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An enzyme-free amplified detection platform is described using the horseradish peroxidase (HRP)-mimicking DNAzyme as an amplifying label. Two hairpin structures that include three-fourths and one-fourth of the HRP-mimicking DNAzyme in caged, inactive configurations are used as functional elements for the amplified detection of the target DNA. In the presence of the analyte DNA, one of the hairpins is opened, and this triggers the autonomous cross-opening of the two hairpins using the strand displacement principle. This leads to the formation of nanowires consisting of the HRP-mimicking DNAzyme. The resulting DNA nanowires act as catalytic labels for the colorimetric or chemiluminescent readout of the sensing processes (the term "enzyme-free" refers to a protein-free catalyst). The analytical platform allows the sensing of the analyte DNA with a detection limit corresponding to 1 × 10(-13) M. The optimized system acts as a versatile sensing platform, and by coaddition of a "helper" hairpin structure any DNA sequence may be analyzed by the system. This is exemplified with the detection of the BRCA1 oncogene with a detection limit of 1 × 10(-13) M.

A systematic study of the amplified optical detection of DNA by Mg(2+)-dependent DNAzyme subunits is described. The use of two DNAzyme subunits and the respective fluorophore/quencher-modified substrate allows the detection of the target DNA with a sensitivity corresponding to 1 × 10(-9) M. The use of two functional hairpin structures that include the DNAzyme subunits in a caged, inactive configuration leads, in the presence of the target DNA, to the opening of one of the hairpins and to the activation of an autonomous cross-opening process of the two hairpins, which affords polymer DNA wires consisting of the Mg(2+)-dependent DNAzyme subunits. This amplification paradigm leads to the analysis of the target DNA with a sensitivity corresponding to 1 × 10(-14) M. The amplification mixture composed of the two hairpins can be implemented as a versatile sensing platform for analyzing any gene in the presence of the appropriate hairpin probe. This is exemplified with the detection of the BRCA1 oncogene.

Communication between cancer cells and the microenvironment appears to be an important determinant of disease prognosis. However, the detailed mechanisms of the interactions between cancer cells and surrounding cells have yet to be clarified. Recent studies on cell fusion have indicated this interaction to be one of the driving forces in cancer progression. Fibroblasts constitute a significant component of the carcinoma stromal compartment. Many of these fibroblasts are thought to differentiate into myofibroblasts, which are characterized by a positive expression of α-smooth muscle actin. Expression of α-smooth muscle actin in osteosarcoma was evaluated, and was observed to be excessive in the multinucleated osteoclast-like giant cells in osteosarcoma tissue, indicating the possibility of cell fusion between cancer cells and myofibroblasts. In order to test the above hypothesis, we first transformed the primary mouse embryonic fibroblast cells into activated myofibroblast cells. Osteosarcoma cells were then co-cultured with mouse myofibroblast cells, and cell fusion was investigated using species-specific chromosomal markers. Expression of α-smooth muscle actin was successfully induced in primary mouse embryonic fibroblast cells. Cells fused spontaneously with a fusion rate of approximately 1-2% and fusion between more than two cells was also observed. Our study demonstrated that fusion between cancer cells and myofibroblasts may contribute to the observed multinucleated giant cells in osteosarcoma. We posit that cell fusion is a novel mechanism for the interaction between cancer cells and the microenvironment.

There is increasing evidence that natural killer (NK) cells play an important role in antitumor immunity following dendritic cell (DC) vaccination. Little is known, however, about the optimal stimulation of DCs that favors NK activation in tumor-bearing hosts. In this study, we demonstrate that treatment with toll-like receptor (TLR) ligands and infection with a mutant vesicular stomatitis virus (VSV-ΔM51) both induced DC maturation. Further, inoculation of these DCs led to robust NK-mediated protection against tumor challenge. Strikingly, only VSV-ΔM51-infected DCs were capable of suppressing the growth of established tumors, suggesting that additional signals provided by viral infection may be required to activate tumoricidal NK cells in tumor-bearing hosts. VSV-ΔM51 infection of DCs induced greater type I interferon (IFN I) production than TLR ligand treatment, and disruption of the IFN I pathway in DCs eliminated their ability to induce NK activation and tumor protection. However, further studies indicated that IFN I alone was not sufficient to activate NK cells, especially in the presence of a tumor, and DC-derived IL-15 was additionally required for tumoricidal NK activation. These results suggest that induction of IFN I by VSV-ΔM51 allows DCs to overcome tumor-associated immunosuppression and facilitate IL-15-mediated priming of tumoricidal NK cells. Thus, the mode of DC maturation should be carefully considered when designing DC-based cancer immunotherapies.

A method to fabricate AuAg bimetallic nanoparticles film by H(2)O(2)-mediated reduction of silver was reported. Gold nanoparticles (Au NPs) were first adsorbed onto the surface of a self-assembled 2-aminoethanethiol monolayer-modified gold film or 3-aminopropyltriethoxysilane (APTES) monolayer-modified quartz slide. Upon further treatment of this modified film with the solution containing silver nitrate (AgNO(3)) and H(2)O(2), silver was deposited on the surface of Au NPs. The size of the AuAg bimetallic particles could be readily tuned by manipulating the concentration of H(2)O(2). Surface plasmon resonance (SPR) was used to investigate the process, the deposition of silver on Au NPs modified gold film resulted in an obvious decrease of depth in the SPR reflectance intensity and minimum angle curves (SPR R-theta curves), which may be utilized for the quantitative SPR detection of the analyte, H(2)O(2). Combination of the biocatalytic reaction that could yield H(2)O(2) by using the enzyme, glucose oxidase, with the deposition of silver may enable the design of a glucose biosensor by SPR technique. Furthermore, we evaluated the AuAg bimetallic nanoparticles film for their ability to be an effective substrate for surface-enhanced Raman scattering (SERS)., (Copyright 2010 Elsevier B.V. All rights reserved.)

Single-walled carbon nanohorns (SWNHs), distinguished by their high purity and distinct structure, were noncovalently functionalized with poly(sodium 4-styrenesulfonate). The functionalized SWNHs were characterized by scanning electron microscopy, atomic force microscopy, ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, and thermogravimetry. Heme protein myoglobin was adsorbed onto surface of functionalized SWNHs to prepare electrochemical biosensor. Surface assembly process and direct electrochemistry of immobilized myoglobin were investigated by electrochemical impedance spectroscopy and cyclic voltammetry, respectively. The proposed biosensor exhibited good electrocatalysis to the reduction of hydrogen peroxide. The response was linear over the range 3-350 microM with a detection limit of 0.5 microM. Good reproducibility and stability of the biosensor were obtained toward hydrogen peroxide detection., (Copyright 2010 Elsevier B.V. All rights reserved.)

Biological systems that are capable of performing computational operations could be of use in bioengineering and nanomedicine, and DNA and other biomolecules have already been used as active components in biocomputational circuits. There have also been demonstrations of DNA/RNA-enzyme-based automatons, logic control of gene expression, and RNA systems for processing of intracellular information. However, for biocomputational circuits to be useful for applications it will be necessary to develop a library of computing elements, to demonstrate the modular coupling of these elements, and to demonstrate that this approach is scalable. Here, we report the construction of a DNA-based computational platform that uses a library of catalytic nucleic acids (DNAzymes), and their substrates, for the input-guided dynamic assembly of a universal set of logic gates and a half-adder/half-subtractor system. We demonstrate multilayered gate cascades, fan-out gates and parallel logic gate operations. In response to input markers, the system can regulate the controlled expression of anti-sense molecules, or aptamers, that act as inhibitors for enzymes.

Myxoma virus (MV) is a highly lethal, rabbit-specific poxvirus that induces a disease called myxomatosis in European rabbits. In an effort to understand the function of predicted immunomodulatory genes we have deleted various viral genes from MV and tested the ability of these knockout viruses to induce lethal myxomatosis. MV encodes a unique 15 kD cytoplasmic protein (M130R) that is expressed late (12h post infection) during infection. M130R is a non-essential gene for MV replication in rabbit, monkey or human cell lines. Construction of a targeted gene knockout virus (vMyx130KO) and infection of susceptible rabbits demonstrate that the M130R knockout virus is attenuated and that loss of M130R expression allows the rabbit host immune system to effectively respond to and control the lethal effects of MV. M130R expression is a bona fide poxviral virulence factor necessary for full and lethal development of myxomatosis.

Mouse embryo fibroblasts (MEFs) are a widely used cell culture system in life sciences, including virology. Here, we show that although primary MEFs are nonpermissive to myxoma virus replication, the corresponding immortalized MEFs support a highly productive myxoma virus infection. We further demonstrate that this permissive phenotype for myxoma virus in immortalized MEFs is due to the immortalization-associated selective block to the cellular alpha/beta interferon induction machinery involved in responding to myxoma virus challenge. Thus, our report presents a clear example, illustrating that a drastic phenotypic alteration can occur with respect to virus infection between primary cells and their immortalized counterparts.

Poxviruses currently are known to disrupt Jak-STAT signal transduction induced by interferon (IFN) through two distinct mechanisms: (1) secreted poxviral IFN decoy receptors that prevent the initiation of IFN signaling from type I or II receptors at the cell surface; and (2) poxviral phosphatase that dephosphorylates STAT1 intracellularly. Here, we report a novel mechanism by which poxviruses can inhibit Jak-STAT signaling in response to type I IFN. Myxoma virus (MV) is a highly species-restricted member of the poxvirus family that infects only rabbits under the natural setting. Interestingly, primary human fibroblasts support a permissive MV infection that is only partially sensitive to the antiviral state induced by type I IFN. In this study we show that when type I IFN is added to primary human fibroblasts following MV infection, the tyrosine phosphorylation of the Janus kinase Tyk2 is specifically blocked, thereby preventing the subsequent activation of downstream STAT1 and STAT2. In stark contrast, type II IFN-induced activation of Jak1, Jak2 and STAT1 remains largely unaffected in MV-infected human fibroblasts. Unlike the de-activation of STAT1 by the poxvirus phosphatase, which is delivered into the cell by the input virions, the Tyk2 inhibition by MV infection requires new viral gene expression. Thus, our study documents a previously unrecognized immune evasion mechanism exploited by a poxvirus to selectively disrupt the type I IFN-Jak-STAT signaling cascade.

We report a sensitively amplified electrochemical aptasensor using adenosine triphosphate (ATP) as a model. ATP is a multifunctional nucleotide that is most important as a "molecular currency" of intracellular energy transfer. In the sensing process, duplexes consisting of partly complementary strand (PCS1), ATP aptamer (ABA) and another partly complementary strand (PCS2) were immobilized onto Au electrode through the 5'-HS on the PCS1. Meanwhile, PCS2 was grafted with the Au nanoparticles (AuNPs) to amplify the detection signals. In the absence of ATP, probe methylene blue (MB) bound to the DNA duplexes and also bound to guanine bases specifically to produce a strong differential pulse voltammetry (DPV) signal. But when ATP exists, the ABA-PCS2 or ABA-PCS1 part duplexes might be destroyed, which decreased the amount of MB on the electrode and led to obviously decreased DPV signal. This phenomenon can be used to detect ATP and get a very sensitive detection limit low to 0.1nM, and the detection range could extend up to 10(-7)M. Compared to the sensing platform without PCS2 grafted AuNPs, amplified function of this sensing system was also evidently proved. Therefore, such PCS1-ABA-PCS2/AuNPs sensing system could provide a promising signal-amplified model for aptamer-based small-molecules detection.

We developed a stable, sensitive electrochemiluminescence (ECL) biosensor based on the synthesis of a new sol-gel material with the ion-exchange capacity sol-gel to coimmobilize the Ru(bpy)(3)(2+) and enzyme. The partial sulfonated (3-mercaptopropyl)-trimethoxysilane sol-gel (PSSG) film acted as both an ion exchanger for the immobilization of Ru(bpy)(3)(2+) and a matrix to immobilize gold nanoparticles (AuNPs). The AuNPs/PSSG/Ru(bpy)(3)(2+) film modified electrode allowed sensitive the ECL detection of NADH as low as 1 nM. Such an ability of AuNPs/PSSG/Ru(bpy)(3)(2+) film to promote the electron transfer between Ru(bpy)(3)(2+) and the electrode suggested a new, promising biocompatible platform for the development of dehydrogenase-based ECL biosensors. With alcohol dehydrogenase (ADH) as a model, we then constructed an ethanol biosensor, which had a linear range of 5 microM to 5.2 mM with a detection limit of 12nM.

A NADH and glucose biosensor based on thionine cross-linked multiwalled carbon nanotubes (MWNTs) and Au nanoparticles (Au NPs) multilayer functionalized indium-doped tin oxide (ITO) electrode were presented in this paper. The effect of light irradiation on the enhancement of bioelectrocatalytic processes of the biocatalytic systems by the photovoltaic effect was investigated. This bioelectrode exhibited excellent catalytic activity of the oxidation towards dihydronicotinamide adenine dinucleotide (NADH). Most interesting, the performance of this NADH sensor could be tuned by the visible light. When the biosensor was performed in the dark, the anodic current increased linearly with NADH concentration over the range from 0.5 to 237 microM with detection limit 0.1 microM and sensitivity 17 nA microM(-1). The sensitivity became 115 nA microM(-1) with detection limit 0.05 microM with the light irradiation. Compared with the reaction in dark, the sensitivity increased around 7 folds while the detection limit decreased 2 folds. The glucose biosensor also exhibited the same behavior. The linear range was from 10 microM to 2.56 mM with the sensitivity of 7.8 microAmM(-1) and detection limit 5.0 microM in the dark. After the light irradiation, the linear range was from 1 microM to 3.25 mM with the sensitivity of 18.5 microA mM(-1) and detection limit 0.7 microM. It indicated a potential to provide an operational access to develop new kinds of photocontrolled dehydrogenase enzyme-based bioelectronics.

In this work, we illustrate a simple chelation-based strategy to trigger DNA release from DNA-incorporated multilayer films, which were fabricated through the layer-by-layer (LbL) assembly of DNA and inorganic zirconium (IV) ion (Zr(4+)). After being incubated in several kinds of chelator solutions, the DNA multilayer films disassembled and released the incorporated DNA. This was most probably due to the cleavage of coordination/electrostatic interactions between Zr(4+) and phosphate groups of DNA. Surface plasmon resonance (SPR), UV-vis spectrometry and atomic force microscopy (AFM) were used to characterize the assembly and the disassembly of the films. By incorporating plasmid DNA (pDNA) into this controllable disassembly system, the multilayer films sustained the consecutive DNA release. The released pDNA retained its integrity and transcriptional activity, and also expressed enhanced green fluorescent protein (EGFP) after being transfected into HEK 293 cells. Besides the simplicity and cost efficiency of this method, the most advantage of this route was that the release of DNA from the films could be modulated by various external conditions, such as the chelator and ionic strength. The Zr(4+)/DNA multilayer films with the ability to precisely control the release rate of DNA might serve as an alternative localized gene delivery system in the perspective of biomedical applications.

A one-compartment glucose/O(2) biofuel cell based on an electrostatic layer-by-layer (LbL) technique on three-dimensional ordered macroporous (3DOM) gold electrode was described. A 3DOM gold electrode was synthesized electrochemically by an inverted colloidal crystal template technique. Then the macroporous gold electrodes were functionalized with Au nanoparticles (AuNPs) and enzyme, glucose dehydrogenase (GDH) or laccase. The (AuNPs/GDH)(n) multilayer modified macroporous gold electrode showed excellent bioelectrocatalytic activity towards glucose. The direct electroreduction towards oxygen was realized at (AuNPs/laccase)(n) films on 3DOM gold electrodes. The maximum power density of the cell with the macroporous film as matrix was 178 microW cm(-2) at 226 mV, which was 16 times larger than that of the biofuel cell with the flat electrode under the same condition. The proposed method is simple and would be applicable to enhance the power output of miniaturized biofuel cell.

A layer-by-layer film composed of DNA and inorganic zirconium ion (Zr(4+)) was fabricated on the surface of gold thin film, and an electric field triggered disintegration of the multilayer film was studied by using electrochemical surface plasmon resonance (EC-SPR). EC-SPR results demonstrated that the film was disassembled upon the application of an electric field and the disassembly rate varied with the applied potential, leading to the controlled release of DNA. The electrodissolution could be switched off by removing the electric potential and reactivated by reapplying the potential. By incorporating plasmid DNA (pDNA) in to this controlled release system, the multilayer film could sustain the consecutive release of pDNA electrochemically. The released pDNA retained its integrity and transfection activity, and expressed enhanced green fluorescent protein (EGFP) after being transfected into HEK 293 cells. The electrochemical systems, with advantages of miniaturization, surface-tailoring, safety, simplicity, convenience, automation, low-cost, and free of immune reactions, made the electrical route a very attractive gene-delivery alternative.

The sensing of pathogen infection and subsequent triggering of innate immunity are key to controlling zoonotic infections. Myxoma virus (MV) is a cytoplasmic DNA poxvirus that in nature infects only rabbits. Our previous studies have shown that MV infection of primary mouse cells is restricted by virus-induced type I interferon (IFN). However, little is known about the innate sensor(s) involved in activating signaling pathways leading to cellular defense responses in primary human immune cells. Here, we show that the complete restriction of MV infection in the primary human fibroblasts requires both tumor necrosis factor (TNF) and type I IFN. We also demonstrate that MV infection of primary human macrophages (pHMs) activates the cytoplasmic RNA sensor called retinoic acid inducible gene I (RIG-I), which coordinately induces the production of both TNF and type I IFN. Of note, RIG-I sensing of MV infection in pHMs initiates a sustained TNF induction through the sequential involvement of the downstream IFN-regulatory factors 3 and 7 (IRF3 and IRF7). Thus, RIG-I-mediated co-induction of TNF and type I IFN by virus-infected pHMs represents a novel innate defense mechanism to restrict viral infection in human cells. These results also reveal a new regulatory mechanism for TNF induction following viral infection.

In this paper, electrochemical surface plasmon resonance (SPR) method was first used to detect enzymatic reaction in bilayer lipid membrane (BLM) based on immobilizing horseradish peroxidase (HRP) in the BLMs supported by the redox polyaniline (PAn) film. By SPR kinetic curve in situ monitoring the redox transformation of PAn film resulted from the reaction between HRP and PAn, the enzymatic reaction of HRP with H(2)O(2) was successfully analyzed by electrochemical SPR spectroscopy. The results show that this BLM supported on PAn film cannot only preserve the bioactivity of HRP immobilized in the membrane, but also provide a channel for the transfer of electrons between HRP and PAn on electrode surface. These characteristics enabled the development of SPR biosensor for sensitively detecting H(2)O(2). H(2)O(2) has been detected by electrochemical SPR spectroscopy in the concentration range of 5 x 10(-5)M to 2 x 10(-3)M. After each of detections, the SPR sensor surface was completely regenerated by electrochemically reducing the oxidized PAn to its reduced state. This method provides a novel route for enhancing the detection of small ligand of enzymatic reaction in BLM by electrochemical SPR spectroscopy.

The authors have recently demonstrated that wild-type myxoma virus (MV) tagged with gfp (vMyxgfp) can generate a tumor-specific infection that productively infects and clears human tumor-derived xenografts when injected intratumorally into human gliomas transplanted into immunodeficient mice (Lun et al, 2005). To expand the understanding of MV tropism in cancer cells from a specific tissue lineage, the authors have screened a series of human glioma cells (U87, U118, U251, U343, U373) for myxoma virus replication and oncolysis. To assess the viral tropism determinants for these infections, the authors have screened myxoma virus knockout constructs that have been deleted for specific host range genes (M-T2, M-T4, M-T5, M11L, and M063), as well as a control MV gene knockout construct with no known host range function (vMyx135KO) but is highly attenuated in rabbits. The authors report wide variation in the ability of various vMyx-hrKOs to replicate and spread in the human glioma cells as measured by early and late viral gene expression. This differential ability to support vMyx-hrKO productive viral replication is consistent with levels of endogenous activated Akt in the various gliomas. The authors have identified one vMyx-hrKO virus (vMyx63KO) and one nonhost range knockout construct (vMyx135KO) that appear to replicate in the gliomas even more efficiently than the wild-type virus and that reduce the viability of the infected gliomas. These knockout viruses also inhibit the proliferation of gliomas in a manner similar to the wild-type virus. Together these data, as well as the fact that these knockout viruses are attenuated in their natural hosts, may represent environmentally safer candidate oncolytic viruses for usage in human trials.

Through electrostatic layer-by-layer (LbL) assembly, negatively charged calf thymus double stranded DNA (CTds-DNA), and positively charged Zr4+ ions were alternately deposited on gold substrate modified with chemisorbed cysteamine. Thus-prepared three-dimensional DNA networks were characterized by surface plasmon resonance (SPR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and infrared reflection-absorption spectroscopy (IR-RAS). SPR spectroscopy indicates that the effective thickness of DNA monolayer in the (DNA/Zr4+)(1) bilayer was 1.5+/-0.1 nm, which corresponds to the surface coverage of 79% of its full packed monolayer. At the same time, a linear increase of film thickness with increasing number of layers was also confirmed by SPR characterizations. The data of XPS and IR-RAS show that Zr4+ ions interact with both the phosphate groups and nitrogenous bases of DNA and load into the framework of DNA. Furthermore, the interactions between this composite film and heme protein cytochrome c (Cyt c) were investigated by SPR spectroscopy and electrochemistry. Compared with the adsorption of Cyt c on DNA monolayer, this composite multilayer film can obviously enhance the amount of immobilized Cyt c confirmed by SPR reflectivity-incident angle (R-theta) curves. Cyclic voltammetry (CV) indicates the Cyt c adsorbed on the composite film is electroactive, and the enhancement of peak current in CV indirectly verifies the increase of the amount of immobilized Cyt c.

Supramolecular organized multilayers were constructed by multiwalled carbon nanotubes modified with ferrocene-derivatized poly(allylamine) redox polymer and glucose oxidase by electrostatic self-assembly. From the analysis of voltammetric signals and fluorescence results, a linear increment of the coverage of enzyme per bilayer was estimated, which demonstrated that the multilayer is constructed in a spatially ordered manner. The cyclic voltammograms obtained from the indium tin oxide (ITO) electrodes coated by the (Fc-PAH@CNT/GOx)n multilayers revealed that bioelectrocatalytic response is directly correlated to the number of deposited bilayers; that is, the sensitivity is tunable by controlling the number of bilayers associated with ITO electrodes. The incorporation of redox-polymer-functionalized carbon nanotubes (CNT) into enzyme films resulted in a 6-10-fold increase in the glucose electrocatalytic current; the bimolecular rate constant of FADH2 oxidation (wiring efficiency) was increased up to 12-fold. Impedance spectroscopy data have yielded the electron diffusion coefficient (De) of this nanostructure to be over 10(-8) cm2 s(-1), which is typically higher than those systems without CNT by at least a factor of 10, indicating that electron transport in the new supramolecular architecture was enhanced by communication of the redox active site of enzyme, redox polymer, and CNT.

Myxoma virus (MV) encodes a cell surface protein (M135R) that is predicted to mimic the host alpha/beta interferon receptor (IFN-alpha/beta-R) and thus prevent IFN-alpha/beta from triggering a host antiviral response. This prediction is based on sequence similarity to B18R, the viral IFN-alpha/beta-R from vaccinia virus (VV), which has been demonstrated to bind and inhibit type I interferons. However, M135R is only half the size of VV B18R. All other poxvirus-encoded IFN-alpha/beta-R homologs align only to the amino-terminal half of M135R. Peptide antibodies raised against M135R were used for immunoblotting and immunofluorescence and indicate that M135R is expressed as an early gene and that the product is a cell surface N-linked glycoprotein that is not secreted. In contrast to the predicted properties of M135R as an inhibitor of type I interferon, all binding and inhibition assays designed to demonstrate whether M135R can interact with IFN-alpha/beta have been negative. However, pathogenesis studies with a targeted M135-knockout MV construct (vMyx135KO) indicate that the deletion of M135R severely attenuates MV pathogenesis in the European rabbit. We propose that M135R is an important immunomodulatory virulence factor for myxomatosis but that the target immune ligand is not from the predicted type I interferon family and remains to be identified.

To meet the requirement of surface plasmon resonance (SPR) sensing, controlling the thickness of the gold film is very important. Here, we report an efficient and simple approach to prepare a SPR-active substrate when the thickness of the gold film is larger than the optimizing 50 nm and smaller than 100 nm. This method is based on anodic electrodissolution of gold in electrolyte containing chloride ions. Using this method, the thickness of gold films can be easily changed at a nanometer scale by controlling the number of potential scans and the concentrations of chloride ions in the electrolyte. At the same time, the influence of gold film thickness on the SPR signal is recorded by SPR in real time. To assess the change of the surface roughness and morphology of gold film through anodic electrodissolution, atomic force microscopy was used. The surface roughness of the same Au film before and after anodic electrodissolution is 1.179 and 2.767 nm, respectively. The change of the surface roughness of Au film brings out a slight angle shift of SPR. This indicates that surface electrodissolution of the gold does not affect the character of the original bulk film and this film can be used for SPR experiments. To confirm our expectation, a simple adsorption experiment of cytochrome c (Cyt c) on the gold film treated with anodic electrodissolution modified by 11-mercaptoundecanic acid was carried out. The angle shift of SPR confirmed the adsorption of Cyt c, and the cyclic voltammetry of Cyt c provided a complementary confirmation for the adsorption of Cyt c. These results show that this approach provides a good way to change the thicker gold film to an optimized thickness of SPR sensing. The great advantage brought by this approach is in that it can convert the waste gold films with greater thicknesses fabricated by the vacuum deposition method or other methods into useful materials as active SPR substrates.

Myxoma virus, a member of the poxvirus family, causes lethal infection only in rabbits, but the mechanism underlying the strict myxoma virus species barrier is not known. Here we show that myxoma virus infection of primary mouse embryo fibroblasts elicited extracellular signal-regulated kinase (Erk) signaling, which was integrated to interferon regulatory factor 3 activation and type I interferon induction. We further show that Erk inactivation or disruption of interferon signaling mediated by the transcription factor STAT1 broke the cellular blockade to myxoma virus multiplication. Moreover, STAT1 deficiency rendered mice highly susceptible to lethal myxoma virus infection. Thus, the Erk-interferon-STAT1 signaling cascade elicited by myxoma virus in nonpermissive primary mouse embryo fibroblasts mediates an innate cellular barrier to poxvirus infection.

Subversion or appropriation of cellular signal transduction pathways is a common strategy employed by viruses to promote an environment within infected cells that supports the viral replicative cycle. Using subsets of 3T3 murine fibroblasts previously shown to differ in their ability to support myxoma virus (MV) replication, we investigated the role of host serine-threonine kinases (STKs) as potential mediators of the permissive phenotype. Both permissive and nonpermissive 3T3 cells supported equivalent levels of virion binding, entry, and early virus gene expression, indicating that MV tropism in 3T3 cells was not determined by receptor-mediated entry. In contrast, late virus gene expression and viral DNA replication were selectively compromised in restrictive 3T3 cells. Addition of specific protein kinase inhibitors, many of which shared the ability to influence the activity of the STKs p21-activated kinase 1 (PAK-1) and Raf-1 attenuated MV replication in permissive 3T3 cells. Western blot detection of the phosphorylated forms of PAK-1 (Thr423) and Raf-1 (Ser338) confirmed activation of these kinases in permissive cells after MV infection or gamma interferon treatment, but the activated forms of both kinases were greatly reduced or absent in restrictive 3T3 cells. The biological significance of these activations was demonstrated by using the autoinhibitory domain of PAK-1 (amino acids 83 to 149), expression of which reduced the efficiency of MV infection in permissive 3T3 cells concurrent with a decrease in PAK-1 activation. In comparison, overexpression of a constitutively active PAK-1 (T423E) mutant increased MV replication in restrictive 3T3 cells. These observations suggest that induced signaling via cellular STKs may play important roles in determining the permissiveness of host cells to poxvirus infection.