114 research outputs found
BID-F1 and BID-F2 Domains of Bartonella henselae Effector Protein BepF Trigger Together with BepC the Formation of Invasome Structures
No full textThe gram-negative, zoonotic pathogen Bartonella henselae (Bhe) translocates seven distinct Bartonella effector proteins (Beps) via the VirB/VirD4 type IV secretion system (T4SS) into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation.Swiss National Science Foundation (grant 31003A-132979)Howard Hughes Medical Institute (grant 5500550)Swiss Initiative for Systems Biology (grant 51RT-0_126008 (InfectX)
Weydert_3D_TI_Supplementary_Figure_Legend_rev – Supplemental material for A 3D Heterotypic Multicellular Tumor Spheroid Assay Platform to Discriminate Drug Effects on Stroma versus Cancer Cells
No full textSupplemental material, Weydert_3D_TI_Supplementary_Figure_Legend_rev for A 3D Heterotypic Multicellular Tumor Spheroid Assay Platform to Discriminate Drug Effects on Stroma versus Cancer Cells by Zoe Weydert, Madhu Lal-Nag, Lesley Mathews-Greiner, Christoph Thiel, Henrik Cordes, Lars Küpfer, Patrick Guye, Jens M. Kelm and Marc Ferrer in SLAS Discovery</p
A translocated effector required for bartonella dissemination from derma to blood safeguards migratory host cells from damage by co-translocated effectors
No full textNumerous bacterial pathogens secrete multiple effectors to modulate host cellular functions. These effectors may interfere with each other to efficiently control the infection process. Bartonellae are Gram-negative, facultative intracellular bacteria using a VirB type IV secretion system to translocate a cocktail of Bartonella effector proteins (Beps) into host cells. Based on in vitro infection models we demonstrate here that BepE protects infected migratory cells from injurious effects triggered by BepC and is required for in vivo dissemination of bacteria from the dermal site of inoculation to blood. Human endothelial cells (HUVECs) infected with a ΔbepE mutant of B. henselae (Bhe) displayed a cell fragmentation phenotype resulting from Bep-dependent disturbance of rear edge detachment during migration. A ΔbepCE mutant did not show cell fragmentation, indicating that BepC is critical for triggering this deleterious phenotype. Complementation of ΔbepE with BepEBhe or its homologues from other Bartonella species abolished cell fragmentation. This cyto-protective activity is confined to the C-terminal Bartonella intracellular delivery (BID) domain of BepEBhe (BID2.EBhe). Ectopic expression of BID2.EBhe impeded the disruption of actin stress fibers by Rho Inhibitor 1, indicating that BepE restores normal cell migration via the RhoA signaling pathway, a major regulator of rear edge retraction. An intradermal (i.d.) model for B. tribocorum (Btr) infection in the rat reservoir host mimicking the natural route of infection by blood sucking arthropods allowed demonstrating a vital role for BepE in bacterial dissemination from derma to blood. While the Btr mutant ΔbepDE was abacteremic following i.d. inoculation, complementation with BepEBtr, BepEBhe or BIDs.EBhe restored bacteremia. Given that we observed a similar protective effect of BepEBhe on infected bone marrow-derived dendritic cells migrating through a monolayer of lymphatic endothelial cells we propose that infected dermal dendritic cells may be involved in disseminating Bartonella towards the blood stream in a BepE-dependent manner
Fic domain catalyzed adenylylation: Insight provided by the structural analysis of the type IV secretion system effector BepA
No full textNumerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N-terminal Fic domain and a C-terminal BID domain, the latter being responsible for T4SS-mediated translocation into host cells. A proteolysis resistant fragment (residues 10 to 302) that includes the Fic domain shows auto-adenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography upon incubation with alpha-[(32)P]-ATP. Its crystal structure, determined to 2.9 A resolution by the SeMet-SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional beta-rich domain at the C-terminus. Upon crystal soaking with ATP/Mg(2+), additional electron density indicated the presence of a PP(i)/Mg(2+) moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. Based on this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg(2+) and target tyrosine. The model is consistent with an in-line nucleophilic attack of the deprotonated side-chain hydroxyl group onto the alpha-phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence independent mechanism of target positioning through antiparallel beta-strand interactions between enzyme and target is suggested
Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes
No full textThe mechanisms underlying the development of complications in type 1 diabetes (T1D) are poorly understood. Disease modeling of induced pluripotent stem cells (iPSCs) from patients with longstanding T1D(disease duration ≥ 50 years) with severe (Medalist +C) or absent to mild
complications (Medalist −C) revealed impaired growth, reprogramming, and differentiation in Medalist +C. Genomics and proteomics analyses suggested differential regulation of DNA damage checkpoint proteins favoring protection from cellular apoptosis in Medalist −C. In silico
analyses showed altered expression patterns of DNA damage checkpoint factors among the Medalist groups to be targets of miR200, whose expression was significantly elevated in Medalist +C serum. Notably, neurons differentiated from Medalist +C iPSCs exhibited enhanced
susceptibility to genotoxic stress that worsened upon miR200 overexpression. Furthermore, knockdown of miR200 in Medalist +C fibroblasts and iPSCs rescued checkpoint protein expression and reduced DNA damage. We propose miR200-regulated DNA damage checkpoint
pathway as a potential therapeutic target for treating complications of diabete
Genetically engineering self-organization of human pluripotent stem cells into a liver bud-like tissue using Gata6
No full textHuman induced pluripotent stem cells (hiPSCs) have potential for personalized and regenerative medicine. While most of the methods using these cells have focused on deriving homogenous populations of specialized cells, there has been modest success in producing hiPSC-derived organotypic tissues or organoids. Here we present a novel approach for generating and then co-differentiating hiPSC-derived progenitors. With a genetically engineered pulse of GATA-binding protein 6 (GATA6) expression, we initiate rapid emergence of all three germ layers as a complex function of GATA6 expression levels and tissue context. Within 2 weeks we obtain a complex tissue that recapitulates early developmental processes and exhibits a liver bud-like phenotype, including haematopoietic and stromal cells as well as a neuronal niche. Collectively, our approach demonstrates derivation of complex tissues from hiPSCs using a single autologous hiPSCs as source and generates a range of stromal cells that co-develop with parenchymal cells to form tissues.National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (NSF CBET-0939511)Synthetic Biology Engineering Research CenterNational Institutes of Health (U.S.) (P50GM098792)Ernst Schering FoundationSwiss National Science Foundatio
Rapid, modular and reliable construction of complex mammalian gene circuits
No full textWe developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up to 64 kb in size comprising 11 transcription units and 33 basic parts. We show robust gene expression control of multiple transcription units by small molecule inducers in human cells with transient transfection and stable chromosomal integration of these circuits. This framework enables development of complex gene circuits for engineering mammalian cells with unprecedented speed, reliability and scalability and should have broad applicability in a variety of areas including mammalian cell fermentation, cell fate reprogramming and cell-based assays.Synthetic Biology Engineering Research Center (SA5284-11210)United States. Defense Advanced Research Projects Agency (HR0011-12-C-0067)United States. Defense Advanced Research Projects Agency (DARPA-BAA-11-23)National Science Foundation (U.S.) (CBET-0939511)National Institutes of Health (U.S.). (5-R01-CA155320-02
Imagerie spectroscopique, imagerie de diffusion et tractographie, dans les épilepsies partielles humaines caractérisées par stéréo-électroencéphalographie
No full textAIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF
Translation of selected passages from Guy Gunaratne's In Our Mad and Furious City with an introduction to the author, the style of the novel and translation challenges
No full textThis thesis focuses on translation of selected parts of In Our Mad And Furious City by Guy Gunaratne, a British author of Sri Lankan origin. The novel tells a story of a few days, during which dramatic events sweep through the lives of people living in a multicultural London area. The thesis deals with the novel's style and its lexical and syntactic specifics, focusing especially on the way characters talk and on various dialects that come through in the novel.Tato diplomová práce se zabývá překladem vybraných částí románu In Our Mad And Furious City Guye Gunaratneho, britského autora srílanského původu. Román je příběhem několika dní, jejichž dramatické události zasáhnou do životů lidí žijících v londýnském multikulturním prostředí. Práce se zabývá stylistickými, lexikálními a syntaktickými vlastnostmi románu, přičemž se zaměřuje především na řeč postav a na to, jak se v ní projevují různé dialekty.Ústav translatologieInstitute of Translation StudiesFaculty of ArtsFilozofická fakult
SPECTROMETRIE DE RMN DANS LES EPILEPSIES TEMPORALES (DES NEUROLOGIE)
No full textAIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF
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