Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these molecular syringes for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells

Studies of electron and proton isochoric heating for fast ignition Studies of electron and proton isochoric heating for fast ignition

Abstract Isochoric heating of inertially confined fusion plasmas by laser driven MeV electrons or protons is an area of great topical interest in the inertial confinement fusion community, particularly with respect to the fast ignition (FI) proposal to use this technique to initiate burn in a fusion capsule. Experiments designed to investigate electron isochoric heating have measured heating in two limiting cases of interest to fast ignition, small planar foils and hollow cones. Data from Cu K α fluorescence, crystal x-ray spectroscopy of Cu K shell emission, and XUV imaging at 68eV and 256 eV are used to test PIC and Hybrid PIC modeling of the interaction. Isochoric heating by focused proton beams generated at the concave inside surface of a hemi-shell and from a sub hemi-shell inside a cone have been studied with the same diagnostic methods plus imaging of proton induced K α . Conversion efficiency to protons has also been measured and modeled. Conclusions from the proton and electron heating experiments will be presented. Recent advances in modeling electron transport and innovative target designs for reducing igniter energy and increasing gain curves will also be discussed. This work was performed under the auspices of the U.S. Department of Energy by University of California Lawrence Livermore National Laboratory under contract No. W-7405-Eng-48, with the additional support of the Ohio State University, the Hertz Foundation and General Atomics. Introduction The Fast Ignition (FI) approach to Inertial Confinement Fusion (ICF) holds particular promise for fusion energy because the independently generated ignition pulse allows ignition with less compression, resulting in (potentially) higher gain. Designing targets able to exploit the FI scheme efficiently requires an understanding of the transport of electrons in prototypical geometries and at relevant densities and temperatures. We present an overview of recent research designed to investigate proton and electron isochoric heating in regimes of interest to fast ignition. This work, which is part of the US Fusion Energy Program, has been conducted through international collaborative experiments carried out on the Callisto and Titan laser facilities at LLNL in the USA, at the Vulcan laser facility in the UK and at the ILE Osaka Gekko PW facility in Japan. In the near term the goals of this program are to test theoretical models of isochoric heating with well diagnosed experiments. In the longer term we aim to carry out tests of the integrated problem where short pulse lasers isochorically heat shock-compressed materials. Finally we plan to carry out fast ignition experiments on ignition scale plasmas on the National Ignition Facility. In cone coupled electron fast ignition the fuel capsule is imploded onto the tip of a hollow cone. The short pulse laser is focused through the cone and relativistic electrons (of a few MeV average energy) are transported from the cone tip over a distance of the order of 100 µm into the assembled DT core at about 300 gcm -3 and confined within a diameter <40µm. The total energy deposited in the hot spot is about 20kJ in 10 ps. Overall coupling efficiency between laser energy and thermal energy in the ignition hot spot should exceed 10% and preferably reach 20% (as seen in the first small scale integrated experiments) to make the scheme practically attractive. Modeling of the laser accelerated electron sources, the transport of energy by electrons and the consequent isochoric heating have advanced considerably (studies into the sensitivity of the electron transport to the incident electron distribution are currently underway, while a number of new target concepts that may reduce the ignition energy and increase the gain curve are also under active investigation) but there is still no well established modeling capability to enable extrapolation from small scale experiments to full scale FI. The processes are complex and challenging from both experimental study and modeling aspects. We have recently used two limiting cases, which address specific issues in electron transport and offer good opportunities for comparison with modeling. (1) Thin foils of small area constrain electrons to reflux between the surfaces in a time short compared to the laser pulse duration so that there is always approximate cancellation of the net injected current by reflux current thus eliminating the effect of Ohmic heating by the return current of cold electrons, which is a dominant effect in initially cold solid targets in the absence of refluxing. (2) In the opposite limit a hollow cone couples the laser to a long thin wire and provides a situation where there is 100% compensation of the fast electron injection into the wire by the cold electron return current thus maximizing Ohmic effects. The geometry is simple in that the area of the current flow is constant and equal to the cross sectional area of the wire