Domain motions, dimerization, and membrane interactions of the murine guanylate binding protein 2.

Guanylate-binding proteins (GBPs) are a group of GTPases that are induced by interferon-[Formula: see text] and are crucial components of cell-autonomous immunity against intracellular pathogens. Here, we examine murine GBP2 (mGBP2), which we have previously shown to be an essential effector protein for the control of Toxoplasma gondii replication, with its recruitment through the membrane of the parasitophorous vacuole and its involvement in the destruction of this membrane likely playing a role. The overall aim of our work is to provide a molecular-level understanding of the mutual influences of mGBP2 and the parasitophorous vacuole membrane. To this end, we performed lipid-binding assays which revealed that mGBP2 has a particular affinity for cardiolipin. This observation was confirmed by fluorescence microscopy using giant unilamellar vesicles of different lipid compositions. To obtain an understanding of the protein dynamics and how this is affected by GTP binding, mGBP2 dimerization, and membrane binding, assuming that each of these steps are relevant for the function of the protein, we carried out standard as well as replica exchange molecular dynamics simulations with an accumulated simulation time of more than 30 µs. The main findings from these simulations are that mGBP2 features a large-scale hinge motion in its M/E domain, which is present in each of the studied protein states. When bound to a cardiolipin-containing membrane, this hinge motion is particularly pronounced, leading to an up and down motion of the M/E domain on the membrane, which did not occur on a membrane without cardiolipin. Our prognosis is that this up and down motion has the potential to destroy the membrane following the formation of supramolecular mGBP2 complexes on the membrane surface.

Essential Role of mGBP7 for Survival of Toxoplasma gondii Infection.

Members of the murine guanylate-binding protein family (mGBP) are induced by interferon gamma (IFN-γ) and have been shown to be important factors in cell-autonomous immunity toward the intracellular pathogen Toxoplasma gondii Previously, we identified that mGBP2 mediates disruption of the parasitophorous vacuole membrane (PVM) and directly assaults the plasma membrane of the parasite. Here, we show that mGBP7-deficient mice are highly susceptible to T. gondii infection. This is demonstrated by the loss of parasite replication control, pronounced development of ascites, and death of the animals in the acute infection phase. Interestingly, live-cell microscopy revealed that mGBP7 recruitment to the PVM occurs after mGBP2 recruitment, followed by disruption of the PVM and T. gondii integrity and accumulation of mGBP7 inside the parasite. This study defines mGBP7 as a crucial effector protein in resistance to intracellular T. gondiiIMPORTANCE Guanylate-binding proteins (GBPs) are induced by the inflammatory cytokine interferon gamma (IFN-γ) and have been shown to be important factors in the defense of the intracellular pathogen Toxoplasma gondii In previous studies, we showed that members of the mouse GBP family, such as mGBP2 and mGBP7, accumulate at the parasitophorous vacuole of T. gondii, which is the replicatory niche of the parasite. In this study, we show that mice deficient in mGBP7 succumb early after infection with T. gondii, showing a complete failure of resistance to the pathogen. On a molecular level, mGBP7 is found directly at the parasite, likely mediating its destruction.

Biochemical and structural characterization of murine GBP7, a guanylate binding protein with an elongated C-terminal tail.

Guanylate-binding proteins (GBPs) constitute a family of interferon-inducible guanosine triphosphatases (GTPases) that are key players in host defense against intracellular pathogens ranging from protozoa to bacteria and viruses. So far, human GBP1 and GBP5 as well as murine GBP2 (mGBP2) have been biochemically characterized in detail. Here, with murine GBP7 (mGBP7), a GBP family member with an unconventional and elongated C-terminus is analyzed. The present study demonstrates that mGBP7 exhibits a concentration-dependent GTPase activity and an apparent GTP turnover number of 20 min-1. In addition, fluorescence spectroscopy analyses reveal that mGBP7 binds GTP with high affinity (KD = 0.22 µM) and GTPase activity assays indicate that mGBP7 hydrolyzes GTP to GDP and GMP. The mGBP7 GTPase activity is inhibited by incubation with γ-phosphate analogs and a K51A mutation interfering with GTP binding. SEC-MALS analyses give evidence that mGBP7 forms transient dimers and that this oligomerization pattern is not influenced by the presence of nucleotides. Moreover, a structural model for mGBP7 is provided by homology modeling, which shows that the GTPase possesses an elongated C-terminal (CT) tail compared with the CaaX motif-containing mGBP2 and human GBP1. Molecular dynamics simulations indicate that this tail has transmembrane characteristics and, interestingly, confocal microscopy analyses reveal that the CT tail is required for recruitment of mGBP7 to the parasitophorous vacuole of Toxoplasma gondii.

Análisis comparativo de la remoción de la dentina / Comparative Analysis of Dentin Removal by two Endodontic Techniques

Fundamento: una adecuada preparación de los conductos radiculares dentales es esencial para garantizar el éxito del tratamiento endodóntico y el de los procedimientos restauradores subsecuentes. Objetivo: comparar dos técnicas de instrumentación endodónticas cuantificando cuál de ellas remueve menos cantidad de dentina. Método: la muestra estuvo constituida por 30 molares mandibulares extraídos y almacenados en cloramina T al 12 %. El grupo uno (n=15) se trató con una técnica manual rotatoria en la que se utilizaron fresas Gates-Glidden, mientras que el grupo dos (n=15) se instrumentó con una técnica rotatoria mediante limas Pro-Taper. Después de la instrumentación radicular en los dos grupos experimentales, se realizaron mediciones en la porción cervical, media y apical de cada uno de los conductos distales. Las radiografías periapicales de evaluación se procesaron con el programa Adobe Photoshop. Para el análisis estadístico se utilizó una prueba t no pareada. Resultados: para el grupo uno las mediciones cervicales, medias y apicales fueron 0,48 mm, 0,27 mm y 0,21 mm respectivamente, mientras que para el grupo dos fueron 0,69 mm, 0,31 mm y 0,24 mm respectivamente. Sólo se encontraron diferencias estadísticamente significativas en la porción cervical de los dos grupos; se observó menor desgaste dentinal en el grupo 1 (p= 0.01). Conclusiones: se encontró una mayor remoción de dentina cervical radicular en los conductos tratados con limas Pro-Taper.

ABSTRACT Background: one of the most important aspects in treating dental root canals is the mechanical root canal preparation. Objective: to compare two techniques for quantifying endodontic instrumentation, searching which one removes the least amount of dentin. Methods: the sample consisted of 30 mandible molars extracted and stored in chloramines T to 12%. The group 1 (n = 15) was treated with a manual rotary technique using Gates-Glidden drills while group 2 (n = 15) was instrumented with a rotational technique using Pro-Taper files. Following instrumentation root in the two experimental groups was measured in the cervical portion, and apical half of each distal canal. The evaluation of periapical radiographs was processed with Adobe Photoshop. The statistical analysis used unpaired t test. Results: for the group 1 the measurements cervical, middle and apical were 0.48 mm, 0.27 mm and 0.21 mm, respectively, while for the group 2 were 0.69 mm, 0.31 mm and 0.24 mm, respectively. Only statistically significant differences in the cervical portion of the two groups were showed, viewing less wear dentin in group 1 (p = 0.01). Conclusions: higher cervical root dentin removal in canals treated with Pro-Taper files was observed.

Microbiología de la enfermedad periodontal / Microbiology of periodontal disease

Las periodontitis son un grupo de patologías que afectan a un alto porcentaje de la población adulta chilena. Esta enfermedad la constituyen distintas entidades clínicas que afectan a los tejidos de soporte de los dientes, esto es, al periodonto de protección (encía) y al periodonto de inserción (hueso alveolar), ocasionando la pérdida de las piezas dentarias. Las patologías señaladas son causadas por la interacción de algunos microorganismos y la respuesta inmune del huésped. Actualmente se describen más de 500 especies como habitantes del área subgingival o el saco periodontal, haciendo de la microbiología periodontal, una de las más complejas áreas de estudio de toda la microbiología clínica. Estudios recientes han clarificado la etiología microbiana de la enfermedad periodontal y la importancia de identificar a personas en riesgo de desarrollarla, mejorando de este modo el diagnóstico y posterior tratamiento.