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Heart ablation using a planar rectangular high intensity ultrasound transducer and MRI guidance
The aim of this study was to evaluate a flat rectangular (3 × 10 mm2) MRI compatible transducer operating at 5 MHz. The main task was to explore the feasibility of creating deep lesions in heart at a depth of at least 15 mm. The size of thermal necrosis in heart tissue was estimated as a function of power and time using a simulation model. The system was then tested in an excised lamb heart. In this study, we were able to create lesions of 15 mm deep with acoustic power of 6 W for an exposure of approximately 1 min. The contrast to noise ratio (CNR) between lesion and heart tissue was evaluated using fast spin echo (FSE). The CNR value was approximately 22 using T1 W FSE. Maximum CNR was achieved with repetition time (TR) between 300 and 800 ms. Using T2W FSE, the corresponding CNR was approximately 13 for the 14 in vivo experiments. The average lesion depth was 11.93 mm with a standard deviation of 0.62 mm. In vivo irradiation conditions were 6 W for 60 s. The size of the lesion in the other two dimensions was close to 3 × 10 mm2 (size of the transducer element)
Up-regulation of flotillins, new marker of metastatic development, induces cell invasion and metastatic development
Flotillin 1 and 2 are two ubiquitous, highly conserved and related proteins present in man
Late Endosomal Cholesterol Accumulation Leads to Impaired Intra-Endosomal Trafficking
Background Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. Methodology/Principal Findings Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2–3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus. Conclusions/Significance These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation
Rab6 and Rab11 Regulate Chlamydia trachomatis Development and Golgin-84-Dependent Golgi Fragmentation
Many intracellular pathogens that replicate in special membrane bound compartments exploit cellular trafficking pathways by targeting small GTPases, including Rab proteins. Members of the Chlamydiaceae recruit a subset of Rab proteins to their inclusions, but the significance of these interactions is uncertain. Using RNA interference, we identified Rab6 and Rab11 as important regulators of Chlamydia infections. Depletion of either Rab6 or Rab11, but not the other Rab proteins tested, decreased the formation of infectious particles. We further examined the interplay between these Rab proteins and the Golgi matrix components golgin-84 and p115 with regard to Chlamydia-induced Golgi fragmentation. Silencing of the Rab proteins blocked Chlamydia-induced and golgin-84 knockdown-stimulated Golgi disruption, whereas Golgi fragmentation was unaffected in p115 depleted cells. Interestingly, p115-induced Golgi fragmentation could rescue Chlamydia propagation in Rab6 and Rab11 knockdown cells. Furthermore, transport of nutrients to Chlamydia, as monitored by BODIPY-Ceramide, was inhibited by Rab6 and Rab11 knockdown. Taken together, our results demonstrate that Rab6 and Rab11 are key regulators of Golgi stability and further support the notion that Chlamydia subverts Golgi structure to enhance its intracellular development
Endocytosis of DNA-Hsp65 Alters the pH of the Late Endosome/Lysosome and Interferes with Antigen Presentation
BACKGROUND: Experimental models using DNA vaccine has shown that this vaccine is efficient in generating humoral and cellular immune responses to a wide variety of DNA-derived antigens. Despite the progress in DNA vaccine development, the intracellular transport and fate of naked plasmid DNA in eukaryotic cells is poorly understood, and need to be clarified in order to facilitate the development of novel vectors and vaccine strategies. METHODOLOGY AND PRINCIPAL FINDINGS: Using confocal microscopy, we have demonstrated for the first time that after plasmid DNA uptake an inhibition of the acidification of the lysosomal compartment occurs. This lack of acidification impaired antigen presentation to CD4 T cells, but did not alter the recruitment of MyD88. The recruitment of Rab 5 and Lamp I were also altered since we were not able to co-localize plasmid DNA with Rab 5 and Lamp I in early endosomes and late endosomes/lysosomes, respectively. Furthermore, we observed that the DNA capture process in macrophages was by clathrin-mediated endocytosis. In addition, we observed that plasmid DNA remains in vesicles until it is in a juxtanuclear location, suggesting that the plasmid does not escape into the cytoplasmic compartment. CONCLUSIONS AND SIGNIFICANCE: Taken together our data suggests a novel mechanism involved in the intracellular trafficking of plasmid DNA, and opens new possibilities for the use of lower doses of plasmid DNA to regulate the immune response
The Interaction Properties of the Human Rab GTPase Family – A Comparative Analysis Reveals Determinants of Molecular Binding Selectivity
Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood.Comparative analysis of the amino acid sequences and the three-dimensional electrostatic and hydrophobic molecular interaction fields of 62 human Rab proteins revealed a wide range of binding properties with large differences between some Rab proteins. This analysis assists the functional annotation of Rab proteins 12, 14, 26, 37 and 41 and provided an explanation for the shared function of Rab3 and 27. Rab7a and 7b have very different electrostatic potentials, indicating that they may bind to different effector proteins and thus, exert different functions. The subfamily V Rab GTPases which are associated with endosome differ subtly in the interaction properties of their switch regions, and this may explain exchange factor specificity and exchange kinetics.We have analysed conservation of sequence and of molecular interaction fields to cluster and annotate the human Rab proteins. The analysis of three dimensional molecular interaction fields provides detailed insight that is not available from a sequence-based approach alone. Based on our results, we predict novel functions for some Rab proteins and provide insights into their divergent functions and the determinants of their binding partner selectivity
Ebolavirus Is Internalized into Host Cells via Macropinocytosis in a Viral Glycoprotein-Dependent Manner
Ebolavirus (EBOV) is an enveloped, single-stranded, negative-sense RNA virus that causes severe hemorrhagic fever with mortality rates of up to 90% in humans and nonhuman primates. Previous studies suggest roles for clathrin- or caveolae-mediated endocytosis in EBOV entry; however, ebolavirus virions are long, filamentous particles that are larger than the plasma membrane invaginations that characterize clathrin- or caveolae-mediated endocytosis. The mechanism of EBOV entry remains, therefore, poorly understood. To better understand Ebolavirus entry, we carried out internalization studies with fluorescently labeled, biologically contained Ebolavirus and Ebolavirus-like particles (Ebola VLPs), both of which resemble authentic Ebolavirus in their morphology. We examined the mechanism of Ebolavirus internalization by real-time analysis of these fluorescently labeled Ebolavirus particles and found that their internalization was independent of clathrin- or caveolae-mediated endocytosis, but that they co-localized with sorting nexin (SNX) 5, a marker of macropinocytosis-specific endosomes (macropinosomes). Moreover, the internalization of Ebolavirus virions accelerated the uptake of a macropinocytosis-specific cargo, was associated with plasma membrane ruffling, and was dependent on cellular GTPases and kinases involved in macropinocytosis. A pseudotyped vesicular stomatitis virus possessing the Ebolavirus glycoprotein (GP) also co-localized with SNX5 and its internalization and infectivity were affected by macropinocytosis inhibitors. Taken together, our data suggest that Ebolavirus is internalized into cells by stimulating macropinocytosis in a GP-dependent manner. These findings provide new insights into the lifecycle of Ebolavirus and may aid in the development of therapeutics for Ebolavirus infection
Rab protein evolution and the history of the eukaryotic endomembrane system
Spectacular increases in the quantity of sequence data genome have facilitated major advances in eukaryotic comparative genomics. By exploiting homology with classical model organisms, this makes possible predictions of pathways and cellular functions currently impossible to address in intractable organisms. Echoing realization that core metabolic processes were established very early following evolution of life on earth, it is now emerging that many eukaryotic cellular features, including the endomembrane system, are ancient and organized around near-universal principles. Rab proteins are key mediators of vesicle transport and specificity, and via the presence of multiple paralogues, alterations in interaction specificity and modification of pathways, contribute greatly to the evolution of complexity of membrane transport. Understanding system-level contributions of Rab proteins to evolutionary history provides insight into the multiple processes sculpting cellular transport pathways and the exciting challenges that we face in delving further into the origins of membrane trafficking specificity
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