Evaluación de la capacidad de centrado y transporte apical de los sistemas Reciproc Blue y XP-endo Shaper / Evaluation of the centering and apical transport capacity of Reciproc Blue and XP-endo Shaper systems

Resumen Objetivo: Comparar la capacidad de centrado y el transporte apical de los sistemas Reciproc Blue y XP-endo Shaper en bloques de resina con fotografías pre y posoperatorias yuxtapuestas, mediante un programa de análisis de imágenes. Materiales y Métodos: Se dividieron al azar 20 tacos de resina en dos grupos (n=10) preparados con XP-endo Sha- per y Reciproc Blue respectivamente. Se tomaron fotografías digitales de los tacos, antes y después de la preparación con cada sistema en una misma posición. Luego las fotografías obtenidas fueron yuxtapuestas mediante el software Photo shop. Se midieron las desviaciones del centro del conducto con respecto a la pared interna y externa del mismo en diversos puntos de referencia preestablecidos. Estos datos fueron analizados estadísticamente con el test de ANOVA. Resultados: Ambos sistemas produjeron transporte y desviación respecto a la trayectoria original del conducto radicular, pero el análisis estadístico (ANOVA Factorial 2x8) no cuantificó diferencias significativas entre ambos sistemas (p=0,4044) Conclusiones: Bajo las condiciones de este trabajo, ambos sistemas producen transporte y desviación respecto a la trayectoria original del conducto radicular.

Abstract Aim: To compare the centering capacity and apical transport of the Reciproc Blue and X-Pendo Shaper system in resin blocks with juxtaposed pre and postoperative photographs, using the Photoshop software. Materials and methods: 20 resin plugs were randomly divided into two groups (n=10) that were prepared with XP-endo Shaper and Reciproc Blue respectively. Digital photographs of the blocks were taken, before and after preparation, with each system in the same position. Then the obtained photographs were juxtaposed using Photoshop software. The deviations of the center of the canal, with respect to its inter nal and external wall, were measured at various pre-established reference points. These data were statistically analysed with the ANOVA test. Results: Both systems produced transport and deviation from the original trajectory of the root canal, but the statistical analysis (2x8 Factorial ANOVA) did not quantify significant differences between both systems (p=0.4044) Conclusions: Under the conditions of this work, both systems produce transport and deviation with respect to the original trajectory of the root canal.

Influencia de la Experiencia del Operador en la Conformación de Canales Radiculares Simulados Utilizando Waveone Gold

Evaluar la influencia del operador sobre el transporte apical y centrado de la instrumentación endodóntica utilizando WaveOne Gold en canales radiculares simulados de resina acrílica. Se asignaron 32 canales simulados de resina acrílica a dos grupos (n = 16). El grupo 1 conformado por estudiantes inexpertos y el grupo 2 por especialistas en el área de endodoncia. Estos canales se instrumentaron hasta un tamaño apical de 25/07 con lima Primary WaveOne Gold. Para evaluar el transporte apical y el centrado de la instrumentación se utilizaron imágenes fotográficas que fueron analizadas mediante los software de edición de imagen Photoshop e Image J. Además fue registrada la incidencia de fractura de instrumentos. Los datos fueron registrados mediante un formulario Google Forms y analizados mediante el software estadístico R. Se evaluó la normalidad de los dato s mediante el test Shapiro-Wilk y para establecer diferencias entre los grupos se realizó la prueba t. El nivel de significancia estadística fue establecido en p < 0,05. La mayor diferencia al evaluar el centrado se evidenció a nivel de los 6 mm y en cuanto al transporte apical la mayor discrepancia fue a nivel de 1 mm. Sin embargo, no se observaron diferencias significativas entre ambos grupos e n ninguno de los puntos evaluados. Se reportó una baja incidencia de fractura de instrumentos. La experiencia del operador no influye en la conformación del canal radicular simulado al evaluar centrado y transporte apical al utilizar limas WaveOne Gold.

To evaluate root canal transportation and centering ability as influenced by operator experience using WaveOne Gold files in simulated acrylic resin root canals. Thirty-two simulated acrylic resin canals were distributed to two groups (n = 16). Group 1 is made up of inexperienced students, and group 2 by specialists in the field of endodontics. These canals were shaped to an apical size of 07/25 with the Primary WaveOne Gold file. Photographic images were used to evaluate apical transport and centering of the instrumentation, which was analyzed using Photoshop and Image J image editing software. File separation was also recorded. The data were recorded using Google Forms and analyzed using the R statistical program. The normality of the data was evaluated using the Shapiro-Wilk test, and the t-test was performed to establish differences between the groups. The level of statistical significance was established as p <0.05. The normality of the data was evaluated using the Shapiro-Wilk test. Then statistical significance was analyzed using the T-test. The most remarkable difference when evaluating centering was evident at the 6 mm level. In terms of apical transport, the major discrepancy was at the 1 mm level. However, no significant differences were observed between both groups in any of the points evaluated. A low incidence of instrument fracture was reported. According to our results, the operator's experience does not influence shaping in simulated root canals in terms of centering ability and apical transportation when using WaveOne Gold files.

The large GTPase Mx1 binds Kif5B for cargo transport along microtubules.

A highly specific transport and sorting machinery directing secretory cargo to the apical or basolateral plasma membrane maintains the characteristic polarized architecture of epithelial cells. This machinery comprises a defined set of transport carriers, which are crucial for cargo delivery to the correct membrane domain. Each carrier is composed of a distinct set of proteins to verify precise routing and cargo selection. Among these components, the dynamin-related GTPase Mx1 was identified on post-Golgi vesicles destined for the apical membrane of MDCK cells. In addition to the presence on late secretory compartments, Mx1 was also detected on compartments of the early secretory pathway. Vesicular structures positive for this GTPase are highly dynamic, and we have studied the influence of the microtubule cytoskeleton on this motility. Live-cell microscopy indicated that microtubule disruption using nocodazole inhibits long-range trafficking of these structures. Mx1 directly or indirectly interacts with α-tubulin and the kinesin motor Kif5B as assessed by coimmunoprecipitation. In agreement with these observations knock out of Mx1 or a mutation in the unstructured L4 loop of Mx1 decreases the efficiency of apical cargo delivery. Interestingly, the L4 loop mutant still interacts with Kif5B; however, it causes vesicle elongation. This suggests that Mx1 aids in vesicle fission and stabilizes the interaction between Kif5B, microtubules and apical transport carriers.

Galectin-3 modulates the polarized surface delivery of ß1-integrin in epithelial cells.

Epithelial cells require a precise intracellular transport and sorting machinery to establish and maintain their polarized architecture. This machinery includes ß-galactoside-binding galectins for targeting of glycoprotein to the apical membrane. Galectin-3 sorts cargo destined for the apical plasma membrane into vesicular carriers. After delivery of cargo to the apical milieu, galectin-3 recycles back into sorting organelles. We analysed the role of galectin-3 in the polarized distribution of ß1-integrin in MDCK cells. Integrins are located primarily at the basolateral domain of epithelial cells. We demonstrate that a minor pool of ß1-integrin interacts with galectin-3 at the apical plasma membrane. Knockdown of galectin-3 decreases apical delivery of ß1-integrin. This loss is restored by supplementation with recombinant galectin-3 and galectin-3 overexpression. Our data suggest that galectin-3 targets newly synthesized ß1-integrin to the apical membrane and promotes apical delivery of ß1-integrin internalized from the basolateral membrane. In parallel, knockout of galectin-3 results in a reduction in cell proliferation and an impairment in proper cyst development. Our results suggest that galectin-3 modulates the surface distribution of ß1-integrin and affects the morphogenesis of polarized cells.

Ankyrin G Expression Regulates Apical Delivery of the Epithelial Sodium Channel (ENaC).

The epithelial sodium channel (ENaC) is the limiting entry point for Na+ reabsorption in the distal kidney nephron and is regulated by numerous hormones, including the mineralocorticoid hormone aldosterone. Previously we identified ankyrin G (AnkG), a cytoskeletal protein involved in vesicular transport, as a novel aldosterone-induced protein that can alter Na+ transport in mouse cortical collecting duct cells. However, the mechanisms underlying AnkG regulation of Na+ transport were unknown. Here we report that AnkG expression directly regulates Na+ transport by altering ENaC activity in the apical membrane. Increasing AnkG expression increased ENaC activity while depleting AnkG reduced ENaC-mediated Na+ transport. These changes were due to a change in ENaC directly rather than through alterations to the Na+ driving force created by Na+/K+-ATPase. Using a constitutively open mutant of ENaC, we demonstrate that the augmentation of Na+ transport is caused predominantly by increasing the number of ENaCs at the surface. To determine the mechanism of AnkG action on ENaC surface number, changes in rates of internalization, recycling, and membrane delivery were investigated. AnkG did not alter ENaC delivery to the membrane from biosynthetic pathways or removal by endocytosis. However, AnkG did alter ENaC insertion from constitutive recycling pathways. These findings provide a mechanism to account for the role of AnkG in the regulation of Na+ transport in the distal kidney nephron.

Complex Polarity: Building Multicellular Tissues Through Apical Membrane Traffic.

The formation of distinct subdomains of the cell surface is crucial for multicellular organism development. The most striking example of this is apical-basal polarization. What is much less appreciated is that underpinning an asymmetric cell surface is an equally dramatic intracellular endosome rearrangement. Here, we review the interplay between classical cell polarity proteins and membrane trafficking pathways, and discuss how this marriage gives rise to cell polarization. We focus on those mechanisms that regulate apical polarization, as this is providing a number of insights into how membrane traffic and polarity are regulated at the tissue level.

Monocarboxylate transporter 1 is up-regulated in Caco-2 cells by the methionine precursor DL-2-hydroxy-(4-methylthio)butanoic acid.

The methionine precursor, DL-2-hydroxy-(4-methylthio)butanoic acid (HMTBA), is a synthetic source of dietary methionine, which is widely used as a poultry nutritional supplement. In the intestinal epithelium, HMTBA transport across the apical membrane is mediated by monocarboxylate transporter 1 (MCT1). The first step in biological utilisation of this methionine precursor is the stereospecific conversion of HMTBA to the corresponding keto acid. In the present study, the regulation of trans-epithelial HMTBA transport was investigated in Caco-2 cell monolayers. Differentiated Caco-2 cells were maintained under control conditions (apical compartment: 0.2 mmol/L L-methionine) or in a HMTBA-enriched medium (2 mmol/L HMTBA). The effect of culture on HMTBA transport was evaluated from apical and basolateral kinetic parameters. MCT1 and MCT4 immuno-localisation and gene expression were investigated by confocal microscopy and real-time quantitative RT-PCR, respectively. The results indicated that apical MCT1 was up-regulated by exposure to HMTBA (1.4-fold increase in Vmax without changes in Km). Moreover, total monolayer MCT1 immunoreactivity increased 1.8-fold in HMTBA-supplemented cultures, this effect mainly being localised at the apical membrane. Functional and immuno-localisation data suggest involvement of MCT1 and MCT4 in basolateral HMTBA transport, although, in this case, no effect was observed for HMTBA-enrichment. Molecular analysis confirmed MCT1 mRNA up-regulation (1.8-fold), with no effect on MCT4 mRNA expression. Thus, exposure to HMTBA up-regulates the trans-epithelial transport of this methionine precursor by increasing the expression and the transport capacity of apical MCT1.

Acylation and cholesterol binding are not required for targeting of influenza A virus M2 protein to the hemagglutinin-defined budozone.

Influenza virus assembles in the budozone, a cholesterol-/sphingolipid-enriched ("raft") domain at the apical plasma membrane, organized by hemagglutinin (HA). The viral protein M2 localizes to the budozone edge for virus particle scission. This was proposed to depend on acylation and cholesterol binding. We show that M2-GFP without these motifs is still transported apically in polarized cells. Employing FRET, we determined that clustering between HA and M2 is reduced upon disruption of HA's raft-association features (acylation, transmembranous VIL motif), but remains unchanged with M2 lacking acylation and/or cholesterol-binding sites. The motifs are thus irrelevant for M2 targeting in cells.