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Bourgeois A.,CNRS Developmental Biology Laboratory | Bourgeois A.,French Institute of Health and Medical Research | Esteves De Lima J.,CNRS Developmental Biology Laboratory | Esteves De Lima J.,French Institute of Health and Medical Research | And 6 more authors.
BMC Developmental Biology | Year: 2015

Background: Components of the limb musculoskeletal system have distinct mesoderm origins. Limb skeletal muscles originate from somites, while the skeleton and attachments (tendons and connective tissues) derive from limb lateral plate. Despite distinct mesoderm origins, the development of muscle, skeleton and attachments is highly coordinated both spatially and temporally to ensure complete function of the musculoskeletal system. A system to study molecular interactions between somitic-derived tissues (muscles) and lateral-plate-derived tissues (skeletal components and attachments) during limb development is missing. Results: We designed a gene delivery system in chick embryos with the ultimate aim to study the interactions between the components of the musculoskeletal system during limb development. We combined the Tol2 genomic integration system with the viral T2A system and developed new vectors that lead to stable and bicistronic expression of two proteins at comparable levels in chick cells. Combined with limb somite and lateral plate electroporation techniques, two fluorescent reporter proteins were co-expressed in stoichiometric proportion in the muscle lineage (somitic-derived) or in skeleton and their attachments (lateral-plate-derived). In addition, we designed three vectors with different promoters to target muscle cells at different steps of the differentiation process. Conclusion: Limb somite electroporation technique using vectors containing these different promoters allowed us to target all myogenic cells, myoblasts or differentiated muscle cells. These stable and promoter-specific vectors lead to bicistronic expression either in somitic-derived myogenic cells or lateral plate-derived cells, depending on the electroporation sites and open new avenues to study the interactions between myogenic cells and tendon or connective tissue cells during limb development. © 2015 Bourgeois et al.

Ivanusic D.,Robert Koch InstituteBerlin | Ivanusic D.,Freie University BerlinBerlin | Heinisch J.J.,University of Osnabruck | Eschricht M.,Robert Koch InstituteBerlin | And 2 more authors.
BioTechniques | Year: 2015

Yeast-based methods are still the workhorse for the detection of proteinprotein interactions (PPIs) in vivo. Yeast two-hybrid (Y2H) systems, however, are limited to screening for a specific group of molecules that interact in a particular cell compartment. For this reason, the splitubiquitin system (SUS) was developed to allow screening of cDNA libraries of full-length membrane proteins for protein-protein interactions in Saccharomyces cerevisiae. Here we demonstrate that a modification of the widely used membrane SUS involving the transmembrane (TM) domain of the yeast receptor Wsc1 increases the stringency of screening and improves the selectivity for proteins localized in the plasma membrane (PM). © 2015 Eaton Publishing Company. All rights reserved.

Richter C.,Humboldt University of Berlin | Schneider C.,Freie University BerlinBerlin | Quick M.T.,Humboldt University of Berlin | Volz P.,Freie University BerlinBerlin | And 5 more authors.
Physical Chemistry Chemical Physics | Year: 2015

Although seminaphtorhodafluor (SNARF) dyes are already widely used to measure pH in cells and at biofilms, their synthesis has low yield and results in an unspecific position of a carboxy-group. The separation of 5′- and 6′-carboxy-SNARF reveals a pKa difference of 0.15, calling into question pH measurements with the (commercially available) mixture. Here we replace the bulky external dicarboxyphenyl ring with a propionate group and evaluate the spectral properties of the new derivative. Proceeding to the ethyl-iodoacetamide, covalent linkage to cysteine protein sites is achieved efficiently as shown with a cyanobacterial phytochrome, extending the scarce application of SNARF in bio-labelling in the current literature. Application in fluorescence lifetime imaging is demonstrated both with the lifetime-based and ratiometric-yield method. © the Owner Societies.

LaRock C.N.,University of California at San Diego | Dohrmann S.,University of California at San Diego | Todd J.,University of California at San Diego | Corriden R.,University of California at San Diego | And 9 more authors.
Cell Host and Microbe | Year: 2015

Summary The antimicrobial peptide LL-37 is generated upon proteolytic cleavage of cathelicidin and limits invading pathogens by directly targeting microbial membranes as well as stimulating innate immune cell function. However, some microbes evade LL-37-mediated defense. Notably, group A Streptococcus (GAS) strains belonging to the hypervirulent M1T1 serogroup are more resistant to human LL-37 than other GAS serogroups. We show that the GAS surface-associated M1 protein sequesters and neutralizes LL-37 antimicrobial activity through its N-terminal domain. M1 protein also binds the cathelicidin precursor hCAP-18, preventing its proteolytic maturation into antimicrobial forms. Exogenous M1 protein rescues M1-deficient GAS from killing by neutrophils and within neutrophil extracellular traps and neutralizes LL-37 chemotactic properties. M1 also binds murine cathelicidin, and its virulence contribution in a murine model of necrotizing skin infection is largely driven by its ability to neutralize this host defense peptide. Thus, cathelicidin resistance is essential for the pathogenesis of hyperinvasive M1T1 GAS. © 2015 Elsevier Inc.

Kowalik B.,Freie University BerlinBerlin | Schubert T.,University of Heidelberg | Wada H.,Ritsumeikan University | Tanaka M.,University of Heidelberg | And 3 more authors.
Journal of Physical Chemistry B | Year: 2015

The phase behavior of membrane lipids plays an important role in the formation of functional domains in biological membranes and crucially affects molecular transport through lipid layers, for instance, in the skin. We investigate the thermotropic chain melting transition from the ordered Lβ phase to the disordered Lα phase in membranes composed of dipalmitoylphosphatidylcholine (DPPC) by atomistic molecular dynamics simulations in which the membranes are subject to variable heating rates. We find that the transition is initiated by a localized nucleus and followed by the propagation of the phase boundary. A two-state kinetic rate model allows characterizing the transition state in terms of thermodynamic quantities such as transition state enthalpy and entropy. The extrapolated equilibrium melting temperature increases with reduced membrane hydration and thus in tendency reproduces the experimentally observed dependence on dehydrating osmotic stress. © 2015 American Chemical Society.

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