Testing of ABs to keratin and p62 in mouse liver and demonstration of equivalent binding properties of ABs used in IF and <i>is</i>PLA.

<p>IF staining of normal mouse liver tissues (Swiss Albino, SWA fed a standard diet) with antibodies to K8 (green), p62 (red) (<b>A</b>) and for K18 (green) and p62 (red) (<b>B</b>). Image (<b>C</b>) shows that the two different ABs directed to K8 (rat-anti-K8 [Troma I] and mouse-anti-K8 [Ks 8.7 Progen]) used for <i>is</i>PLA and IF or DIIF recognize identical structures in liver from a DDC fed SWA mouse. Scale bars: 20 µm.</p

<i>is</i>PLA for 12 week-DDC treated mice.

<p>Images (<b>A, B</b>) show <i>is</i>PLA signal (red) using ABs to p62 and K8 (<b>A</b>) and p62 and K18 (<b>B</b>). (<b>C</b>) shows a combined visualization of p62 and K8 in <i>is</i>PLA (red) and K8 in IF (green). (<b>D</b>) shows a combined visualization of p62 and K18 in <i>is</i>PLA (red) and K18 IF (green). In images (<b>C</b>) and (<b>D</b>) note that even very small <i>is</i>PLA positive structures are visible (arrowhead), demonstrating the high sensitivity of <i>is</i>PLA. However, there were also larger MDB-like keratin aggregates that are negative for <i>is</i>PLA stain (empty arrow), suggesting the absence of p62. Scale bars: 20 µm.</p

Influence of channel amplification on demonstration of antigen colocalization in <i>is</i>PLA and IF in 12 week-DDC treated <i>krt18^−/−</i> mice.

<p>(<b>A, B, C</b>) Show different levels of digital amplification of the green channel (IF for K8) and constant amplification of the red channel showing <i>is</i>PLA for K8 and p62. Arrows indicate a MDB-like aggregate that was constantly positive for K8 but negative for p62. Arrowheads indicate MDBs which were positive in <i>is</i>PLA for p62 and K8 but IF showed only presence of K8 (yellow merged signal) in <i>is</i>PLA positive MDBs after maximal amplification of the green channel. Scale bars: 20 µm.</p

Technical controls performed on mice with and without DDC treatment.

<p>In order to complement the biological controls (knockout mice) we performed a number of technical controls to make sure that no cross reactions between the different reagents might lead to false results.</p

Testing of ABs to keratin and p62 in mouse liver cells after DDC treatment.

<p>Immunofluorescence stain on SWA mouse livers after 12 weeks DDC treatment using ABs to K8 (green), p62 (red) (<b>A</b>), and K18 (green), p62 (red) ABs (<b>B</b>). For image (<b>A</b>), note the presence of small (arrowhead) and large (empty arrow) MDBs. Additionally, small granules positive for p62 only can be observed (arrow). For image (<b>B</b>), note that small MDB-like keratin aggregates can be observed that are negative for p62 (empty arrow). Scale bars: 20 µm.</p

Schematic drawing of different antibody binding and visualization steps of <i>is</i>PLA combined with IF.

<p>(<b>A</b>) Primary ABs of the <i>is</i>PLA bind to two different proteins, which are expected to colocalize. (<b>B</b>) Secondary ABs conjugated with oligonucleotides bind to the primary ABs (PLA Probe PLUS and PLA Probe MINUS). (<b>C</b>) Two more oligonucleotides (blue) hybridize to the two PLA probes. (<b>D</b>) Ligase (yellow) joins the two added oligonucleotides to form a closed circle. (<b>E</b>) Polymerase (yellow) induces a rolling circle amplification (RCA) using the ligated circle as a template. (<b>F</b>) Fluorescence labeled oligonucleotides (red) hybridize to the RCA product. (<b>G</b>) Primary ABs of the IF (purple) bind to one of the proteins, which is also targeted by <i>is</i>PLA. (<b>H</b>) Secondary ABs of the IF bind to the primary ABs of the IF. They are fluorescence labeled in red while the <i>is</i>PLA signals are green.</p

(Des)Amor a la mexicana: de las críticas a la gloria : Un recorrido discursivo del diario Clarín sobre la selección campeona del Mundial 1986

En el siguiente Trabajo Integrador Final se explicita el recorrido discursivo del diario Clarín sobre la Selección Argentina en el período comprendido entre mayo y julio de 1986. Para ello, se utilizaron las herramientas que propicia el análisis de contenido, una técnica que permitió describir y repensar sobre la construcción de sentido elaborada por el medio de comunicación mediante la clasificación de los artículos periodísticos en diferentes categorías. Además, se reflexiona en torno a las entrevistas realizadas a los diferentes actores vinculados con el conjunto argentino, con el objetivo de discernir cuáles eran las modalidades utilizadas por el multimedio a la hora de las conferencias de prensa y los reportajes, y cómo los mismos se utilizaban para reforzar los sentidos creados en el suplemento deportivo. Complementariamente se examina la línea editorial de Clarín con el fin de descubrir cuál era la ideología del medio en relación a la Selección que, al final de la historia, terminaría consagrándose campeona mundial.Facultad de Periodismo y Comunicación Socia

Human relevant fungi cultivated, treated with PAXgene and formalin and investigated for viability.

<p>Human relevant fungi cultivated, treated with PAXgene and formalin and investigated for viability.</p

Loss of p62 impairs the formation of large MDBs.

<p>Double immunofluorescence staining with K8/K18 (green) and p62 (red) antibodies was performed on liver sections of <i>p62</i>^<i>f/f</i>, <i>p62</i>^<i>-/-</i>, <i>p62hep</i>^<i>+/+</i>and <i>p62hep</i>^<i>-/</i> mice under normal diet (panel 1). To detect MDBs, double immunofluorescence staining with M_M120-1 (red) and K8/18 antibodies (green) was performed on liver sections of 8 weeks DDC-intoxicated mice (n ≥10). Note that MDBs were found in all genotypes. However, <i>p62</i>^<i>f/f</i> and <i>p62hep</i>^<i>+/+</i> mice developed large aggregates while only small inclusions were observed in <i>p62</i>^<i>-/-</i> and <i>p62hep</i>^<i>-/-</i> mice (panels 2–5) (scale bar = 20μm). To confirm the absence of p62 in MDBs present in p62-deficient livers, various combinations of double immunofluorescence stainings with p62 (green) + M_M120 (red), K8/K18 (green) + p62 (red) and ubiquitin (green) + p62 (red) antibodies were performed on DDC-treated liver of the different genotypes (panels 5–7. The arrows are used to indicate few MDBs among all the MDBs positive for keratin, M_M120-1 or p62 to underline the observations (panels 2–7).</p

Schematic drawing of the different role of p62 and keratin in two distinct hypothetical pathways of MDB formation.

<p><b>(A1)</b> In the first pathway alcohol abuse or metabolic alterations associated with obesity in the context of human alcoholic or non-alcoholic steatohepatitis (ASH or NASH), and DDC or griseofulvin administration in mice trigger the upregulation of keratins (K8 and K18) with, increased K8:K18 ratio and cross β-sheet conformation of K8, which leads to the formation of small ‟early” MDBs with filamentous ultrastructure and M_M120-1 antigen positivity. <b>(A2)</b> The coalescence of small MDBs to form large, compact ‟mature” MDBs occurs via incorporation of p62. Binding of p62 to MDBs also promotes the recruitment of NBR1 and other proteins to MDBs. <b>(B1)</b> In the second pathway (found in hepatocellular carcinoma and idiopathic copper toxicosis) p62-containing IHBs, which are negative for keratin and do not show cross β-sheet conformation, may progress to <b>(B2)</b> hybrid inclusions (showing mixed features of both IHBs and MDBs) by incorporation of keratins (K8 and K18). <b>(B3)</b> The hybrid inclusions may transform to ‟mature” MDBs upon further incorporation of K8 and K18.</p