Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers

Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of six amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together, these data allow us to develop a general model for activation of TpoR and explain the critical role of the JM W515 residue in the regulation of the activity of the receptor

Antimicrobial susceptibility testing of mycobacterium tuberculosis complex isolates - the EUCAST broth microdilution reference method for MIC determination

Scope:Several methods are used worldwide for antibiotic susceptibility testing (AST) for theMycobac-terium tuberculosiscomplex (MTBC). The variability in the results obtained with these methods hamperssetting epidemiological cut-off (ECOFF) values and clinical breakpoints according to EUCAST guidelines.Methods for susceptibility testing and determination of the minimal inhibitory concentrations (MICs)need to be standardized for MTBC isolates for old and new agents. Our objective was to establish astandardized reference method for MIC determination for MTBC.Methods:The EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared pro-tocols of MIC determination with regard to medium, inoculum preparation, antituberculous agentpreparation, incubation, reading of the results and interpretation.Recommendations:The EUCAST reference method of MIC determination for MTBC is the broth micro-dilution method in Middlebrook 7H9-10% OADC medium. Thefinal inoculum is a 105CFU/mL suspension,obtained from a 10 2dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonieswith glass beads before suspending them in sterile water. The culture is maintained in a U-shaped 96-well polystyrene microtitre sterile plate with a lid incubated at 36 ±1 C. Reading is done using aninverted mirror as soon as the 1:100 diluted control (i.e. 103CFU/mL suspension) shows visual growth.The MIC, expressed in mg/L, is the lowest concentration that inhibits visual growth.MycobacteriumtuberculosisH37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within therange 0.03e0.12 for isoniazid, 0.12e0.5 for levofloxacin and 0.25e1 mg/L for amikacin.Conclusions:The EUCAST reference method for MTBC was endorsed by EUCAST after public consultationand will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference methodis not primarily intended to be used under routine conditions and the AST methods will need to b

Multicentre testing of the EUCAST broth microdilution reference method for MIC determination on mycobacterium tuberculosis

Objectives: the first objective of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee for antimycobacterial susceptibility testing (AMST), launched in 2016, was to set a reference method for determining the MICs of antituberculous agents, since many protocols are used worldwide and a consensus one is needed for the determination of microbiological breakpoints. Methods: during 2017 and 2018, MIC determination protocols were evaluated prospectively in a multicentre study within the four AMST laboratories. MIC results were obtained for isoniazid, levofloxacin and amikacin on the reference strain Mycobacterium tuberculosis H37Rv ATCC 27294. Broth microdilution (BMD) in Middlebrook 7H9 and solid medium dilution (SMD) in Middlebrook 7H10 were performed using two inoculum concentrations. MICs were interpreted with regard to visual and 99% inhibition after 7, 14 or 21 days of incubation for BMD and 21 days for SMD. Results: following the EUCAST reference protocol, intra- and inter-assay agreements were within ±1 MIC dilution for >95% of the observations for the three drugs in both methods. MIC values, presented as MIC mode (range) for BMD and SMD respectively, were: 0.03 (0.015-0.06) mg/L and 0.12 (0.06-0.25) mg/L for isoniazid, 0.25 mg/L (0.25-0.5) and 0.5 mg/L (0.12-0.5) for levofloxacin, and 0.5 mg/L (0.5-1.0) and 0.5 mg/L (0.5-1.0) for amikacin. Conclusions: both SMD and BMD were reproducible and eligible as a reference method for MIC determination of the Mycobacterium tuberculosis complex (MTBC). BMD was finally selected as the EUCAST reference method. From now on it will be used to set epidemiological cut-off values and clinical breakpoints of new and old antituberculous agents

Validation platform implementation description – D5.2

Deliverable D5.2 del projecte OneFITPostprint (published version

Protocols, performance assessment and consolidation on interfaces for standardization – D3.3

The following document presents a detailed description of the protocol for the “ Control Channels for the Cooperation of the Cognitive Management System ” (C4MS) which provides the necessary means to enable proper management of Opportunistic Networks. Additionally, the document defines the methodology that was applied for the purpose of signalling evaluation. The protocol overview presented in section 2 of the main document, provides the C4MS principles. The section includes, among others, the description of the protocol identifiers, procedures, protocol state machines and message format as well as the security asp ects. Section 3 provides a high-level description of the data structures defined within the scope of OneFIT project. The data structures are classified into five categories, i.e.: Profiles, Context, Decisions,Knowledge and Policies. The high level description is complemented by some detailed data structures in the Appendix to D3.3 Section 3[10]. Section 4 provides details on the evaluation methodology applied for the purpose of C4MS performance assessment. The section presents the evaluation plan along with a description of metrics that are to be exploited in the scope of WP3. Section 5 and Section 6 are composed of the signalling evaluation results. Section 5 focuses on the estimation of the signalling load imposed by ON management in different ON phases. Additionally some results for the initialization phase (not explicitly mentioned in the previous phases of the project)and security related aspects are also depicted. Section 6 on the other hand is focused on the evaluation of the signalling traffic generated by different ON related algorithms. Conclusions to the document are drawn in section 7. Detailed description of the C4MS procedures, implementation options based on IEEE 802.21, DIAMTER and 3GPP are depicted in the appendix to the D3.3[10] . Additionally, the appendix incorporates the detailed definition of the information data structures and final set of Message Sequence Charts (MSCs) provided for the OneFIT project.Peer ReviewedPreprin

Business scenarios, technical challenges and system requirements - D2.1

Deliverable D2.1 del projecte Europeu OneFIT (ICT-2009-257385)Preprin

Performance assessment & synergic operation of algorithmic solutions enabling opportunistic networks– D4.2

Deliverable D4.2 del projecte europeu OneFITPeer ReviewedPostprint (updated version

Results analysis and validation - D5.3

Deliverable D5.3 del projecte OneFITPostprint (author’s final draft