Differentially optimized cell-free buffer enables robust expression from unprotected linear DNA in exonuclease-deficient extracts

International audienceThe use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time-and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from σ70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions across two different laboratories. Finally, we illustrate the use of the ΔrecBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology

Symbolic Supervisory Control of Infinite Transition Systems Under Partial Observation Using Abstract Interpretation

We propose algorithms for the synthesis of state-feedback controllers with partial observation of infinite state discrete event systems modelled by Symbolic Transition Systems. We provide models of safe memoryless controllers both for potentially deadlocking and deadlock free controlled systems. The termination of the algorithms solving these problems is ensured using abstract interpretation techniques which provide an overapproximation of the transitions to disable. We then extend our algorithms to controllers with memory and to online controllers. We also propose improvements in the synthesis of controllers in the finite case which, to our knowledge, provide more permissive solutions than what was previously proposed in the literature. Our tool SMACS gives an empirical validation of our methods by showing their feasibility, usability and efficiency. © Springer Science+Business Media, LLC 2011.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Adiponectin reduces hepatic stellate cell migration by promoting tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion

Hepatic stellate cells (HSC) are central players in liver fibrosis that when activated, proliferate, migrate to sites of liver injury, and secrete extracellular matrix. Obesity, a known risk factor for liver fibrosis is associated with reduced levels of adiponectin, a protein that inhibits liver fibrosis in vivo and limits HSC proliferation and migration in vitro. Adiponectin-mediated activation of adenosine monophosphate-activated kinase (AMPK) inhibits HSC proliferation, but the mechanism by which it limits HSC migration to sites of injury is unknown. Here we sought to elucidate how adiponectin regulates HSC motility. Primary rat HSCs were isolated and treated with adiponectin in migration assays. The in vivo actions of adiponectin were examined by treating mice with carbon tetrachloride for 12 weeks and then injecting them with adiponectin. Cell and tissue samples were collected and analyzed for gene expression, signaling, and histology. Serum from patients with liver fibrosis was examined for adiponectin and tissue inhibitor of metalloproteinase-1 (TIMP-1) protein. Adiponectin administration into mice increased TIMP-1 gene and protein expression. In cultured HSCs, adiponectin promoted TIMP-1 expression and through binding of TIMP-1 to the CD63/β1-integrin complex reduced phosphorylation of focal adhesion kinase to limit HSC migration. In mice with liver fibrosis, adiponectin had similar effects and limited focal adhesion kinase phosphorylation. Finally, in patients with advanced fibrosis, there was a positive correlation between serum adiponectin and TIMP-1 levels. In sum, these data show that adiponectin stimulates TIMP-1 secretion by HSCs to retard their migration and contributes to the anti-fibrotic effects of adiponectin

Measurement of the Ωc0\Omega_c^0 lifetime at Belle II

We report on a measurement of the $\Omega_c^0$ lifetime using $\Omega_c^0 \to \Omega^-\pi^+$ decays reconstructed in $e^+e^-\to c\bar{c}$ data collected by the Belle II experiment and corresponding to $207~{\rm fb^{-1}}$ of integrated luminosity. The result, $\rm\tau(\Omega_c^0)=243\pm48( stat)\pm11(syst)~fs$, agrees with recent measurements indicating that the $\Omega_c^0$ is not the shortest-lived weakly decaying charmed baryon

Measurement of the Ωc0\Omega_c^0 lifetime at Belle II

We report on a measurement of the $\Omega_c^0$ lifetime using $\Omega_c^0 \to \Omega^-\pi^+$ decays reconstructed in $e^+e^-\to c\bar{c}$ data collected by the Belle II experiment and corresponding to $207~{\rm fb^{-1}}$ of integrated luminosity. The result, $\rm\tau(\Omega_c^0)=243\pm48( stat)\pm11(syst)~fs$, agrees with recent measurements indicating that the $\Omega_c^0$ is not the shortest-lived weakly decaying charmed baryon

Test of light-lepton universality in τ\tau decays with the Belle II experiment

International audienceWe present a measurement of the ratio $R_\mu = \mathcal{B}(\tau^-\to \mu^-\bar\nu_\mu\nu_\tau) / \mathcal{B}(\tau^-\to e^-\bar\nu_e\nu_\tau)$ of branching fractions $\mathcal{B}$ of the $\tau$ lepton decaying to muons or electrons using data collected with the Belle II detector at the SuperKEKB $e^+e^-$ collider. The sample has an integrated luminosity of 362 fb$^{-1}$ at a centre-of-mass energy of 10.58 GeV. Using an optimised event selection, a binned maximum likelihood fit is performed using the momentum spectra of the electron and muon candidates. The result, $R_\mu = 0.9675 \pm 0.0007 \pm 0.0036$, where the first uncertainty is statistical and the second is systematic, is the most precise to date. It provides a stringent test of the light-lepton universality, translating to a ratio of the couplings of the muon and electron to the $W$ boson in $\tau$ decays of $0.9974 \pm 0.0019$, in agreement with the standard model expectation of unity