Mass transport of lipopolysaccharide induced H2O2 detected by an intracellular carbon nanoelectrode sensor.

Hydrogen peroxide is a key component of the innate immune response, regulating how a cell responds to a bacterial threat; however, being transient in nature makes it extremely difficult to detect. We show the development of an improved biosensor capable of the rapid detection of the hydrogen peroxide produced intracellularly in response to both smooth and rough lipopolysaccharides (LPS) structures. The arising signal and mass transport behaviour to the electrodes were characterised. This response was detected utilising a single walled carbon nanotube-based sensor that has been functionalised with an osmium complex for specificity and detecting the change in intracellular concentrations of hydrogen peroxide through chronoamperometry. This was conducted within murine macrophage (RAW264.7) cells and using ultra-pure LPS extracted from two different serotypes of bacteria (0111:B4 and Re495). This allowed the comparison of the immune response when infected with different structures of LPS. We demonstrate that the hydrogen peroxide signal can be electrochemically detected within 3 seconds post injection. Combining the nature of the mass transport of hydrogen peroxide and concentration characteristics, a bacterial 'fingerprint' was identified. The impact of this work will be demonstrated in allowing us to develop a rapid diagnostic for bacterial detection.

Drumming-associated anthrax incidents: exposures to low levels of indoor environmental contamination.

Two fatal drumming-related inhalational anthrax incidents occurred in 2006 and 2008 in the UK. One individual was a drum maker and drummer from the Scottish Borders, most likely infected whilst playing a goat-skin drum contaminated with Bacillus anthracis spores; the second, a drummer and drum maker from East London, likely became infected whilst working with contaminated animal hides.We have collated epidemiological and environmental data from these incidents and reviewed them alongside three similar contemporaneous incidents in the USA. Sampling operations recovered the causative agent from drums and drum skins and from residences and communal buildings at low levels. From these data, we have considered the nature of the exposures and the number of other individuals likely to have been exposed, either to the primary infection events or to subsequent prolonged environmental contamination (or both).Despite many individual exposures to widespread low-level spore contamination in private residences and in work spaces for extended periods of time (at least 1 year in one instance), only one other individual acquired an infection (cutaneous). Whilst recognising the difficulty in making definitive inferences from these incidents to specific residual contamination levels, and by extending the risk to public health, we believe it may be useful to reflect on these findings when considering future incident management risk assessments and decisions in similar incidents that result in low-level indoor contamination.

Comparison of a novel microcrystalline tyrosine adjuvant with aluminium hydroxide for enhancing vaccination against seasonal influenza.

BACKGROUND: Vaccination against seasonal influenza strains is recommended for "high risk" patient groups such as infants, elderly and those with respiratory or circulatory diseases. However, efficacy of the trivalent influenza vaccine (TIV) is poor in many cases and in the event of an influenza pandemic, mono-valent vaccines have been rapidly developed and deployed. One of the main issues with use of vaccine in pandemic situations is the lack of a suitable quantity of vaccine early enough during the pandemic to exert a major influence on the transmission of virus and disease outcome. One approach is to use a dose-sparing regimen which inevitably involves enhancing the efficacy using adjuvants. METHODS: In this study we compare the use of a novel microcrystalline tyrosine (MCT) adjuvant, which is currently used in a niche area of allergy immunotherapy, for its ability to enhance the efficacy of a seasonal TIV preparation. The efficacy of the MCT adjuvant formulation was compared to alum adjuvanted TIV and to TIV administered without adjuvant using a ferret challenge model to determine vaccine efficacy. RESULTS: The MCT was found to possess high protein-binding capacity. In the two groups where TIV was formulated with adjuvant, the immune response was found to be higher (as determined by HAI titre) than vaccine administered without adjuvant and especially so after challenge with a live influenza virus. Vaccinated animals exhibited lower viral loads (as determined using RT-PCR) than control animals where no vaccine was administered. CONCLUSIONS: The attributes of each adjuvant in stimulating single-dose protection against a poorly immunogenic vaccine was demonstrated. The properties of MCT that lead to the reported effectiveness warrants further exploration in this and other vaccine targets - particularly where appropriate immunogenic, biodegradable and stable alternative adjuvants are sought.

Electrochemical communication with the inside of cells using micro-patterned vertical carbon nanofibre electrodes.

With the rapidly increasing demands for ultrasensitive biodetection, the design and applications of new nano-scale materials for development of sensors based on optical and electrochemical transducers have attracted substantial interest. In particular, given the comparable sizes of nanomaterials and biomolecules, there exist plenty of opportunities to develop functional nanoprobes with biomolecules for highly sensitive and selective biosensing, shedding new light on cellular behaviour. Towards this aim, herein we interface cells with patterned nano-arrays of carbon nanofibers forming a nanosensor-cell construct. We show that such a construct is capable of electrochemically communicating with the intracellular environment.

A comprehensive study on the role of the Yersinia pestis virulence markers in an animal model of pneumonic plague.

We determined the role of Yersinia pestis virulence markers in an animal model of pneumonic plague. Eleven strains of Y. pestis were characterized using PCR assays to detect the presence of known virulence genes both encoded by the three plasmids as well as chromosomal markers. The virulence of all Y. pestis strains was compared in a mouse model for pneumonic plague. The presence of all known virulence genes correlated completely with virulence in the Balb/c mouse model. Strains which lacked HmsF initially exhibited visible signs of disease whereas all other strains (except wild-type strains) did not exhibit any disease signs. Forty-eight hours post-infection, mice which had received HmsF(-) strains regained body mass and were able to control infection; those infected with strains possessing a full complement of virulence genes suffered from fatal disease. The bacterial loads observed in the lung and other tissues reflected the observed clinical signs as did the cytokine changes measured in these animals. We can conclude that all known virulence genes are required for the establishment of pneumonic plague in mammalian animal models, the role of HmsF being of particular importance in disease progression.

Differential identification of Bacillus anthracis from environmental Bacillus species using microarray analysis.

AIMS: To determine whether microarray analysis could be employed for the differential identification of a range of environmental Bacillus sp. from four strains of Bacillus anthracis. METHODS AND RESULTS: Oligonucleotide probes were designed that were specific to virulence factor genes of B. anthracis (pag, lef and cap), the variable number tandem repeat region of the B. anthracis vrrA gene and to the 16S-23S rRNA intergenic transcribed spacer region (ITS) and pleiotropic regulator (plcR) regions of the Bacillus cereus subgroup species. Generic probes were also designed to hybridize with conserved regions of the 16S rRNA genes of Bacillus (as a positive control), Neisseria sp., Pseudomonas sp., Streptococcus sp., Mycobacterium sp. and to all members of the Enterobacteriaceae to allow simultaneous detection of these bacteria. Identification of B. anthracis was found to rely entirely on hybridization of DNA specific to regions of the pag, lef and cap genes. Cross-reaction was observed between B. anthracis and other Bacillus species with all the other Bacillus probes tested. Results obtained using microarray hybridizations were confirmed using conventional microbiological techniques and found to have very high comparability. CONCLUSIONS: Microarray-based assays are an effective method for the identification of B. anthracis from mixed-culture environmental samples without problems of false-positivity that have been observed with conventional PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of environmental Bacillus sp. by conventional PCR is prone to potential for reporting false-positives. This study provides a method for the exclusion of such isolates.

Novel in vitro functional assays for the determination of anthrax toxin components.

The characterisation and evaluation of the UK licensed human anthrax vaccine depends on several in vivo tests that determine its safety and potency. Assays for the determination of functionally active and/or immunoreactive toxin components and S-layer proteins have been developed and applied to the characterisation of anthrax vaccine. These technologies may support production of consistent and effective vaccines, and may ultimately reduce the requirements for in vivo testing.

Tyrosine-1290 of tetanus neurotoxin plays a key role in its binding to gangliosides and functional binding to neurones.

Tetanus toxin acts by blocking the release of glycine from inhibitory neurones within the spinal cord. An initial stage in the toxin's action is binding to acceptors on the nerve surface and polysialogangliosides are a component of these acceptor moieties. Using site-directed mutagenesis, we identify tyrosine-1290 of tetanus toxin as a key residue that is involved in ganglioside binding. This residue, which is located at the centre of a shallow pocket on the beta-trefoil domain of the tetanus H(c) fragment, is also shown to play a key role in the functional binding of tetanus toxin to spinal cord neurones leading to the inhibition of neurotransmitter release.

Biophysical targeting of adenovirus vectors for gene therapy.

Advances in understanding the interaction of animal viruses with their cognate receptors has led to improvements in the development of cell-specific, targeted viral vectors. Research strategies to generate safe, non-inflammatory viral vectors that are capable of delivering a therapeutic gene to a specific population of cells are currently underway in many laboratories. One approach in the utilization of this cell targeting activity is to ablate the natural interaction of the virus with its native receptor, although this is not an absolute requirement. The initial development of 'viral targeting strategies' was based on the view that by modifying the viral protein/receptor interaction, it would be possible to redirect virus vectors to new host cells. As the understanding of virus/cell interactions increased it was observed, however, that many viruses can use different entry mechanisms for cell attachment and penetration. Adenovirus vectors have been used extensively for the delivery of genes to cells. The entry mechanism for adenoviruses into cells has recently been studied and is relatively well understood, however, there are many aspects of cell receptor/virus interactions, which have still to be elucidated. The single high-affinity receptor on mammalian cells for adenovirus type 5 is recognized as the coxsackie and adenovirus receptor. However, in the absence of coxsackie and adenovirus receptor other receptors are used. A thorough understanding of the biology of adenoviruses is essential in the further development of their use as vectors for cell targeting. One strategy is to modify the viral capsid, either through coating the surface using bispecific antibodies, or by chemically crosslinking the targeting ligand onto the virion surface. Another approach is to genetically modify the virus by incorporating the targeting ligand into the viral 'spike' (fiber) protein. This involves manipulating the adenovirus genome and generating a new targeting ligand on the surface of the fiber protein using recombinant DNA technology. The penton base protein has also received attention as a means of directing adenoviruses via insertion of novel targeting ligands.

An immunological approach to detect phosphate stress in populations and single cells of photosynthetic picoplankton.

In the marine cyanobacterium Synechococcus sp. strain WH7803, PstS is a 32-kDa cell wall-associated phosphate-binding protein specifically synthesized under conditions of restricted inorganic phosphate (P1) availability (D. J. Scanlan, N. H. Mann, and N. G. Carr, Mol. Microbiol. 10:181-191, 1993). We have assessed its use as a potential diagnostic marker for the P status of photosynthetic picoplankton. Expression of PstS in Synechococcus sp. strain WH7803 was observed when the P1 concentration fell below 50 nM, demonstrating that the protein is induced at concentrations of P1 typical of oligotrophic conditions. PstS expression could be specifically detected by use of standard Western blotting (immunoblotting) techniques in natural mesocosm samples under conditions in which the N/P ratio was artificially manipulated to force P depletion. In addition, we have developed an immunofluorescence assay that can detect PstS expression in single Synechococcus cells both in laboratory cultures and natural samples. We show that antibodies raised against PstS cross-react with P-depleted Prochlorococcus cells, extending the use of these antibodies to both major groups of prokaryotic photosynthetic picoplankton. Furthermore, DNA sequencing of a Prochlorococcus pstS homolog demonstrated high amino acid sequence identity (77%) with the marine Synechococcus sp. strain WH7803 protein, including those residues in Escherichia coli PstS known to be directly involved in phosphate binding.

ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803.

Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP+ and, to a lesser extent, NAD+, suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [32P]NAD+ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.

Rubisco but not Rubisco activase is clustered in the carboxysomes of the cyanobacterium Synechococcus sp. PCC 7942: Mud-induced carboxysomeless mutants.

The Mud technology of Groisman and Casadaban was adapted to the cyanobacterium Synechococcus sp. PCC 7942. A new high-CO2-requiring (hcr) mutant, hcr Mu28 was isolated following the integration of the Mud element 89 bp upstream of ORFI, at the 5'-flanking region of the rbc operon, which encodes RuBP carboxylase/oxygenase (Rubisco). The integration involved a 7 bp duplication that formed a direct repeat at the integration site, as previously shown in Escherichia coli. The mutant was devoid of apparent carboxysome bodies, which are considered to be important for the availability of CO2 for Rubisco. Immunolabelling studies demonstrated that Rubisco was distributed throughout hcr Mu28 cells, while in the wild type (WT) and in the carboxysome aberrant mutant hcr O221, Rubisco was markedly associated with the carboxysomes. Rubisco activase, however, was evenly distributed throughout the cytosol of the hcr and WT cells, without any preferential association with the apparent carboxysomes.

Bicarbonate Concentration by Synechocystis PCC6803 : Modulation of Protein Phosphorylation and Inorganic Carbon Transport by Glucose.

The ability of the cyanobacterium Synechocystis PCC6803 to transport inorganic carbon in the form of bicarbonate rapidly decreased following a shift from bicarbonate-limited growth to either excess bicarbonate supply or to photoheterotrophic growth on glucose. Nonmetabolizable analogs of glucose did not exert this effect. The rate at which the bicarbonate uptake rate declined was too rapid to be accounted for by dilution of the activity by culture growth and suggested that posttranslational modification may be involved. Several proteins that were unphosphorylated during bicarbonate-limited growth became phosphorylated during the shifts to high CO(2) conditions and to photoheterotrophic growth. A similar alteration in the profile of phosphopolypeptides was observed following a shift into the dark. The changes in protein phosphorylation were not blocked by chloramphenicol or rifampicin.