An x-ray penumbral imager for measurements of electron-temperature profiles in inertial confinement fusion implosions at OMEGA.

Hot-spot shape and electron temperature (Te) are key performance metrics used to assess the efficiency of converting shell kinetic energy into hot-spot thermal energy in inertial confinement fusion implosions. X-ray penumbral imaging offers a means to diagnose hot-spot shape and Te, where the latter can be used as a surrogate measure of the ion temperature (Ti) in sufficiently equilibrated hot spots. We have implemented a new x-ray penumbral imager on OMEGA. We demonstrate minimal line-of-sight variations in the inferred Te for a set of implosions. Furthermore, we demonstrate spatially resolved Te measurements with an average uncertainty of 10% with 6 µm spatial resolution.

High-resolution x-ray radiography with Fresnel zone plates on the University of Rochester's OMEGA Laser Systems.

Experiments performed at the Laboratory for Laser Energetics with a continuous-wave (cw) x-ray source and on the OMEGA and OMEGA EP Laser Systems [Boehly et al., Opt. Commun. 133, 495 (1997) and Waxer et al., Opt. Photonics News 16, 30 (2005)] have utilized a Fresnel zone plate (FZP) to obtain x-ray images with a spatial resolution as small as ∼1.5 µm. Such FZP images were obtained with a charge-coupled device or a framing camera at energies ranging from 4.5 keV to 6.7 keV using x-ray line emission from both the cw source and high-intensity, laser-beam-illuminated metal foils. In all cases, the resolution test results are determined from patterns and grids backlit by these sources. The resolutions obtained are shown to be due to a combination of the spectral content of the x-ray sources and detector resolution limited by the magnification of the images (14× to 22×). High-speed framing cameras were used to obtain FZP images with frame times as short as ∼30 ps. Double-shell implosions on OMEGA were backlit by laser-irradiated Fe foils, thus obtaining a framing-camera-limited, FZP-image resolution of ∼3 µm-4 µm.

Cherenkov detector analysis for implosions with multiple nuclear reactions.

Nuclear reactions that produce γ rays occur in inertial fusion implosions and are commonly measured with Cherenkov detectors. Typically a detector is primarily sensitive to a single reaction, but in some implosions, multiple fusion reactions can occur and are combined in the data. We discuss an analysis technique using multiple thresholded detectors to reproduce the individual burn histories from reactions like DT and HT fusion, which is applicable to separated-reactant mix experiments. Requirements for this technique and resulting analysis uncertainties are quantified using synthetic data.

Diffusion-dominated mixing in moderate convergence implosions.

High-Z material mixed into the fuel degrades inertial fusion implosions and can prevent ignition. Mix is often assumed to be dominated by hydrodynamic instabilities, but we report Omega data, using shells with ∼150nm deuterated layers to gain unprecedented resolution, which give strong evidence that the dominant mix mechanism is diffusion for these moderate temperature (≲6 keV) and convergence (∼12) implosions. Small-scale instability-driven or turbulent mix is negligible.

Primary T cells for mRNA-mediated immunotoxin delivery.

Immune cells become increasingly attractive as delivery system for immunotoxins in cancer therapy to reduce the intrinsic toxicity and severe side effects of chimeric protein toxins. In this study, we investigated the potential of human primary T cells to deliver a secreted immunotoxin through transient messenger RNA (mRNA) transfection. The chimeric protein toxin was directed toward the neovasculature of cancer cells by fusing a truncated version of Pseudomonas exotoxin A (PE38) to human vascular endothelial growth factor (VEGF) and to the single chain variable fragment (scFv) of anti-Her2/neu. Protocols for the transient transfection of human embryonic kidney cells (HEK293) as well as activated primary human T cells were established. Transient transfection with mRNA coding for the immunotoxins e23-PE38, VEGF-PE38 and its attenuated variant VEGF-PE38D yielded efficient expression and secretion. Mass spectrometry analysis endorsed that a fraction of VEGF-PE38D was properly translocated into the endoplasmic reticulum. Furthermore, cytotoxic activity of immunotoxin secreting T cells toward cancer cells was confirmed in co-culture with ovarian adenocarcinoma cells in the presence of a bispecific antibody (bsAb), highlighting the potential of primary T cells for mRNA-mediated immunotoxin delivery.

Simultaneous measurement of the HT and DT fusion burn histories in inertial fusion implosions.

Measuring the thermonuclear burn history is an important way to diagnose inertial fusion implosions. Using the gas Cherenkov detectors at the OMEGA laser facility, we measure the HT fusion burn in a H2+T2 gas-fueled implosion for the first time. Using multiple detectors with varied Cherenkov thresholds, we demonstrate a technique for simultaneously measuring both the HT and DT burn histories from an implosion where the total reaction yields are comparable. This new technique will be used to study material mixing and kinetic phenomena in implosions.

Demonstration of Fuel Hot-Spot Pressure in Excess of 50 Gbar for Direct-Drive, Layered Deuterium-Tritium Implosions on OMEGA.

A record fuel hot-spot pressure P_{hs}=56±7 Gbar was inferred from x-ray and nuclear diagnostics for direct-drive inertial confinement fusion cryogenic, layered deuterium-tritium implosions on the 60-beam, 30-kJ, 351-nm OMEGA Laser System. When hydrodynamically scaled to the energy of the National Ignition Facility, these implosions achieved a Lawson parameter ∼60% of the value required for ignition [A. Bose et al., Phys. Rev. E 93, 011201(R) (2016)], similar to indirect-drive implosions [R. Betti et al., Phys. Rev. Lett. 114, 255003 (2015)], and nearly half of the direct-drive ignition-threshold pressure. Relative to symmetric, one-dimensional simulations, the inferred hot-spot pressure is approximately 40% lower. Three-dimensional simulations suggest that low-mode distortion of the hot spot seeded by laser-drive nonuniformity and target-positioning error reduces target performance.

Coexpression of human perforin improves yeast-mediated delivery of DNA and mRNA to mammalian antigen-presenting cells.

Previous studies underlined the capacity of recombinant yeast as efficient vehicle for the targeted delivery of functional nucleic acids as well as proteinaceous antigens to mammalian antigen-presenting cells (APCs). To improve this yeast-mediated cargo transport into APCs, we investigated the impact of coexpression of the human membrane-perturbing protein perforin in comparison with bacterial listeriolysin O (LLO) on the yeast-based delivery of DNA, mRNA and proteins to mammalian APCs. In contrast to LLO, a cholesterol-dependent pore-forming toxin of Listeria, intracellular expression of human perforin in Saccharomyces cerevisiae had no impact on yeast cell viability, while its coexpression significantly increased translocation of ovalbumin and subsequent activation of ovalbumin-specific T lymphocytes. Likewise, perforin improved the expression of the model antigen enhanced green fluorescent protein after yeast-mediated DNA and mRNA delivery, whereas LLO was only able to enhance DNA delivery. Taken together, our data show that human perforin, besides bacterial hemolysins, represents a promising means to improve the yeast-mediated delivery of functional nucleic acids and proteins to mammalian APCs.

Gamma-hydroxybutyrate, acting through an anti-apoptotic mechanism, protects native and amyloid-precursor-protein-transfected neuroblastoma cells against oxidative stress-induced death.

Clinical observations suggested that gamma-hydroxybutyrate (GHB) protects nerve cells against death but the direct proofs are missing. Here, we combined several approaches to investigate GHB capacity to protect human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced death. To increase the patho-physiological relevancy of our study, we used native SH-SY5Y cells and SH-SY5Y cells stably transfected with the wild-type amyloid-precursor-protein (APPwt) or control-vector-pCEP4. Trypan Blue exclusion and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide) assays combined with pharmacological analyses showed that H2O2 reduced native and genetically modified cell viability and APPwt-transfected cells were the most vulnerable. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and activated caspase-3 staining assessed by flow cytometry revealed a basally elevated apoptotic signal in APPwt-transfected cells. Reverse-transcription, real-time quantitative polymerase chain reaction (qPCR) and Western blotting showed that mRNA and protein basal ratios of apoptotic modulators Bax/Bcl-2 were also high in APPwt-transfected cells. GHB efficiently and dose-dependently rescued native and genetically modified cells from H2O2-induced death. Interestingly, GHB, which strongly decreased elevated basal levels of TUNEL-staining, activated caspase 3-labeling and Bax/Bcl-2 in APPwt-transfected cells, also counteracted H2O2-evoked increased apoptotic markers in native and genetically modified SH-SY5Y cells. Since GHB did not promote cell proliferation, anti-apoptotic action through the down-regulation of Bax/Bcl-2 ratios and/or caspase 3 activity appears as a critical mechanism involved in GHB-induced protection of SH-SY5Y cells against APPwt-overexpression- or H2O2-evoked death. Altogether, these results, providing multi-parametric evidence for the existence of neuroprotective action of GHB, also open interesting perspectives for the development of GHB analog-based strategies against neurodegeneration or nerve cell death.

MiRNA-29: a microRNA family with tumor-suppressing and immune-modulating properties.

MicroRNAs (miRNAs) are ubiquitously expressed small, non-coding RNAs that negatively regulate gene expression at a post-transcriptional level. So far, over 1000 miRNAs have been identified in human cells and their diverse functions in normal cell homeostasis and many different diseases have been thoroughly investigated during the past decade. MiR-29, one of the most interesting miRNA families in humans to date, consists of three mature members miR-29a, miR-29b and miR-29c, which are encoded in two genetic clusters. Members of this family have been shown to be silenced or down-regulated in many different types of cancer and have subsequently been attributed predominantly tumor-suppressing properties, albeit exceptions have been described where miR-29s have tumor-promoting functions. MiR-29 targets expression of diverse proteins like collagens, transcription factors, methyltransferases and others, which may partake in abnormal migration, invasion or proliferation of cells and may favor development of cancer. Furthermore, members of the miR-29 family can be activated by interferon signaling, which suggests a role in the immune system and in host pathogen interactions, especially in response to viral infections. In this review, we summarize current knowledge on the genomic organization and regulation of the miR-29 family and we provide an overview of its implication in cancer suppression and promotion as well as in host immune responses. The numerous remarkable properties of these miRNAs and their often altered expression patterns might make the miR-29 family promising biomarkers and therapeutic targets for various diseases in future.

Delivery of functional DNA and messenger RNA to mammalian phagocytic cells by recombinant yeast.

Among the different vaccination approaches, DNA/RNA vaccination represents a promising means in particular for the induction of effective cellular immune responses conferred by CD8-positive T lymphocytes. To achieve such immune responses, there is a need for novel delivery systems that allow the introduction of nucleic acids to the cytosol of immune cells. We show, for the first time, the delivery of functional DNA and messenger RNA (mRNA) to mammalian antigen-presenting cells, including murine macrophages and human dendritic cells, using the yeast Saccharomyces cerevisiae as the delivery vehicle. After transfer of the particular nucleic acid, subsequent antigen processing and presentation were demonstrated in a human system. Remarkably, release of DNA/mRNA does not require additional 'helper' proteins such as listeriolysin. In conclusion, the yeast-based system described here is superior to many bacterial and viral systems in terms of efficacy, safety and targeting suggesting 'mycofection' as a promising approach for the development of a novel type of live vaccines.

Modeling the National Ignition Facility neutron imaging system.

Numerical modeling of the neutron imaging system for the National Ignition Facility (NIF), forward from calculated target neutron emission to a camera image, will guide both the reduction of data and the future development of the system. Located 28 m from target chamber center, the system can produce two images at different neutron energies by gating on neutron arrival time. The brighter image, using neutrons near 14 MeV, reflects the size and symmetry of the implosion "hot spot." A second image in scattered neutrons, 10-12 MeV, reflects the size and symmetry of colder, denser fuel, but with only ∼1%-7% of the neutrons. A misalignment of the pinhole assembly up to ±175 µm is covered by a set of 37 subapertures with different pointings. The model includes the variability of the pinhole point spread function across the field of view. Omega experiments provided absolute calibration, scintillator spatial broadening, and the level of residual light in the down-scattered image from the primary neutrons. Application of the model to light decay measurements of EJ399, BC422, BCF99-55, Xylene, DPAC-30, and Liquid A suggests that DPAC-30 and Liquid A would be preferred over the BCF99-55 scintillator chosen for the first NIF system, if they could be fabricated into detectors with sufficient resolution.

Spacer-elongated cell wall fusion proteins improve cell surface expression in the yeast Saccharomyces cerevisiae.

Fusion proteins for cell surface expression in the yeast Saccharomyces cerevisiae were constructed that consisted of the N-terminal leader sequence of Kre1p, followed by the nine amino acid viral epitope hemagglutinin (HA), and the carboxyterminal anchoring domain of either Cwp2p or Flo1p. All fusions were constitutively expressed under transcriptional control of the phosphoglycerate kinase promoter and immunofluorescence analysis indicated that in each construct the HA peptide was correctly anchored to the outer yeast cell surface. Successful solubilization of the cell wall fusions by laminarinase treatment indicated that the fusions are covalently linked to cell wall beta-1,3- D-glucans in vivo. FACS analyses further demonstrated that 70% of the yeast cell population expressed the corresponding cell wall fusion. Neither the number of positive cells within the population nor the distribution of the fusion at the single-cell level were negatively affected by replacing the "heterologous" Kre1p leader by the "native" Cwp2p leader. Insertion of a 350 amino acid Ser/Thr-rich spacer sequence into the fusions led to a dramatic increase in HA peptide accessibility on the yeast cell surface. Our data show that FACS analyses represent a valuable means for investigating cell surface expression, and indicate that artificial-spacer-elongated cell wall fusions might raise novel possibilities for cell surface expression of heterologous proteins in yeast.

Expression, processing and high level secretion of a virus toxin in fission yeast.

The virally encoded K28 toxin of Saccharomyces cerevisiae kills sensitive yeast cells in a multi-step receptor-mediated fashion by cell cycle arrest and inhibition of DNA synthesis. In vivo, the toxin is translated as a 38 kDa preprotoxin (pptox) which is enzymatically processed to the biologically active alpha/beta heterodimer during passage through the yeast secretory pathway. Here, we demonstrate that Schizosaccharomyces pombe, a yeast from which no natural toxin-secreting killer strains are known, is perfectly capable of expressing a killer phenotype. Episomal as well as integrating K28 pptox gene cassettes were constructed that allowed a tightly thiamine-regulated killer phenotype expression under transcriptional control of the Sch. pombe nmt1 promotor. Northern analysis of the toxin-coding transcript as well as Western analysis of the secreted toxin indicated that fission yeast is capable of expressing a correctly processed and fully functional virus toxin. Moreover, toxin secretion in recombinant Sch. pombe was at least ten-fold higher than in any natural and/or recombinant Sac. cerevisiae killer strain, indicating that pptox-derived vectors might be attractive in the fast growing field of heterologous protein expression and secretion in yeast.

Kinetically controlled cross-metathesis reactions with high E-olefin selectivities.

[reaction: see text] Homoallylic alcohols with anti-allylic substituents display enhanced E-olefin selectivity in cross-metathesis (CM) reactions with allyltrimethylsilane. The high selectivity can be explained via a five-membered chelate intermediate in which the hydroxyl group of the homoallylic alcohol coordinates to the ruthenium metal center of Grubbs' catalyst.

Endocytotic uptake and retrograde transport of a virally encoded killer toxin in yeast.

We demonstrate that a virally encoded yeast 'killer' toxin is entering its eukaryotic target cell by endocytosis, subsequently travelling the yeast secretory pathway in reverse to exhibit its lethal effect. The K28 killer toxin is a secreted alpha/beta heterodimer that kills sensitive yeasts in a receptor-mediated fashion by blocking DNA synthesis in the nucleus. In vivo processing of the toxin precursor results in a protein whose beta-C-terminus carries the endoplasmic reticulum (ER) retention signal HDEL, which, as we show here, is essential for retrograde toxin transport. Yeast end3/4 mutants as well as cells lacking the HDEL receptor (Deltaerd2) or mutants defective in Golgi-to-ER protein recycling (erd1) are toxin resistant because the toxin can no longer enter and/or retrograde pass the cell. Site-directed mutagenesis further indicated that the toxin's beta-HDEL motif ensures retrograde transport, although in a toxin-secreting yeast the beta-C-terminus is initially masked by an R residue (beta-HDELR) until Kex1p cleavage uncovers the toxin's targeting signal in a late Golgi compartment. Prevention of Kex1p processing results in high-level secretion of a biologically inactive protein incapable of re-entering the secretory pathway. Finally, we present evidence that ER-to-cytosol toxin export is mediated by the Sec61p translocon and requires functional copies of the lumenal ER chaperones Kar2p and Cne1p.

Retroviral vector targeting to human immunodeficiency virus type 1-infected cells by receptor pseudotyping.

We report the generation of retroviral vectors based on Moloney murine leukemia virus that specifically transduce cells infected with T-cell-tropic human immunodeficiency virus type 1 (HIV-1). This vector was pseudotyped with T-cell-tropic HIV-1 receptors CD4 and CXCR4. We demonstrate that transduction is contingent upon HIV-1 gp120 and gp41 expression.

Wave optics simulation of atmospheric turbulence and reflective speckle effects in CO2 lidar.

Laser speckle can influence lidar measurements from a diffuse hard target. Atmospheric optical turbulence will also affect the lidar return signal. We present a numerical simulation that models the propagation of a lidar beam and accounts for both reflective speckle and atmospheric turbulence effects. Our simulation is based on implementing a Huygens-Fresnel approximation to laser propagation. A series of phase screens, with the appropriate atmospheric statistical characteristics, are used to simulate the effect of atmospheric turbulence. A single random phase screen is used to simulate scattering of the entire beam from a rough surface. We compare the output of our numerical model with separate CO(2) lidar measurements of atmospheric turbulence and reflective speckle. We also compare the output of our model with separate analytical predictions for atmospheric turbulence and reflective speckle. Good agreement was found between the model and the experimental data. Good agreement was also found with analytical predictions. Finally, we present results of a simulation of the combined effects on a finite-aperture lidar system that are qualitatively consistent with previous experimental observations of increasing rms noise with increasing turbulence level.

LFG: an anti-apoptotic gene that provides protection from Fas-mediated cell death.

Programmed cell death regulates a number of biological phenomena, and the apoptotic signal must itself be tightly controlled to avoid inappropriate cell death. We established a genetic screen to search for molecules that inhibit the apoptotic signal from the Fas receptor. Here we report the isolation of a gene, LFG, that protects cells uniquely from Fas but not from the mechanistically related tumor necrosis factor alpha death signal. LFG is widely distributed, but remarkably is highly expressed in the hippocampus. LFG can bind to the Fas receptor, but does not regulate Fas expression or interfere with binding of an agonist antibody. Furthermore LFG does not inhibit binding of FADD to Fas.

Unique double-stranded RNAs responsible for the anti-Candida activity of the yeast Hanseniaspora uvarum.

Killer strains of the yeast Hanseniaspora uvarum contain cytoplasmic double-stranded RNAs (dsRNAs) of 4.7-kbp L and 1.0-kbp M species, which were shown to be separately packaged into icosahedral virus-like particles exhibiting RNA-dependent RNA polymerase activity. The L genome of the H. uvarum L-dsRNA virion HuV-L was shown to encode a 77-kDa major capsid protein. Peptide maps of the purified HuV coat protein and the 81-kDa major capsid protein from K1 killer viruses of Saccharomyces cerevisiae revealed distinctly different peptide patterns, suggesting significant sequence divergence at the level of the capsid-coding L-dsRNAs. In vitro transcripts from purified HuV-L particles showed no cross-hybridization to denatured L(A), L(B), or L(C), indicating that L from H. uvarum represents a unique L-dsRNA species. Weak, but clearly detectable cross-hybridization of the 1.0-kb dsRNA of HuV-M, encoding the secreted 18-kDa anti-Candida toxin, to the toxin-coding M genomes of S. cerevisiae K1, K2, and K28 killers indicated partial sequence homology among all of the M-dsRNAs tested.