Characterization of PA-N terminal domain of Influenza A polymerase reveals sequence specific RNA cleavage.

Influenza virus uses a unique cap-snatching mechanism characterized by hijacking and cleavage of host capped pre-mRNAs, resulting in short capped RNAs, which are used as primers for viral mRNA synthesis. The PA subunit of influenza polymerase carries the endonuclease activity that catalyzes the host mRNA cleavage reaction. Here, we show that PA is a sequence selective endonuclease with distinct preference to cleave at the 3' end of a guanine (G) base in RNA. The G specificity is exhibited by the native influenza polymerase complex associated with viral ribonucleoprotein particles and is conferred by an intrinsic G specificity of the isolated PA endonuclease domain PA-Nter. In addition, RNA cleavage site choice by the full polymerase is also guided by cap binding to the PB2 subunit, from which RNA cleavage preferentially occurs at the 12th nt downstream of the cap. However, if a G residue is present in the region of 10-13 nucleotides from the cap, cleavage preferentially occurs at G. This is the first biochemical evidence of influenza polymerase PA showing intrinsic sequence selective endonuclease activity.

Structural analysis of specific metal chelating inhibitor binding to the endonuclease domain of influenza pH1N1 (2009) polymerase.

It is generally recognised that novel antiviral drugs, less prone to resistance, would be a desirable alternative to current drug options in order to be able to treat potentially serious influenza infections. The viral polymerase, which performs transcription and replication of the RNA genome, is an attractive target for antiviral drugs since potent polymerase inhibitors could directly stop viral replication at an early stage. Recent structural studies on functional domains of the heterotrimeric polymerase, which comprises subunits PA, PB1 and PB2, open the way to a structure based approach to optimise inhibitors of viral replication. In particular, the unique cap-snatching mechanism of viral transcription can be inhibited by targeting either the PB2 cap-binding or PA endonuclease domains. Here we describe high resolution X-ray co-crystal structures of the 2009 pandemic H1N1 (pH1N1) PA endonuclease domain with a series of specific inhibitors, including four diketo compounds and a green tea catechin, all of which chelate the two critical manganese ions in the active site of the enzyme. Comparison of the binding mode of the different compounds and that of a mononucleotide phosphate highlights, firstly, how different substituent groups on the basic metal binding scaffold can be orientated to bind in distinct sub-pockets within the active site cavity, and secondly, the plasticity of certain structural elements of the active site cavity, which result in induced fit binding. These results will be important in optimising the design of more potent inhibitors targeting the cap-snatching endonuclease activity of influenza virus polymerase.

Glycyrrhizin, the main active compound in liquorice, attenuates pro-inflammatory responses by interfering with membrane-dependent receptor signalling.

The triterpene glycoside glycyrrhizin is the main active compound in liquorice. It is used as a herbal medicine owing to its anticancer, antiviral and anti-inflammatory properties. Its mode of action, however, remains widely unknown. In the present study, we aimed to elucidate the molecular mechanism of glycyrrhizin in attenuating inflammatory responses in macrophages. Using microarray analysis, we found that glycyrrhizin caused a broad block in the induction of pro-inflammatory mediators induced by the TLR (Toll-like receptor) 9 agonist CpG-DNA in RAW 264.7 cells. Furthermore, we found that glycyrrhizin also strongly attenuated inflammatory responses induced by TLR3 and TLR4 ligands. The inhibition was accompanied by decreased activation not only of the NF-kappaB (nuclear factor kappaB) pathway but also of the parallel MAPK (mitogen-activated protein kinase) signalling cascade upon stimulation with TLR9 and TLR4 agonists. Further analysis of upstream events revealed that glycyrrhizin treatment decreased cellular attachment and/or uptake of CpG-DNA and strongly impaired TLR4 internalization. Moreover, we found that the anti-inflammatory effects were specific for membrane-dependent receptor-mediated stimuli, as glycyrrhizin was ineffective in blocking Tnfa (tumour necrosis factor alpha gene) induction upon stimulation with PMA, a receptor- and membrane-independent stimulus. These observations suggest that the broad anti-inflammatory activity of glycyrrhizin is mediated by the interaction with the lipid bilayer, thereby attenuating receptor-mediated signalling.

Applicability of the bioluminescence inhibition test in the 96-well microplate format for PAH-solutions and elutriates of PAH-contaminated soils.

The use of conventional plastic microplates for a miniaturised luminescent bacteria test may result in an underestimation of the toxicity for poorly water soluble highly adsorbing toxicants such as PAHs. In this study, the suitability of microplates for testing elutriates of PAH-contaminated soils was investigated. The LUMIStox test was performed as the standard test in the miniaturised format using contaminated soil elutriates and aqueous solutions of four selected PAHs (viz. naphthalene (NAP), acenaphthene (ACE), fluorene (FLU), and phenanthrene (PHE)). For the aqueous PAH-solutions, we observed reduced light inhibition values for the miniaturised bioassay when using black microplates made of polypropylene (PP) and polystyrene (PS) compared to the standard LUMIStox test. This phenomenon was most likely due to adsorption of toxicants to the microplate surfaces with PAHs of lower water solubility being significantly more affected; however, after minimizing the exposure of samples to plastic surfaces, polystyrene microplates revealed equivalent performance (>80% 'relative' light inhibition) to the standard glass cuvette test system. For soil elutriates, black microplates again exhibited slightly lower light inhibition values while white plates made of PS and Barex resulted in a pronounced overestimation of toxicity for a coloured soil elutriate. In general, microplates were applicable for testing elutriates of PAH-contaminated soils. In cases where samples are coloured or turbid, the application of black microplates is recommended.

Priming effects on PAH degradation and ecotoxicity during a phytoremediation experiment.

An experiment was conducted to distinguish priming effects from the effects of phytoremediation of a creosote-polluted soil. The concentration of 13 polycyclic aromatic hydrocarbons (PAHs), and their combined soil toxicity (using four bioassays), was determined on recently excavated, homogenized soil and on such soil subjected to a time-course phytoremediation experiment with lucerne. The results showed a high priming effect, with minor positive and synergistic effects of planting and fertilization on PAH degradation rates. At the end of the experiment, PAH degradation reached 86% of the initial 519 mg PAHs kg(-1). Two of the four toxicity tests (bioluminescence inhibition and ostracod growth inhibition) corroborated the chemical data for residual PAHs, and indicated a significant reduction in soil toxicity. We conclude that priming effects can easily surpass treatment effects, and that an unintentional pre-incubation that ignores these effects can jeopardize the full quantitative assessment of in situ bioremediation of contaminated soil.

Behavior of PAHs during cold storage of historically contaminated soil samples.

The stability of historically polycyclic aromatic hydrocarbon (PAH)-contaminated soils during cold storage was investigated. Samples from two former manufactured gas plants exhibited quantitative recoveries of PAHs over the whole period of sample holding at 4 degrees C in the dark (8-10 months), whereas significant losses of PAHs were observed for soils received from a former railroad sleeper preservation plant with low molecular weight compounds being notably more affected compared to heavier PAHs. Already after 2 weeks of holding time, 3-ring PAHs in one of theses samples were down to 29-73% of the initial concentration and significant losses were observed for up to 5-ring compounds. Dissipation of PAHs was found to be predominantly due to aerobic microbial metabolism since sodium azide poisoned samples showed quantitative recoveries for all PAHs over the entire storage time of 3 months. A similar stabilizing effect was observed for freezing at -20 degrees C as means of preservation. Except for acenaphthene, no significant loss for any of the PAHs was observed over 6 weeks of holding time. Eventually, selected chemical, physical, and biological parameters of two soils were investigated and identified as potential indicators for the stability of PAH-contaminated soil samples.

Toxicity testing of 16 priority polycyclic aromatic hydrocarbons using Lumistox.

Hazard assessment of industrial sites contaminated with coal tar and its products usually focuses on selected pollutants such as the 16 polycyclic aromatic hydrocarbons (PAHs) prioritized by the U.S. Environmental Protection Agency (U.S. EPA). The aim of this study was to investigate to which extent these 16 PAHs contribute to the Vibrio fischeri bioluminescence inhibition measured by the acute Lumistox luminescent bacteria test. Five of the 16 PAHs-naphthalene (NAP), acenaphthylene (ACY), acenaphthene (ACE), fluorene (FLU), and phenanthrene (PHE)-revealed inhibiting effects when measuring saturated aqueous solutions of these compounds. However, in elutriates of PAH-contaminated soils, the amount of leached PAHs was very low, and the 16 PAHs did not considerably contribute to the observed bioluminescence inhibition. Nevertheless, bioluminescence inhibition was higher for elutriates with increased PAH concentration indicating the presence of other toxicants that co-occur with the 16 PAHs. No evidence was observed for increased bioluminescence inhibition due to synergistic effects among PAHs as calculated on the basis of toxic units for an aqueous solution containing all 16 priority PAHs. Data suggest that the U.S. EPA PAHs play only a minor role in causing acute toxicity to V. fischeri when exposed to aqueous elutriates of PAH-contaminated soils.

Sequential supercritical fluid extraction (SSFE) for estimating the availability of high molecular weight polycyclic aromatic hydrocarbons in historically polluted soils.

Sequential supercritical fluid (CO2) extraction (SSFE) was applied to eight historically contaminated soils from diverse sources with the aim to elucidate the sorption-desorption behavior of high molecular weight polycyclic aromatic hydrocarbons (PAHs). The method involved five extraction phases applying successively harsher conditions by increasing fluid temperature and density mobilizing target compounds from different soil particle sites. Two groups of soils were identified based on readily desorbing (available) PAH fractions obtained under mildest extraction conditions (e.g., readily desorbing fractions of fluoranthene and pyrene significantly varied between the soils ranging from <10 to >90%). Moreover, extraction behavior strongly correlated with molecular weight revealing decreasing available PAH fractions with increasing weight. Physicochemical soil parameters such as particle size distribution and organic dry mass were found to have no distinct effect on the sorption-desorption behavior of PAHs in the different soils. However, PAH profiles significantly correlated with readily available pollutant fractions; soils with relatively less mobile PAHs had higher proportions of five- and six-ring PAHs and vice versa. Eventually, biodegradability corresponded well with PAH recoveries under the two mildest extraction phases. However, a quantitative relationship was only established for soils with biodegradable PAHs. Out of eight soils, five showed no biodegradation including the four soils with the lowest fraction of readily desorbing PAHs. Only one soil (which was found to be highly toxic to Vibrio fischeri) did not match the overall pattern showing no PAH biodegradability but large fractions of highly mobile PAHs, concluding that mass transfer limitations may only be one of many factors governing biodegradability of PAHs.

Qualification of a microfluidics-based electrophoretic method for impurity testing of monoclonal antibodies.

In this work, we present a comprehensive evaluation of the Agilent Bioanalyzer, a microfluidics-based electrophoretic device that was used for impurity testing of a monoclonal antibody (mAb). We compared the system to SDS-PAGE, both operated under non-reducing conditions and found a significant improvement of accuracy for the Bioanalyzer. In addition, the latter exhibited a larger assay range and lower limit of quantitation (LOQ) based on a predefined total error limit of +/-30%. However, during method qualification applying a three-factor nested design with two operators performing duplicate measurements per day, each on 4 different days, we observed unpredictable recurring quantitative outliers using the chip-based system. In-depth analysis on multiple runs with various chip lots confirmed the above finding and indicated that most likely on-chip dye labeling and/or post-column background fluorescence elimination are not compatible with the large size of the intact antibody as similar findings were observed for myosin used as upper marker for time correction. Interestingly, after reducing the intact antibody into light and heavy chain, we resolved the outlier issue. Eventually, requalification of the micro-fabricated analytical device under reducing conditions revealed only 1 out of 32 quality control samples (QCs) exceeding the +/-30% total error limits.

Pre-clinical safety evaluation of pyrrolidine dithiocarbamate.

Pyrrolidine dithiocarbamate (PDTC) was examined for its potential in the intranasal treatment of human rhinovirus infections. Prior to clinical testing, a comprehensive non-clinical programme was performed to evaluate the general toxicity of PDTC. The animal experiments included investigations in rodents with study durations ranging from single dose to repeated dosing over a period of 28 days. The routes of administration were intranasal, inhalative, oral and intravenous for single-dose toxicity and pharmacokinetic studies, and intranasal for repeated dose studies. Blood and tissue samples were obtained from PDTC-treated rats to analyse pharmacokinetics and tissue distribution. Accumulation of selected metals due to PDTC treatment was examined in liver, brain, nerves and fat tissues.

Glycyrrhizin inhibits influenza A virus uptake into the cell.

We investigated the mechanism by which glycyrrhizin (GL), the main active component of licorice roots, protects cells from infection with influenza A virus (IAV). We found that GL treatment leads to a clear reduction in the number of IAV-infected human lung cells as well as a reduction in the CCID50 titer by 90%. The antiviral effect, however, was limited to one or two virus replication cycles. Analysis of different GL treatment protocols suggested that the antiviral effect of GL was limited to an early step in the virus replication cycle. A direct inhibitory action of GL on IAV particles could be excluded and GL did not interact with virus receptor binding either. The antiviral effect of GL was abolished by treatment 1h after virus infection, whereas pre-treatment and treatment during and after virus adsorption led to a reduction in the cytopathic effect, reduced viral RNA within the cells and in the cell supernatants, and reduced viral hemagglutination titers. Detailed virus uptake analyses unambiguously demonstrated reduced virus uptake in various GL-treated cells. These observations lead to the conclusion, that the antiviral activity of GL is mediated by an interaction with the cell membrane which most likely results in reduced endocytotic activity and hence reduced virus uptake. These insights might help in the design of structurally related compounds leading to potent anti-influenza therapeutics.

Analysis of polycyclic aromatic hydrocarbons in soil: minimizing sample pretreatment using automated Soxhlet with ethyl acetate as extraction solvent.

A simplified sample pretreatment method for industrially PAH-contaminated soils applying automated Soxhlet (Soxtherm) with ethyl acetate as extraction solvent is presented. Laborious pretreatment steps such as drying of samples, cleanup of crude extracts, and solvent exchange were allowed to be bypassed without notable performance impact. Moisture of the soil samples did not significantly influence recoveries of PAHs at a wide range of water content for the newly developed method. However, the opposite was true for the standard procedure using the more apolar 1:1 (v/v) n-hexane/acetone solvent mixture including postextraction treatments recommended by the U.S. EPA. Moreover, ethyl acetate crude extracts did not appreciably effect the chromatographic performance (HPLC-(3D)FLD), which was confirmed by a comparison of the purity of PAH spectra from both pretreatment methods. Up to 20% (v/v) in acetonitrile, ethyl acetate proved to be fully compatible with the mobile phase of the HPLC whereas the same concentration of n-hexane/acetone in acetonitrile resulted in significant retention time shifts. The newly developed pretreatment method was applied to three historically contaminated soils from different sources with extraction efficiencies not being significantly different compared to the standard procedure. Finally, the certified reference soil CRM 524 was subjected to the simplified procedure resulting in quantitative recoveries (>92%) for all PAHs analyzed.