRNAs (R1, R2), or a pool of four SMU1 siRNAs (Sp). Total cell extracts were prepared at 36 hours post-transfection, and analyzed by western blotting using antibodies specific for RED (left upper panel), SMU1 (right upper panel) and GAPDH (lower panels). The residual levels of SMU1 and RED in silenced cells relative to control cells are indicated. The data are representative of two independent experiments. <b>B</b>. A luminescence-based cell viability assay was performed on RED and SMU1 siRNA-treated cells at 36 hours post-transfection. The data are expressed as the mean +/− SD of triplicates and are representative of two independent experiments. A.U.: arbitrary units. <b>C</b>. A549 cells were transfected with control (NT, solid lines), RED (dashed lines) or SMU1 (dotted lines) siRNAs. At 36 hours post-transfection, they were infected with WSN influenza virus (circles), VSV (triangles), or Adenovirus 5 (squares) at a m.o.i. of 10⁻³, 10⁻⁴ and 6 pfu/cell, respectively. Supernatants of WSN- and VSV-infected cells were collected at 24 and 48 hours post-infection and were titrated by plaque assay. Lysates of AdV5 infected cell were prepared at 24 and 48 hpi and were titrated by immunostaining. The data are expressed as the mean +/− SD of triplicates (WSN, VSV) or duplicates (AdV5), and are representative of two (WSN, VSV) or one (AdV5) independent experiments. <b>D</b>. A549 cells were transfected with control (NT) or with the indicated RED siRNAs. At 36 hours post-transfection, they were infected with the WSN virus (circles) or VSV (triangles) at a m.o.i. of 10⁻³ or 10⁻⁴ pfu/cell, respectively. Supernatants were collected at 48 hours post-infection and were titrated by plaque assay. The data are expressed as the mean +/− SD of triplicates.</p

Collision detection on transmission lines with optical interferometer

V diplomski nalogi skušamo ugotoviti, v kolikšni meri je možno zaznavati in klasificirati trke na jeklenicah daljnovodov z optičnim interferometrom. Na začetku predstavimo osnovne pojme interferometrije in opišemo uporabljen optični interferometer. V jedru diplomske naloge natančneje opišemo eksperimentalni protokol in obdelavo signalov. Nadaljujemo z implementacijo algoritmov za segmentacijo in klasifikacijo zajetih signalov ter predstavimo dobljene rezultate. Segmentacijo izvedemo v domeni števila prehodov signala skozi ničlo, za klasifikacijo pa uporabimo večplastno nevronsko mrežo z algoritmom vzvratnega učenja. Rezultati študije nakazujejo, da sta implementirani segmentacija in klasifikacija uspešni v 77 % izvedenih trkov različnih predmetov.We analyse feasibility of collision detection on transmission lines with optical interferometer. We first provide a brief introduction into interferometry, along with a description of the optical interferometer used for measurements in this study. Afterwards, we describe the conducted experimental protocol and signal processing methodology. The focus is on implementation of algorithms for signal segmentation and collision classification. We used zero-crossing algorithm to transform signals into segmentation domain. Classification of collisions is done with a multilayer neural network trained by the backpropagation algorithm. The results demonstrate an average success rate of 77% for segmentation and classification of collision with five different objects

Sistematización de la experiencia de un ambiente de aprendizaje enriquecido por TIC durante la práctica clínica en fisioterapia cardiopulmonar en un hospital de nivel II de la ciudad de Cali

Esta investigación se centra en la caracterización de la experiencia de 4 estudiantes de fisioterapia de IX semestre de la Institución Universitaria Escuela Nacional del Deporte (IUEND) durante la implementación de un ambiente de aprendizaje enriquecido con Tecnologías de la Información y la Comunicación (TIC) en la práctica clínico – asistencial en Salud Cardiopulmonar; la cual se fundamenta en el hacer y pone a prueba las bases conceptuales del ciclo de fundamentación; todo esto con el fin de identificar las experiencias significativas que facilitan el aprendizaje y desarrollo de competencias clínicas, además analizar si este tipo de estrategias de enseñanza -aprendizaje permite al estudiante y al docente asesor superar inconvenientes propios de la práctica clínica como: optimizar tiempos de atención a pacientes, estudio independiente y trabajo colaborativo, retomar e integrar gran cantidad de conceptos y procedimientos aprendidos en IV semestre con las nuevas experiencias y la realidad del paciente; y a la vez cumplir con funciones administrativas propios del rol del fisioterapeuta asistencial (estadística, indicadores, desarrollo de guías, etc.) que dificultan el proceso de aprendizaje; concluyendo que los ambientes mediados por TIC pueden lograr superar estas dificultades y favorecer finalmente el aprendizaje significativo (juicio clínico), en el que se fundamenta el ciclo de práctica profesional

assay information of the mouse tissue samples for HeliScopeCAGE

a tab delimited flat file (SDRF file) describing the experimental details for mouse tissue samples for the standard protocol of the HeliScopeCAGE protoco