Reversible Photocontrol of Deoxyribozyme-Catalyzed RNA Cleavage under Multiple-Turnover Conditions

Lights, camera, action! Photoswitchable nucleoside analogues containing o-, m-, or p-azobenzenes can be inserted in the catalytic core of RNA-cleaving 10-23 deoxyribozymes by replacing a nonconserved residue (see picture). Irradiation of the modified deoxyribozymes at 366 nm enhances RNA cleavage rates up to ninefold, thus achieving the rates observed for the unmodified deoxyribozyme.

Passive Control of Plume Interference on Slender Axisymmetric Bodies

The physics of the plume-induced shock and separation, particularly at high plume to exit pressure ratios with and without shock-turbulent boundary-layer control methods, were studied using computational techniques. Mass-averaged Navier-Stokes equations with a two-equation turbulence model were solved by using a fully implicit finite volume scheme and time.marching algorithm. The control methodologies for shock interactions included a porous tail and a porous extension attached at the nozzle exit or trailing edge. The porous tail produced a weaker shock and fixed the shock position on the control surface. The effect of the porous extension on shock interactions was mainly to restrain the plume from strongly underexpanding during a change in flight conditions. These techniques could give an additional dimension to the design and control of supersonic missiles.

Cognitive inhibition and interference in dissociative indentity disorder: the effects of anxiety on specific executive functions:The effects of anxiety on specific executive functions

Using an experimentally based, computer-presented task, this study assessed cognitive inhibition and interference in individuals from the dissociative identity disorder (DID; n=12), generalized anxiety disorder (GAD; n=12) and non-clinical (n=12) populations. Participants were assessed in a neutral and emotionally negative (anxiety provoking) context, manipulated by experimental instructions and word stimuli. The DID sample displayed effective cognitive inhibition in the neutral but not the anxious context. The GAD sample displayed the opposite findings. However, the interaction between group and context failed to reach significance. There was no indication of an attentional bias to non-schema specific negative words in any sample. Results are discussed in terms of the potential benefit of weakened cognitive inhibition during anxious arousal in dissociative individuals.

IMPACT OF RNA VIRUSES ON THE REGULATION OF IL-23 IN MOUSE AND HUMAN MODELS OF INFECTION.

Interluekin-23 (IL-23) is a pro-inflammatory cytokine critical to the regulation of innate and adaptive immune responses. The main role for this cytokine is in the proliferation and differentiation of the IL-17 producing CD4 T helper cell, Th17. Virus infection deregulates IL-23 expression and function, but little is known about the mechanism behind this phenomena. Here, I demonstrate a reduction of Toll like receptor (TLR) ligand-induced IL-23 expression in lymphocytic choriomeningitis virus (LCMV)-infected bone marrow-derived dendritic cells (BMDCs), indicating that a function of these cells is disrupted during virus infection. I propose a mechanism of TLR ligand-induced IL-23 expression inhibition upon LCMV infection via the deactivation of p38, AP-1, and NF-κB. Further analysis revealed a direct relationship between LCMV infection with the IL-10 and SOCS3 expression. To understand IL-23 function, I characterized IL-23-induced JAK/STAT signalling pathway and IL-23 receptor expression on human CD4 T cells. My results demonstrate that IL-23 induces activation of p-JAK2, p-Tyk2, p-STAT1, p-STAT3, and p-STAT4 in CD4 T cells. For the first time I show that IL-23 alone induces the expression of its own receptor components, IL-12Rβ1 and IL-23Rα, in CD4 T cells. Blocking JAK2, STAT1, and STAT3 activation with specific inhibitors detrimentally effected expression of IL-23 receptor demonstrating that activation of JAK/STAT signalling is important for IL-23 receptor expression. I also addressed the effect of viral infection on IL-23 function and receptor expression in CD4 T cells using cells isolated from HIV positive individuals. These studies were based on earlier reports that the expression of IL-23 and the IL-23 receptor are impaired during HIV infection. I demonstrate that the phosphorylation of JAK2, STAT1, and STAT3 induced by IL-23, as well as IL-23 receptor expression are deregulated in CD4 T cells isolated from HIV positive individuals. This study has furthered the understanding of how the expression and function of IL-23 is regulated during viral infections.

Rational Attenuation of a Morbillivirus by Modulating the Activity of the RNA-Dependent RNA Polymerase

Negative-strand RNA viruses encode a single RNA-dependent RNA polymerase (RdRp) which transcribes and replicates the genome. The open reading frame encoding the RdRp from a virulent wild-type strain of rinderpest virus (RPV) was inserted into an expression plasmid. Sequences encoding enhanced green fluorescent protein (EGFP) were inserted into a variable hinge of the RdRp. The resulting polymerase was autofluorescent, and its activity in the replication/transcription of a synthetic minigenome was reduced. We investigated the potential of using this approach to rationally attenuate a virus by inserting the DNA sequences encoding the modified RdRp into a full-length anti-genome plasmid from which a virulent virus (rRPV(KO)) can be rescued. A recombinant virus, rRPV(KO)L-RRegfpR, which grew at an indistinguishable rate and to an identical titer as rRPV(KO) in vitro, was rescued. Fluorescently tagged polymerase was visible in large cytoplasmic inclusions and beneath the cell membrane. Subcutaneous injection of 10(4) TCID(50) of the rRPV(KO) parental recombinant virus into cattle leads to severe disease symptoms (leukopenia/diarrhea and pyrexia) and death by 9 days postinfection. Animals infected with rRPV(KO)L-RRegfpR exhibited transient leukopenia and mild pyrexia, and the only noticeable clinical signs were moderate reddening of one eye and a slight ocular-nasal discharge. Viruses that expressed the modified polymerase were isolated from peripheral blood lymphocytes and eye swabs. This demonstrates that a virulent morbillivirus can be attenuated in a single step solely by modulating RdRp activity and that there is not necessarily a correlation between virus growth in vitro and in vivo.

Dynamics of Viral RNA Synthesis during Measles Virus Infection

We propose a reference model of the kinetics of a viral RNA-dependent RNA polymerase (vRdRp) activities and its regulation during infection of eucaryotic cells. After measles virus infects a cell, mRNAs from all genes immediately start to accumulate linearly over the first 5 to 6 h and then exponentially until approximately 24 h. The change from a linear to an exponential accumulation correlates with de novo synthesis of vRdRp from the incoming template. Expression of the virus nucleoprotein (N) prior to infection shifts the balance in favor of replication. Conversely, inhibition of protein synthesis by cycloheximide favors the latter. The in vivo elongation speed of the viral polymerase is approximately 3 nucleotides/s. A similar profile with fivefold-slower kinetics can be obtained using a recombinant virus expressing a structurally altered polymerase. Finally, virions contain only encapsidated genomic, antigenomic, and 5'-end abortive replication fragment RNAs.

Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis

Replication of the giant RNA genome of severe acute respiratory syndrome (SARS) coronavirus (CoV) and synthesis of as many as eight subgenomic (sg) mRNAs are mediated by a viral replicase-transcriptase of outstanding complexity that includes an essential endoribonuclease activity. Here, we show that the CoV replicative machinery, unlike that of other RNA viruses, also uses an exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms of SARS-CoV nsp14 were shown to act on both ssRNAs and dsRNAs in a 3'5' direction. The activity depended on residues that are conserved in the DEDD exonuclease superfamily. The protein did not hydrolyze DNA or ribose-2'-O-methylated RNA substrates and required divalent metal ions for activity. A range of 5'-labeled ssRNA substrates were processed to final products of 8–12 nucleotides. When part of dsRNA or in the presence of nonlabeled dsRNA, the 5'-labeled RNA substrates were processed to significantly smaller products, indicating that binding to dsRNA in cis or trans modulates the exonucleolytic activity of nsp14. Characterization of human CoV 229E ExoN active-site mutants revealed severe defects in viral RNA synthesis, and no viable virus could be recovered. Besides strongly reduced genome replication, specific defects in sg RNA synthesis, such as aberrant sizes of specific sg RNAs and changes in the molar ratios between individual sg RNA species, were observed. Taken together, the study identifies an RNA virus ExoN activity that is involved in the synthesis of multiple RNAs from the exceptionally large genomic RNA templates of CoVs.

Simulation of quantum random walks using the interference of a classical field

We suggest a theoretical scheme for the simulation of quantum random walks on a line using beam splitters, phase shifters, and photodetectors. Our model enables us to simulate a quantum random walk using of the wave nature of classical light fields. Furthermore, the proposed setup allows the analysis of the effects of decoherence. The transition from a pure mean-photon-number distribution to a classical one is studied varying the decoherence parameters.