Inhibition of type I interferon production via suppressing IKK-gamma expression: a new strategy for counteracting host antiviral defense by influenza A viruses?

Blockage of the induction of type I interferons (IFNs) is essential for the success of influenza virus proliferation in host cells. Several molecular mechanisms by which influenza viruses inhibit IFN induction have been characterized. Here we report a potentially new strategy influenza viruses employ to inhibit IFN production during viral infection. Through a two-dimensional gel electrophoresis based proteomic approach, we found that the expression of IκB kinase-gamma (IKKγ) was suppressed by influenza A virus infection in human lung epithelial A549 cells. Silencing of cellular IKKγ by small interfering RNA led to enhanced replication of influenza viruses. Concomitantly, overexpression of IKKγ resulted in increased production of IFNα/ß, whereas influenza virus infection completely eliminated the IKKγ-overexpression-induced production of IFNα/ß. Our results suggest that IKKγ and influenza virus are mutually inhibitory, and influenza viruses may inhibit IFN production through suppressing the expression of IKKγ during viral infection.

Influence of increasing glycine concentrations in reduced crude protein diets fed to broilers from 0 to 48 days.

Two experiments investigated broiler growth performance and processing characteristics when fed increasing Gly concentrations in reduced CP diets fed from 0 to 48 d. In experiment 1, birds were allocated to 1 of 4 dietary treatments: a control (CTL) diet containing feed-grade L-Met, L-Lys, and L-Thr, a reduced CP (RCP) diet with additions of feed-grade L-Val and L-Ile, or the RCP diet with moderate (M Gly) or high Gly (H Gly) inclusion levels to achieve a total Gly + Ser of 100 or 112%, respectively, of the CTL diet. Birds in experiment 2 were assigned to 1 of 6 dietary treatments: a CTL diet, a RCP diet, or a low CP (LCP) diet without or with added Gly to achieve 88, 100, 112, or 124% total Gly + Ser concentrations of the RCP diet. For experiment 1, 0 to 14 d broiler performance was similar (P > 0.05) among dietary treatments. From 0 to 48 d, broilers fed the H Gly diet had the lowest (P = 0.006) body weight gain (BWG) and highest (P = 0.003) feed conversion ratio (FCR). Feeding either the RCP or M Gly diet resulted in similar (P > 0.05) growth and processing characteristics to the CTL. For experiment 2, increasing Gly levels in the LCP diet linearly reduced (P ≤ 0.027) 0 to 14 d FI and FCR. From 0 to 48 d, broilers had similar (P > 0.05) performance when fed the CTL or RCP diet, but had a higher (P < 0.001) FCR when fed the LCP88 diet. Increasing Gly levels linearly reduced (P = 0.033) FCR. Total breast meat yield was negatively affected (P ≤ 0.020) when feeding the LCP88 diet and did not respond to Gly levels. In conclusion, effects of increasing total Gly + Ser levels on 0 to 48 d broiler performance are likely dependent on the content of dietary CP and other potentially interacting nutrients.

Perchlorate detection at nanomolar concentrations by surface-enhanced Raman scattering.

Perchlorate (ClO4-) has emerged as a widespread environmental contaminant and has been detected in various food products and even in human breast milk and urine. This research developed a sensing technique based on surface-enhanced Raman scattering (SERS) for rapid screening and monitoring of this contaminant in groundwater and surface water. The technique was found to be capable of detecting ClO4- at concentrations as low as 10(-9) M (or approximately 0.1 microg/L) by using 2-dimethylaminoethanethiol (DMAE) modified gold nanoparticles as a SERS substrate. Quantitative analysis of ClO4- was validated with good reproducibility by using both simulated and contaminated groundwater samples. When coupled with a portable Raman spectrometer, this technique has the potential to be used as an in situ, rapid screening tool for perchlorate in the environment.

Fabrication of highly ordered TiO2 nanotube arrays using an organic electrolyte.

Anodization of titanium in a fluorinated dimethyl sulfoxide (DMSO) and ethanol mixture electrolyte is investigated. The prepared anodic film has a highly ordered nanotube-array surface architecture. Using a 20 V anodization potential (vs Pt) nanotube arrays having an inner diameter of 60 nm and 40 nm wall thickness are formed. The overall length of the nanotube arrays is controlled by the duration of the anodization, with nanotubes appearing only after approximately 48 h; a 72 h anodization results in a nanotube array approximately 2.3 mum in length. The photoelectrochemical response of the nanotube-array photoelectrodes is studied using a 1 M KOH solution under both UV and visible (AM 1.5) illumination. Enhanced photocurrent density is observed for samples obtained in the organic electrolyte, with an UV photoconversion efficiency of 10.7%.

Detection of viable Salmonella typhimurium by impedance measurement of electrode capacitance and medium resistance.

Three-electrode electrochemical impedance technique was investigated for detection of Salmonella typhimurium by monitoring the growth of bacteria in selenite cystine (SC) broth supplemented with trimethylamine oxide hydrochloride (TMAO.HCl) and mannitol (M). The change in the system impedance during the growth of bacteria was studied using frequency spectral scanning. It was found that the impedance at low frequencies (<10 kHz) mainly came from the double-charged layer capacitance, reflecting the changes at the electrode interface and the adsorption on the electrode surface. While at high frequencies (>10 kHz), the system impedance mainly depended on the medium resistance. The adsorption of bacteria on the electrode surface was detected by measuring low frequency impedance, and verified with Faradic impedance spectroscopy. Enumeration of S. typhimurium using a low frequency (1 Hz) capacitance measurement and a high frequency (1 MHz) resistance measurement were compared. The detection times were determined for quantitative analysis based on the growth curves of bacteria referring to either the medium resistance or electrode capacitance. The regression equations for the detection times (t(d), h) and the initial cell number (N, cells.ml(-1)) were t(d)=-1.24logN+13.4 with R(2)=0.98 and t(d)=-1.40logN+14.46 with R(2)=0.97 for the medium resistance and electrode capacitance methods, respectively.

A staphylococcal enterotoxin B magnetoelastic immunosensor.

A magnetoelastic immunosensor for detection of staphylococcal enterotoxin B (SEB) is described. The magnetoelastic sensor is a newly developed mass/elasticity-based transducer of high sensitivity having a material cost of approximately $0.001/sensor. Affinity-purified rabbit anti-SEB antibody was covalently immobilized on magnetoelastic sensors, of dimensions 6 mm x 2 mm x 28 microm. The affinity reaction of biotin-avidin and biocatalytic precipitation are used to amplify antigen-antibody binding events on the sensor surface. Horseradish peroxidase (HRP) and alkaline phosphatase were examined as the labeled enzymes to induce biocatalytic precipitation. The alkaline phosphatase substrate, 5-bromo-4-chloro-3-indolyl phosphate (BCIP) produces a dimer, which binds tightly to the sensor surface, inducing a change in sensor resonance frequency. The biosensor demonstrates a linear shift in resonance frequency with staphylococcal enterotoxin B concentration between 0.5 and 5 ng/ml, with a detection limit of 0.5 ng/ml.

A magnetoelastic bioaffinity-based sensor for avidin.

A magnetoelastic bioaffinity sensor coupled with biocatalytic precipitation is described for avidin detection. The non-specific adsorption characteristics of streptavidin on different functionalized sensor surfaces are examined. It is found that a biotinylated poly(ethylene glycol) (PEG) interface can effectively block non-specific adsorption of proteins. Coupled with the PEG immobilized sensor surface, alkaline phosphatase (AP) labeled streptavidin is used to track specific binding on the sensor. This mass-change-based signal is amplified by the accumulation on the sensor of insoluble products of 5-bromo-4-chloro-3-indolyl phosphate catalyzed by AP. The resulting mass loading on the sensor surface in turn shifts the resonance frequency of the magnetoelastic sensors, with an avidin detection limit of approximately 200 ng/ml.

Label-free capacitive immunosensor based on quartz crystal Au electrode for rapid and sensitive detection of Escherichia coli O157:H7.

A label-free capacitive immunosensor based on quartz crystal Au electrode was developed for rapid and sensitive detection of Escherichia coli O157:H7. The immunosensor was fabricated by immobilizing affinity-purified anti-E. coli O157:H7 antibodies onto self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (MPA) on the surface of a quartz crystal Au electrode. Bacteria suspended in solution became attached to the immobilized antibodies when the immunosensor was tested in liquid samples. The change in capacitance caused by the bacteria was directly measured by an electrochemical detector. An equivalent circuit was introduced to simulate the capacitive immunosensor. The immunosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The experimental results indicated that the capacitance change was linearly correlated with the cell concentration of E. coli O157:H7. The immunosensor was able to discriminate between cellular concentrations of 10(2)-10(5) cfu mL(-1) and has applications in detecting pathogens in food samples. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were also employed to characterize the stepwise assembly of the immunosensor.

TiO2 nanowire bundle microelectrode based impedance immunosensor for rapid and sensitive detection of Listeria monocytogenes.

A novel TiO 2 nanowire bundle microelectrode based immunosensor was demonstrated as a more sensitive, specific, and rapid technology for detection of Listeria monocytogenes. TiO 2 nanowire bundle was prepared through a hydrothermal reaction of alkali with TiO 2 powder and connected to gold microelectrodes with mask welding. Monoclonal antibodies were immobilized on the surface of a TiO 2 nanowire bundle to specifically capture L. monocytogenes. Impedance change caused by the nanowire-antibody-bacteria complex was measured and correlated to bacterial number. This nanowire bundle based immunosensor could detect as low as 10 (2) cfu/ml of L. monocytogenes in 1 h without significant interference from other foodborne pathogens.

Determination of technetium and its speciation by surface-enhanced Raman spectroscopy.

Technetium-99 (Tc) is an important radionuclide of concern, and there is a great need for its detection and speciation analysis in the environment. For the first time, we report that surface-enhanced Raman spectroscopy (SERS) is capable of detecting an inorganic radioactive anion, pertechnetate (TcO4-), at approximately 10(-7) M concentration levels. The technique also allows the detection of various species of Tc such as oxidized Tc(VII) and reduced and possibly complexed Tc(IV) species by use of gold nanoparticles as a SERS substrate. The primary Raman scattering band of Tc(VII) occurs at about 904 cm-1, whereas reduced Tc(IV) and its humic and ethylenediaminetetraacetic acid (EDTA) complexes show scattering bands at about 866 and 870 cm-1, respectively. Results also indicate that Tc(IV)-humic complexes are unstable and reoxidize to TcO4- upon exposure to oxygen. This study demonstrates that SERS could potentially offer a new tool and opportunity in studying Tc and its speciation and interactions in the environment at low concentrations.

Surface-enhanced Raman spectroscopy for uranium detection and analysis in environmental samples.

Techniques for rapid screening of uranium in environmental samples are needed, and this study entails the development of surface-enhanced Raman scattering (SERS) for analyzing uranium in aqueous media with improved sensitivity and reproducibility. A new SERS substrate based on (aminomethyl)phosphonic acid (APA)-modified gold nanoparticles was found to give greater than three orders of magnitude SERS enhancement compared with unmodified bare gold nanoparticles. Intensities of uranyl band at about 830 cm(-1) were proportional to the concentrations of uranium in solution, especially at relatively low concentrations (<10(-5) M). A detection limit of approximately 8x10(-7) M was achieved with a good reproducibility since the measurement was performed directly in dispersed aqueous suspension. Without pretreatment, the technique was successfully employed for detecting uranium in a highly contaminated groundwater with a low pH, high dissolved salts (e.g., nitrate, sulfate, calcium and aluminum) and total organic carbon.

Controlled fabrication of nanopillar arrays as active substrates for surface-enhanced Raman spectroscopy.

Highly ordered gold nanopillar arrays were fabricated using anodized aluminum oxide (AAO) templates. Nanopillars with a dimension of 110 +/- 15 nm in vertical height and 75 +/- 10 nm in base diameter were formed with a density of 150 microm(-2). The ordered nanopillar arrays give reproducible surface-enhanced Raman scattering (SERS) at a detection limit of 10(-8) M using thionine as probing molecules. The enhancement by the Au nanopillar arrays was comparable with or better than that of dispersed gold nanoparticle SERS substrates. This work demonstrates a new technique for producing highly ordered and reproducible SERS substrates potentially applicable for chemical and biological assay.

In situ bioreduction of uranium (VI) to submicromolar levels and reoxidation by dissolved oxygen.

Groundwater within Area 3 of the U.S. Department of Energy (DOE) Environmental Remediation Sciences Program (ERSP) Field Research Center at Oak Ridge, TN (ORFRC) contains up to 135 microM uranium as U(VI). Through a series of experiments at a pilot scale test facility, we explored the lower limits of groundwater U(VI) that can be achieved by in-situ biostimulation and the effects of dissolved oxygen on immobilized uranium. Weekly 2 day additions of ethanol over a 2-year period stimulated growth of denitrifying, Fe(III)-reducing, and sulfate-reducing bacteria, and immobilization of uranium as U(IV), with dissolved uranium concentrations decreasing to low levels. Following sulfite addition to remove dissolved oxygen, aqueous U(VI) concentrations fell below the U.S. Environmental Protection Agengy maximum contaminant limit (MCL) for drinking water (< 30/microg L(-1) or 0.126 microM). Under anaerobic conditions, these low concentrations were stable, even in the absence of added ethanol. However, when sulfite additions stopped, and dissolved oxygen (4.0-5.5 mg L(-1)) entered the injection well, spatially variable changes in aqueous U(VI) occurred over a 60 day period, with concentrations increasing rapidly from < 0.13 to 2.0 microM at a multilevel sampling (MLS) well located close to the injection well, but changing little at an MLS well located further away. Resumption of ethanol addition restored reduction of Fe(III), sulfate, and U(VI) within 36 h. After 2 years of ethanol addition, X-ray absorption near-edge structure spectroscopy (XANES) analyses indicated that U(IV) comprised 60-80% of the total uranium in sediment samples. Atthe completion of the project (day 1260), U concentrations in MLS wells were less than 0.1 microM. The microbial community at MLS wells with low U(VI) contained bacteria that are known to reduce uranium, including Desulfovibrio spp. and Geobacter spp., in both sediment and groundwater. The dominant Fe(III)-reducing species were Geothrix spp.

Surface-enhanced Raman scattering for perchlorate detection using cystamine-modified gold nanoparticles.

Perchlorate (ClO4-) has recently emerged as a widespread environmental contaminant found in groundwater and surface water, and there is a great need for rapid detection and monitoring of this contaminant. This study presents a new technique using cystamine-modified gold nanoparticles as a substrate for surface-enhanced Raman scattering (SERS) detection of perchlorate at low concentrations. A detection limit of 5x10(-6) M (0.5 mg/L) has been achieved using this method without sample preconcentration. This result was attributed to a strong plasmon enhancement by gold metal surfaces and the electrostatic attraction of ClO4- onto positively charged, cystamine-modified gold nanoparticles at a low pH. The methodology also was found to be reproducible, quantitative, and not susceptible to significant interference from the presence of anions such as sulfate, phosphate, nitrate and chloride at concentrations <1 mM, making it potentially suitable for rapid screening and routine analysis of perchlorate in environmental samples.

Development of gold-silica composite nanoparticle substrates for perchlorate detection by surface-enhanced Raman spectroscopy.

Surface-enhanced Raman spectroscopy (SERS) holds promise for rapid, in situ detection of perchlorate (ClO4-) in the environment if sensitive and reproducible SERS substrates can be developed. In this study, new, functionalized gold-silica (Au-SiO2) composite nanoparticles were synthesized and used as SERS substrates for ClO4- detection. These nanoparticles were composed of a silica core with Au nanoparticles grafted onto the SiO2 spheres by in situ chemical reduction of AuCl4- or physisorption of Au colloids. Chemical coupling agents with such functional groups as -N+(CH3)3 and -NH3+/-NH2 were used to enhance perchlorate sorption onto the substrate and therefore the detection of ClO4-. These new substrates were found to be optically stable and provide a greatly enhanced surface plasmon or SERS, resulting in a detection limit as low as 10(-6) M ClO4- (0.1 mg/L) in water.

Detection of alkaline phosphatase using surface-enhanced Raman spectroscopy.

A new approach was developed to detect the activity of alkaline phosphatase (ALP) enzyme at ultralow concentrations using a surface-enhanced Raman scattering (SERS) technique. The approach is based on the use of gold nanoparticles as a SERS material whereas 5-bromo-4-chloro-3-indolyl phosphate (BCIP) is used as a substrate of ALP. The enzymatic hydrolysis of BCIP led to the formation of indigo dye derivatives, which were found to be highly SERS active. For the first time, we were able to detect ALP at a concentration of approximately 4 x 10(-15) M or at single-molecule levels when ALP was incubated with BCIP for 1 h in the Tris-HCl buffer. The same technique also was successfully employed to detect surface-immobilized avidin, and a detection limit of 10 ng/mL was achieved. This new technique allows the detection of both free and labeled ALP as a Raman probe in enzyme immunoassays, immunoblotting, and DNA hybridization assays at ultralow concentrations.

Immunobiosensor chips for detection of Escherichia coil O157:H7 using electrochemical impedance spectroscopy.

Impedance biosensor chips were developed for detection of Escherichia coli O157:H7 based on the surface immobilization of affinity-purified antibodies onto indium tin oxide (ITO) electrode chips. The immobilization of antibodies onto ITO chips was carried out using an epoxysilane monolayer to serve as a template for chemical anchoring of antibodies. The surface characteristics of chips before and after the binding reaction between the antibodies and antigens were characterized by atomic force microscopy (AFM). The patterns of the epoxysilanes monolayer, antibodies, and E. coli cells were clearly observed from the AFM images. Alkaline phosphatase as the labeled enzyme to anti-E. coli O157:H7 antibody was used to amplify the binding reaction of antibody-antigen on the chips. The biocatalyzed precipitation of 5-bromo-4-chloro-3-indolyl phosphate by alkaline phosphatase on the chips in pH 10 PBS buffer containing 0.1 M MgCl2 increased the electron-transfer resistance for a redox probe of Fe(CN)6(3-/4-) at the electrode-solution interface or the electrode resistance itself. Electrochemical impedance spectroscopy and cyclic voltammetric method were employed to follow the stepwise assembly of the systems and the electronic transduction for the detection of E. coli. The biosensor could detect the target bacteria with a detection limit of 6 x 10(3) cells/mL. A linear response in the electron-transfer resistance for the concentration of E. coli cells was found between 6 x 10(4) and 6 x 10(7) cells/mL.

Magnetoelastic immunosensors: amplified mass immunosorbent assay for detection of Escherichia coli O157:H7.

A mass-sensitive magnetoelastic immunosensor for detection of Escherichia coli O157:H7 is described, based on immobilization of affinity-purified antibodies attached to the surface of a micrometer-scale magnetoelastic cantilever. Alkaline phosphatase is used as a labeled enzyme to the anti-E. coli O157:H7 antibody, amplifying the mass change associated with the antibody-antigen binding reaction by biocatalytic precipitation of 5-bromo-4-chloro-3-indolyl phosphate in a pH 10.0 PBS solution. The detection limit of the biosensor is 10(2) E. coli O157:H7 cells/mL. A linear change in the resonance frequency of the biosensor was found to E. coli O157:H7 concentrations ranging from 10(2) to 10(6) cells/mL.

A wireless, remote query glucose biosensor based on a pH-sensitive polymer.

This paper describes a wireless, remote query glucose biosensor using a ribbonlike, mass-sensitive magnetoelastic sensor as the transducer. The glucose biosensor is fabricated by first coating the magnetoelastic sensor with a pH-sensitive polymer and upon it a layer of glucose oxidase (GOx). The pH-responsive polymer swells or shrinks, thereby changing mass, respectively, in response to increasing or decreasing pH values. The GOx-catalyzed oxidation of glucose produces gluconic acid, inducing the pH-responsive polymer to shrink, which in turn decreases the polymer mass. In response to a time-varying magnetic field, a magnetoelastic sensor mechanically vibrates at a characteristic resonance frequency, the value of which inversely depends on sensor mass loading. As the magnetoelastic films are magnetostrictive, the vibrations launch magnetic flux that can be remotely detected using a pickup coil. Hence, changes in the resonance frequency of a passive magnetoelastic transducer are detected on a remote query basis, without the use of physical connections to the sensors.The sensitivity of the glucose biosensors decreases with increasing ionic strength; at physiological salt concentrations, 0.6 mmol/L of glucose can be measured. At glucose concentrations of 1-10 mmol/L, the biosensor response is reversible and linear, with the detection limit of 0.6 mmol/L corresponding to an error in resonance frequency determination of 20 Hz. Since no physical connections between the sensor and the monitoring instrument are required, this sensor can potentially be applied to in vivo and in situ measurement of glucose concentrations.