Green Tea Catechin, Epigallocatechin Gallate, Suppresses Signaling by the dsRNA Innate Immune Receptor RIG-I

The Innate immune system constitutes the first line of defense against pathogen infections. The Retinoic acid-inducible gene I (RIG-I) receptor recognizes triphosphorylated ssRNAs and dsRNA to initiate downstream signaling of interferon response. However, unregulated activity of these receptors could lead to autoimmune diseases. We seek to identify small molecules that can specifically regulate RIG-I signaling.Epigallocatechin gallate (EGCG), a polyphenolic catechin present in green tea, was identified in a small molecule screen. It was found to bind RIG-I and inhibits its signaling at low micromolar concentrations in HEK293T cells. Furthermore, EGCG dose-dependently inhibited the ATPase activity of recombinant RIG-I but did not compete with RIG-I interaction with RNA or with ATP. EGCG did not inhibit signaling by Toll-like receptors 3, 4, 9 or constitutive signaling by the adapter protein IPS-1. Structure activity relationship analysis showed that EGCG, its epimer GCG and a digallate-containing compound, theaflavin 3,3' digallate (TFDG) were potent RIG-I inhibitors. EGCG also inhibited IL6 secretion and IFN- β mRNA synthesis in BEAS-2B cells, which harbors intact endogenous RIG-I signaling pathway.EGCG and its derivatives could have potential therapeutic use as a modulator of RIG-I mediated immune responses

A Cell-Based Assay for RNA Synthesis by the HCV Polymerase Reveals New Insights on Mechanism of Polymerase Inhibitors and Modulation by NS5A

RNA synthesis by the genotype 1b hepatitis C virus (HCV) polymerase (NS5B) transiently expressed in Human embryonic kidney 293T cells or liver hepatocytes was found to robustly stimulate RIG-I-dependent luciferase production from the interferon β promoter in the absence of exogenously provided ligand. This cell-based assay, henceforth named the 5BR assay, could be used to examine HCV polymerase activity in the absence of other HCV proteins. Mutations that decreased de novo initiated RNA synthesis in biochemical assays decreased activation of RIG-I signaling. In addition, NS5B that lacks the C-terminal transmembrane helix but remains competent for RNA synthesis could activate RIG-I signaling. The addition of cyclosporine A to the cells reduced luciferase levels without affecting agonist-induced RIG-I signaling. Furthermore, non-nucleoside inhibitor benzothiadiazines (BTDs) that bind within the template channel of the 1b NS5B were found to inhibit the readout from the 5BR assay. Mutation M414T in NS5B that rendered the HCV replicon resistant to BTD was also resistant to BTDs in the 5BR assay. Co-expression of the HCV NS5A protein along with NS5B and RIG-I was found to inhibit the readout from the 5BR assay. The inhibition by NS5A was decreased with the removal of the transmembrane helix in NS5B. Lastly, NS5B from all six major HCV genotypes showed robust activation of RIG-I in the 5BR assay. In summary, the 5BR assay could be used to validate inhibitors of the HCV polymerase as well as to elucidate requirements for HCV-dependent RNA synthesis

Quantum Noise Randomized Ciphers

We review the notion of a classical random cipher and its advantages. We sharpen the usual description of random ciphers to a particular mathematical characterization suggested by the salient feature responsible for their increased security. We describe a concrete system known as AlphaEta and show that it is equivalent to a random cipher in which the required randomization is effected by coherent-state quantum noise. We describe the currently known security features of AlphaEta and similar systems, including lower bounds on the unicity distances against ciphertext-only and known-plaintext attacks. We show how AlphaEta used in conjunction with any standard stream cipher such as AES (Advanced Encryption Standard) provides an additional, qualitatively different layer of security from physical encryption against known-plaintext attacks on the key. We refute some claims in the literature that AlphaEta is equivalent to a non-random stream cipher.Comment: Accepted for publication in Phys. Rev. A; Discussion augmented and re-organized; Section 5 contains a detailed response to 'T. Nishioka, T. Hasegawa, H. Ishizuka, K. Imafuku, H. Imai: Phys. Lett. A 327 (2004) 28-32 /quant-ph/0310168' & 'T. Nishioka, T. Hasegawa, H. Ishizuka, K. Imafuku, H. Imai: Phys. Lett. A 346 (2005) 7

LL37 and Cationic Peptides Enhance TLR3 Signaling by Viral Double-stranded RNAs

BACKGROUND:Toll-like Receptor 3 (TLR3) detects viral dsRNA during viral infection. However, most natural viral dsRNAs are poor activators of TLR3 in cell-based systems, leading us to hypothesize that TLR3 needs additional factors to be activated by viral dsRNAs. The anti-microbial peptide LL37 is the only known human member of the cathelicidin family of anti-microbial peptides. LL37 complexes with bacterial lipopolysaccharide (LPS) to prevent activation of TLR4, binds to ssDNA to modulate TLR9 and ssRNA to modulate TLR7 and 8. It synergizes with TLR2/1, TLR3 and TLR5 agonists to increase IL8 and IL6 production. This work seeks to determine whether LL37 enhances viral dsRNA recognition by TLR3. METHODOLOGY/PRINCIPAL FINDINGS:Using a human bronchial epithelial cell line (BEAS2B) and human embryonic kidney cells (HEK 293T) transiently transfected with TLR3, we found that LL37 enhanced poly(I:C)-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3. The presence of LL37 also increased the cytokine response to rhinovirus infection in BEAS2B cells and in activated human peripheral blood mononuclear cells. Confocal microscopy determined that LL37 could co-localize with TLR3. Electron microscopy showed that LL37 and poly(I:C) individually formed globular structures, but a complex of the two formed filamentous structures. To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling. This is the first demonstration that LL37 and other RNA-binding peptides with cell penetrating motifs can activate TLR3 signaling and facilitate the recognition of viral ligands. CONCLUSIONS/SIGNIFICANCE:LL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA

Viral Double-Strand RNA-Binding Proteins Can Enhance Innate Immune Signaling by Toll-Like Receptor 3

Toll-like Receptor 3 (TLR3) detects double-stranded (ds) RNAs to activate innate immune responses. While poly(I:C) is an excellent agonist for TLR3 in several cell lines and in human peripheral blood mononuclear cells, viral dsRNAs tend to be poor agonists, leading to the hypothesis that additional factor(s) are likely required to allow TLR3 to respond to viral dsRNAs. TLR3 signaling was examined in a lung epithelial cell line by quantifying cytokine production and in human embryonic kidney cells by quantifying luciferase reporter levels. Recombinant 1b hepatitis C virus polymerase was found to enhance TLR3 signaling in the lung epithelial BEAS-2B cells when added to the media along with either poly(I:C) or viral dsRNAs. The polymerase from the genotype 2a JFH-1 HCV was a poor enhancer of TLR3 signaling until it was mutated to favor a conformation that could bind better to a partially duplexed RNA. The 1b polymerase also co-localizes with TLR3 in endosomes. RNA-binding capsid proteins (CPs) from two positive-strand RNA viruses and the hepadenavirus hepatitis B virus (HBV) were also potent enhancers of TLR3 signaling by poly(I:C) or viral dsRNAs. A truncated version of the HBV CP that lacked an arginine-rich RNA-binding domain was unable to enhance TLR3 signaling. These results demonstrate that several viral RNA-binding proteins can enhance the dsRNA-dependent innate immune response initiated by TLR3

Dynamics of folding in Semiflexible filaments

We investigate the dynamics of a single semiflexible filament, under the action of a compressing force, using numerical simulations and scaling arguments. The force is applied along the end to end vector at one extremity of the filament, while the other end is held fixed. We find that, unlike in elastic rods the filament folds asymmetrically with a folding length which depends only on the bending stiffness and the applied force. It is shown that this behavior can be attributed to the exponentially falling tension profile in the filament. While the folding time depends on the initial configuration, at late time, the distance moved by the terminal point of the filament and the length of the fold shows a power law dependence on time with an exponent 1/2.Comment: 13 pages, Late

Poly-Thymidine Oligonucleotides Mediate Activation of Murine Glial Cells Primarily Through TLR7, Not TLR8

The functional role of murine TLR8 in the inflammatory response of the central nervous system (CNS) remains unclear. Murine TLR8 does not appear to respond to human TLR7/8 agonists, due to a five amino acid deletion in the ectodomain. However, recent studies have suggested that murine TLR8 may be stimulated by alternate ligands, which include vaccinia virus DNA, phosphothioate oligodeoxynucleotides (ODNs) or the combination of phosphothioate poly-thymidine oligonucleotides (pT-ODNs) with TLR7/8 agonists. In the current study, we analyzed the ability of pT-ODNs to induce activation of murine glial cells in the presence or absence of TLR7/8 agonists. We found that TLR7/8 agonists induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines in mixed glial cultures. In contrast, pT-ODNs alone induced only low level expression of two cytokines, CCL2 and CXCL10. The combination of pT-ODNs along with TLR7/8 agonists induced a synergistic response with substantially higher levels of proinflammatory cytokines and chemokines compared to CL075. This enhancement was not due to cellular uptake of the agonist, indicating that the pT-ODN enhancement of cytokine responses was due to effects on an intracellular process. Interestingly, this response was also not due to synergistic stimulation of both TLR7 and TLR8, as the loss of TLR7 abolished the activation of glial cells and cytokine production. Thus, pT-ODNs act in synergy with TLR7/8 agonists to induce strong TLR7-dependent cytokine production in glial cells, suggesting that the combination of pT-ODNs with TLR7 agonists may be a useful mechanism to induce pronounced glial activation in the CNS

Crystal structure of RIG-I C-terminal domain bound to blunt-ended double-strand RNA without 5′ triphosphate

RIG-I recognizes molecular patterns in viral RNA to regulate the induction of type I interferons. The C-terminal domain (CTD) of RIG-I exhibits high affinity for 5′ triphosphate (ppp) dsRNA as well as blunt-ended dsRNA. Structures of RIG-I CTD bound to 5′-ppp dsRNA showed that RIG-I recognizes the termini of dsRNA and interacts with the ppp through electrostatic interactions. However, the structural basis for the recognition of non-phosphorylated dsRNA by RIG-I is not fully understood. Here, we show that RIG-I CTD binds blunt-ended dsRNA in a different orientation compared to 5′ ppp dsRNA and interacts with both strands of the dsRNA. Overlapping sets of residues are involved in the recognition of blunt-ended dsRNA and 5′ ppp dsRNA. Mutations at the RNA-binding surface affect RNA binding and signaling by RIG-I. These results provide the mechanistic basis for how RIG-I recognizes different RNA ligands

Robust water repellent ZnO nanorod array by Swift Heavy Ion Irradiation: Effect of Electronic Excitation Induced Local Chemical State Modification

Tailoring the surface properties by varying the chemistry and roughness could be of interest for self-cleaning applications. We demonstrate the transformation of hydrophobic ZnO Nano rod (NR) array into superhydrophobic nature by changing the local chemical state and without altering the surface roughness by swift heavy ion (SHI) irradiation. The aligned ZnO NR arrays were irradiated using 150 MeV Ag ions with different fluences from 5E10 to 3E12 ions/cm2. The observed static water contact angles of ZnO NRs samples were 103° ± 3°, 152° ± 4°,161° ± 3°, 164° ± 2°, 167° ± 2°,154 ± 3° and 151° ± 2° for the pristine, ion fluencies of 1E11, 3E11, 5E11, 7E11, 1E12 and 3E12 ions cm−2, respectively. The change in local surface chemistry via formation of surface oxygen related defects due to electronic excitations induced by ion irradiation determine the water dewetting properties. It is found that surface oxygen related defects could be tuned by varying the fluence of the SHIs. Durability tests show that the SHI induced surface oxygen-deficient ZnO NRs have the stable superhydrophobic behavior for more than a year